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POLYNUCLEOTIDES AND POLYPEPTIDE SEQUENCES INVOLVED IN
CANCER
FIELD OF THE INVENTION
The present invention relates to polynucleotide and polypeptide sequences
which are differentially expressed in cancer compared to normal cells. The
present
invention more particularly relates to the use of these sequences in the
diagnosis,
prognosis or treatment of cancer and in the detection of cancer cells.
BACKGROUND OF THE INVENTION
Among gynecologic malignancies, ovarian cancer accounts for the highest
tumor-related mortality in women in the United States (Jemal et al., 2005). It
is the
fourth leading cause of cancer-related death in women in the U.S (Menon et
al., 2005).
The American Cancer Society estimated a total of 22,220 new cases in 2005 and
attributed 16,210 deaths to the disease (Bonome et al., 2005). For the past 30
years,
the statistics have remained largely the same - the majority of women who
develop
ovarian cancer will die of this disease (Chambers and Vanderhyden, 2006). The
disease carries a 1:70 lifetime risk and a mortality rate of >60% (Chambers
and
Vanderhyden, 2006). The high mortality rate is due to the difficulties with
the early
detection of ovarian cancer when the malignancy has already spread beyond the
ovary. Indeed, >80% of patients are diagnosed with advanced staged disease
(stage
III or IV) (Bonome et al., 2005). These patients have a poor prognosis that is
reflected
in <45% 5-year survival rate, although 80% to 90% will initially respond to
chemotherapy (Berek et at., 2000). This increased success compared to 20% 5-
year
survival rate years earlier is, at least in part, due to the ability to
optimally debulk tumor
tissue when it is confined to the ovaries, which is a significant prognostic
factor for
ovarian cancer (Bristow R. E., 2000 and Brown et al., 2004). In patients who
are
diagnosed with early disease (stage I), the 5-yr survival ranges from >90
(Chambers
and Vanderhyden, 2006).
Ovarian cancer comprises a heterogeneous group of tumors that are derived
from the surface epithelium of the ovary or from surface inclusions. They are
classified
into serous, mucinous, endometrioid, clear cell, and Brenner (transitional)
types
corresponding to the different types of epithelia in the organs of the female
reproductive tract (Shih and Kurman, 2005). Of these, serous tumors account
for
¨60% of the ovarian cancer cases diagnosed. Each histologic subcategory is
further
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divided into three groups: benign, intermediate (borderline tumor or low
malignancy
potential (LMP)), and malignant, reflecting their clinical behavior (Seidman
et al.,
2002). LMP represents 10% to 15% of tumors diagnosed as serous and is a
conundrum as they display atypical nuclear structure and metastatic behavior,
yet they
s are considerably less aggressive than high-grade serous tumors. The 5-
year survival
for patients with LMP tumors is 95% in contrast to a <45% survival for
advanced high-
grade disease over the same period (Berek et al., 2000).
Despite improved knowledge of the etiology of the disease, aggressive
cytoreductive surgery, and modern combination chemotherapy, there has been
only
to little change in mortality. Poor outcomes have been attributed to (1)
lack of adequate
screening tests for early disease detection, in combination with only subtle
presentation of symptoms at this stage - diagnosis is frequently being made
only after
progression to later stages, at which point the peritoneal dissemination of
the cancer
limits effective treatment and (2) the frequent development of resistance to
standard
15 chemotherapeutic strategies limiting improvement in the 5-year survival
rate of
patients. The initial chemotherapy regimen for ovarian cancer includes the
combination
of carboplatin (Paraplatin) and paclitaxel (taxol). Years of clinical trials
have proved
this combination to be most effective after effective surgery - reduces tumor
volume in
about 80% of the women with newly diagnosed ovarian cancer and 40% to 50% will
20 have complete regression - but studies continue to look for ways to
improve it. Recent
abdominal infusion of chemotherapeutics to target hard-to-reach cells in
combination
with intravenous delivery has increased the effectiveness. However, severe
side
effects often lead to an incomplete course of treatment Some other
chemotherapeutic
agents include doxorubicin, cisplatin, cyclophosphamide, bleomycin, etoposide.
25 vinblastine, topotecan hydrochloride, ifosfamide, 5-fluorouracil and
melphalan The
excellent survival rates for women with early stage disease receiving
chemotherapy
provide a strong rationale for research efforts to develop strategies to
improve the
detection of ovarian cancer. Furthermore, the discovery of new ovarian cancer-
related
biomarkers will lead to the development of more effective therapeutic
strategies with
30 minimal side effects for the future treatment of ovarian cancer.
Presently, the diagnosis of ovarian cancer is accomplished, in part, through
routine analysis of the medical history of patients and by performing
physical,
ultrasound and x-ray examinations, and hematological screening. Two
alternative
strategies have been reported for early hematological detection of serum
biomarkers.
35 One approach is the analysis of serum samples by mass spectrometry to
find proteins
or protein fragments of unknown identity that detect the presence or absence
of cancer
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(Mor et al., 2005 and Kozak et al., 2003). However, this strategy is expensive
and not
broadly available. Alternatively, the presence or absence of known
proteins/peptides in
the serum is being detected using antibody microarrays, ELISA, or other
similar
approaches. Serum testing for a protein biomarker called CA-125 (cancer
antigen-125)
has long been widely performed as a marker for ovarian cancer. However,
although
ovarian cancer cells may produce an excess of these protein molecules, there
are
some other cancers, including cancer of the fallopian tube or endometrial
cancer
(cancer of the lining of the uterus), 60% of people with pancreatic cancer,
and 20%-
25% of people with other malignancies with elevated levels of CA-125. The CA-
125
i0 test only
returns a true positive result for about 50% of Stage I ovarian cancer
patients
and has a80% chance of returning true positive results from stage II, III, and
IV ovarian
cancer patients. The other 20% of ovarian cancer patients do not show any
increase in
CA-125 concentrations. In addition, an elevated CA-125 test may indicate other
benign
activity not associated with cancer, such as menstruation, pregnancy, or
endometriosis. Consequently, this test has very limited clinical application
for the
detection of early stage disease when it is still treatable, exhibiting a
positive predictive
value (PPV) of <10%. And, even with the addition of ultrasound screening to CA-
125,
the PPV only improves to around 20% (Kozak et al., 2003). Thus, this test is
not an
effective screening test.
/0 Other studies
have yielded a number of biomarker combinations with
increased specificity and sensitivity for ovarian cancer relative to CA-125
alone
(McIntosh et al., 2004, Woolas et al., 1993, Schorge et., 2004). Serum
biomarkers that
are often elevated in women with epithelial ovarian cancer, but not
exclusively, include
carcinoembryonic antigen, ovarian cystadenocarcinoma antigen, lipidassociated
sialic
acid, NB/70,TAG72.3, CA-15.3, and CA-125. Unfortunately, although this
approach
has increased the sensitivity and specificity of early detection, published
biomarker
combinations still fail to detect a significant percentage of stage I/11
epithelial ovarian
cancer. Another study (Elieser et al., 2005) measured serum concentrations of
46
biomarkers including CA-125 and amongst these, 20 proteins in combination
correctly
recognized more than 98% of serum samples of women with ovarian cancer
compared
to other benign pelvic disease. Although other malignancies were not included
in this
study, this multimarker panel assay provided the highest diagnostic power for
early
detection of ovarian cancer thus far.
Additionally, with the advent of differential gene expression analysis
technologies, for example DNA microarrays and subtraction methods, many groups
have now reported large collections of genes that are upregulated in
epithelial ovarian
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cancer (United States Patent Application published under numbers; 20030124579,
20030087250, 20060014686, 20060078941, 20050095592, 20050214831,
20030219760, 20060078941, 20050214826). However, the clinical utilities with
respect to ovarian cancer of one or combinations of these genes are not as yet
fully
determined.
There is a need for new tumor biomarkers for improving diagnosis and/or
prognosis of cancer. In addition, due to the genetic diversity of tumors, and
the
development of chemoresistance by many patients, there exists further need for
better
and more universal therapeutic approaches for the treatment of cancer.
Molecular
targets for the development of such therapeutics may preferably show a high
degree of
specificity for the tumor tissues compared to other somatic tissues, which
will serve to
minimize or eliminate undesired side effects, and increase the efficacy of the
therapeutic candidates.
This present invention tries to address these needs and other needs.
SUMMARY OF THE INVENTION
In accordance with the present invention, there is provided new
polynucleotide sequences and new polypeptide sequences as well as
compositions,
antibodies specific for these sequences, vectors and cells comprising a
recombinant
form of these new sequences.
The present invention also provides methods of detecting cancer cells using
single or multiple polynucleotides and/or polypeptide sequences which are
specific to
these tumor cells. Some of
the polynucleotides and/or polypeptides sequences
provided herein are differentially expressed in ovarian cancer compared to
normal
cells and may also be used to distinguish between malignant ovarian cancer and
an
ovarian cancer of a low malignancy potential and/or a normal state (individual
free of
ovarian cancer).
Also encompassed by the present invention are diagnostic methods,
prognostic methods, methods of detection, kits, arrays, librairies and assays
which
comprises one or more polypeptide and/or polynucleotide sequences or
antibodies
described herein as well as new therapeutic avenues for cancer treatment.
The Applicant has come to the surprising discovery that polynucleotide and/or
polypeptide sequences .described herein are preferentially upregulated in
malignant
ovarian cancer compared to low malignancy potential ovarian cancer and/or
compared
to normal cells. More
interestingly, some of these sequences appear to be
overexpressed in late stage ovarian cancer.
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The Applicant has also come to the surprising discovery that some of the
sequences described herein are not only expressed in ovarian cancer cells but
in other
cancer cells such as cells from breast cancer, prostate cancer, renal cancer,
colon
cancer, lung cancer, melanoma, leukemia and from cancer of the central nervous
system. As such, several of these sequences, either alone or in combination
may
represent universal tumor markers. Therefore, some NSEQs and PSEQs described
herein not only find utility in the field of ovarian cancer detection and
treatment but also
in the detection and treatment of other types of tumors
Therefore, using NSEQs or PSEQs of the present invention, one may readily
identify a cell as being cancerous. As such NSEQs or PSEQs may be used to
identify
a cell as being a ovarian cancer cell, a prostate cancer cell, a breast cancer
cell, a lung
cancer cell, a colon cancer cell, a renal cancer cell, a cell from a melanoma,
a
leukemia cell or a cell from a cancer of the central nervous system.
Even more particularly, NSEQs or PSEQs described herein may be used to
identify a cell as being a malignant ovarian cancer or a low malignant
potential ovarian
cancer.
The presence of some NSEQs or PSEQs in ovarian cancer cell may
preferentially be indicative that the ovarian cancer is of the malignant type.
Some
NSEQs or PSEQs of the present invention may also more particularly indicate
that the
cancer is a late-stage malignant ovarian cancer.
The NSEQs or PSEQs may further be used to treat cancer or to identify
compounds useful in the treatment of cancer including, ovarian cancer (i.e,
LMP
and/or malignant ovarian cancer), prostate cancer, breast cancer, lung cancer,
colon
cancer, renal cancer, melanoma, leukemia or cancer of the central nervous
system.
As used herein and in some embodiments of the invention, the term "NSEQ"
refers generally to polynucleotides sequences comprising or consisting of any
one of
SEQ. ID. NOs:1 to 49, and 169 (e.g., an isolated form) or comprising or
consisting of a
fragment of any one of SEQ. ID. NOs: 1 to 49 and 169. The term "NSEQ" more
particularly refers to a polynucleotide sequence comprising or consisting of a
transcribed portion of any one of SEQ. ID. NOs:1 to 49 and 169, which may be,
for
example, free of untranslated or untranslatable portion(s) (i.e., a coding
portion of any
one of SEQ ID Nos.: 1-49 and 169). The term "NSEQ" additionally refers to a
sequence substantially identical to any one of the above and more particularly
substantially identical to polynucleotide sequence comprising or consisting of
a
transcribed portion of any one of SEQ. ID. NOs:1 to 49 and 169, which may be,
for
example, free of untranslated or untranslatable portion(s). The term
"NSEQ"
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additionally refers to a nucleic acid sequence region of any one of SEQ. ID.
NOs:1 to
49 and 169 which encodes or is able to encode a polypeptide. The term "NSEQ"
also
refers to a polynucleotide sequence able to encode any one of the polypeptides
described herein or a polypeptide fragment of any one of the above. Finally,
the term
"NSEQ" refers to a sequence substantially complementary to any one of the
above.
In other embodiments of the invention such as those which relate to detection
and/or treatment of cancers other than ovarian cancer, NSEQ may also relates
to SEQ
ID NO. :50 including any polynucleotide comprising or consisting of SEQ. ID.
NO:50
(e.g., an isolated form) or comprising or consisting of a fragment of any one
of SEQ.
ID. NO:50, such as a polynucleotide sequence comprising or consisting of a
transcribed portion of any one of SEQ. ID. NO:50, which may be, for example,
free of
untranslated or untranslatable portion(s) (i.e., a coding portion of SEQ. ID.
NO:50).
The term "NSEQ" additionally refers to a sequence substantially identical to
any one of
the above and more particularly substantially identical to polynucleotide
sequence
comprising or consisting of a transcribed portion of SEQ. ID. NO:50, which may
be, for
example, free of untranslated or untranslatable portion(s). The term "NSEQ"
additionally refers to a nucleic acid sequence region of SEQ. ID. NO:50 which
encodes
or is able to encode a polypeptide. Finally, the term "NSEQ" refers to a
sequence
substantially complementary to any one of the above.
As such, in embodiments of the invention NSEQ encompasses, for example,
SEQ. ID. NOs:1 to 49, 50 and 169 and also encompasses polynucleotide sequences
which comprises, are designed or derived from SEQ. ID. NOs:1 to 49, 50 or 169.
Non-
limiting examples of such sequences includes, for example, SEQ ID NOs.: 103-
150 or
151-152.
The term "inhibitory NSEQ" generally refers to a sequence substantially
complementary to any one of SEQ. ID. NOs:1 to 49, 50 or 169, substantially
complementary to a fragment of any one of SEQ. ID. Nos: 1 to 49, 50 or 169,
substantially complementary to a sequence substantially identical to SEQ. ID.
NOs:1
to 49, 50 or 169 and more particularly, substantially complementary to a
transcribed
portion of any one of SEQ. ID. NOs:1 to 49, 50 or 169 (e.g., .which may be
free of
unstranslated or untranslatable portion) and which may have attenuating or
even
inhibitory action againts the transcription of a mRNA or against expression of
a
polypeptide encoded by a corresponding SEQ ID NOs.:1 to 49, 50 or 169.
Suitable
"inhibitory NSEQ" may have for example and without limitation from about 10 to
about
30 nucleotides, from about 10 to about 25 nucleotides or from about 15 to
about 20
nucleotides.
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As used herein the term "PSEQ" refers generally to each and every
polypeptide sequences mentioned herein such as, for example, any polypeptide
sequences encoded (putatively encoded) by any one of NSEQ described herein
(e.g.,
any one of SEQ. ID. NOs:1 to 49 or 169) including their isolated or
substantially
purified form. Therefore, in embodiments of the invention, a polypeptide
comprising or
consisting of any one of SEQ. ID. NOs:51 to 88 or 170 including variants
(e.g., an
isolated natural protein variant), analogs, derivatives and fragments thereof
are
collectively referred to herein as "PSEQ". In other embodiments of the
invention, such
as those related to detection and/or treatment of cancers other than ovarian
cancer,
PSEQ also refers to polypeptide comprising or consisting of SEQ ID NO.:89
including
variants (e.g., an isolated natural protein variant), analogs, derivatives and
fragments.
Some of the NSEQs or PSEQs described herein have been previously
characterized for purposes other than those described herein. As such
dignostics and
therapeutics which are known to target those NSEQs or PSEQs (e.g., antibodies
and/or inhibitors) may thus now be applied for inhibition of these NSEQ or
PSEQ in the
context of treatment of ovarian cancer, prostate cancer, renal cancer, colon
cancer,
lung cancer, melanoma, leukemia or cancer of the central nervous system. The
use
of these known therapeutics and diagnostics for previously undisclosed utility
such as
those described herein is encompassed by the present invention.
70 For example,
antibodies capable of binding to folate receptor-1 may thus be
used for specific binding of tumor cells other than ovarian cancer cells, such
as breast
cancer, prostate cancer, renal cancer, colon cancer, lung cancer, melanoma,
leukemia
and from cancer of the central nervous system. As such the use of antibodies
and/or
inhibitors of folate receptor-1 (e.g.. CB300638, C8300945 which are
Cyclopenta[glquinazoline-based Thymidylate Synthase Inhibitor, those described
in
US20040242606, US20050009851, etc.) in the use of treatment of prostate
cancer,
renal cancer, colon cancer, lung cancer, melanoma, leukemia and cancer of the
central nervous system is encompassed by the present invention
NON-LIMITATIVE EXEMPLARY EMBODIMENTS OF THE INVENTION
Use of NSEQ as a Screening Tool
The NSEQ described herein may be used either directly or in the
development of tools for the detection and isolation of expression products
(mRNA,
mRNA precursor, hnRNA, etc.), of genomic DNA or of synthetic products (cDNA,
PCR
fragments, vectors comprising NSEQ etc.). NSEQs may also be used to prepare
suitable tools for detecting an encoded polypeptide or protein. NSEQ may thus
be
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used to provide an encoded polypeptide and to generate an antibody specific
for the
polypeptide.
Those skilled in the art will also recognize that short oligonucleotides
sequences may be prepared based on the polynucleotide sequences described
herein. For example, oligonucleotides having 10 to 20 nucleotides or more may
be
prepared for specifically hybridizing to a NSEQ having a substantially
complementary
sequence and to allow detection, identification and isolation of nucleic
sequences by
hybridization. Probe sequences of for example, at least 10-20 nucleotides may
be
prepared based on a sequence found in any one of SEQ ID NO.:1 to 49, 50 or 169
and more particularly selected from regions that lack homology to undesirable
sequences. Probe sequences of 20 or more nucleotides that lack such homology
may
show an increased specificity toward the target sequence. Useful hybridization
conditions for probes and primers are readily determinable by those of skill
in the art.
Stringent hybridization conditions encompassed herewith are those that may
allow
hybridization of nucleic acids that are greater than 90% homologous but which
may
prevent hybridization of nucleic acids that are less than 70% homologous. The
specificity of a probe may be determined by whether it is made from a unique
region, a
regulatory region, or from a conserved motif. Both probe specificity and the
stringency
of diagnostic hybridization or amplification (maximal, high, intermediate, or
low)
reactions depend on whether or not the probe identifies exactly complementary
sequences, allelic variants, or related sequences. Probes designed to detect
related
sequences may have, ,for example, at least 50% sequence identity to any of the
selected polynucleotides.
Furthermore, a probe may be labelled by any procedure known in the art, for
example by incorporation of nucleotides linked to a "reporter molecule". A
"reporter
molecule", as used herein, may be a molecule that provides an analytically
identifiable
signal allowing detection of a hybridized probe. Detection may be either
qualitative or
quantitative. Commonly used reporter molecules include fluorophores, enzymes,
biotin, chemiluminescent molecules, bioluminescent molecules, digoxigenin,
avidin,
streptavidin or radioisotopes. Commonly used enzymes include horseradish
peroxidase, alkaline phosphatase, glucose oxidase and 6-galactosidase, among
others. Enzymes may be conjugated to avidin or streptavidin for use with a
biotinylated probe. Similarly, probes may be conjugated to avidin or
streptavidin for
use with a biotinylated enzyme. Incorporation of a reporter molecule into a
DNA probe
may be effected by any method known to the skilled artisan, for example by
nick
translation, primer extension, random oligo priming, by 3' or 5' end labeling
or by other
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means. In addition, hybridization probes include the cloning of nucleic acid
sequences
into vectors for the production of mRNA probes. Such vectors are known in the
art, are
commercially available, and may be used to synthesize RNA probes in vitro. The
labelled polynucleotide sequences may be used in Southern or northern
analysis, dot
blot, or other membrane-based technologies; in PCR technologies; and in micro
arrays
utilizing samples from subjects to detect altered expression. Oligonucleotides
useful as
probes for screening of samples by hybridization assays or as primers for
amplification
may be packaged into kits. Such kits may contain the probes or primers in a
pre-
measured or predetermined amount, as well as other suitably packaged reagents
and
materials needed for the particular hybridization or amplification protocol.
The expression of mRNAs identical or substantially identical to the NSEQs of
the present invention may thus be detected and/or isolated using methods which
are
known in the art. Exemplary embodiment of such methods includes, for example
and
without limitation, hybridization analysis using oligonucleotide probes,
reverse
5 transcription and in vitro nucleic acid amplification methods.
Such procedures may therefore, permit detection of mRNAs in ovarian cells
(e.g., ovarian cancer cells) or in any other cells expressing such mRNAs.
Expression
of mRNA in a tissue-specific or a disease-specific manner may be useful for
defining
the tissues and/or particular disease state. One of skill in the art may
readily adapt the
NSEQs for these purposes.
It is to be understood herein that the NSEQs may hybridize to a substantially
complementary sequence found in a test sample (e.g., cell, tissue, etc.).
Additionally,
a sequence substantially complementary to NSEQ (including fragments) may bind
a
NSEQ and substantially identical sequences found in a test sample (e.g., cell,
tissue,
etc.).
Polypeptide encoded by an isolated NSEQ, polypeptide variants, polypeptide
analogs or polypeptide fragments thereof are also encompassed herewith. The
polypeptides whether in a premature, mature or fused form, may be isolated
from
lysed cells, or from the culture medium, and purified to the extent needed for
the
intended use. One of skill in the art may readily purify these proteins,
polypeptides and
peptides by any available procedure. For example, purification may be
accomplished
by salt fractionation, size exclusion chromatography, ion exchange
chromatography,
reverse phase chromatography, affinity chromatography and the like.
Alternatively,
PSEQ may be made by chemical synthesis.
Natural variants may be identified through hybridization screening of a
nucleic
acid library or polypeptide library from different tissue, cell type,
population, species,
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etc using the NSEQ and derived tools.
Use of NSEQ for Development of an Expression System
In order to express a polypeptide, a NSEQ able to encode any one of a PSEQ
described herein may be inserted into an expression vector, i.e., a vector
that contains
the elements for transcriptional and translational control of the inserted
coding
sequence in a particular host. These elements may include regulatory
sequences,
such as enhancers, constitutive and inducible promoters, and 5' and 3' un-
translated
regions. Methods that are well known to those skilled in the art may be used
to
construct such expression vectors. These methods include in vitro recombinant
DNA
techniques, synthetic techniques, and in vivo genetic recombination.
A variety of expression vector/host cell systems known to those of skill in
the
art may be utilized to express a polypeptide or RNA from NSEQ. These include,
but
are not limited to, microorganisms such as bacteria transformed with
recombinant
bacteriophage, plasmid, or cosmid DNA expression vectors; yeast transformed
with
yeast expression vectors; insect cell systems infected with baculovirus
vectors; plant
cell systems transformed with viral or bacterial expression vectors; or animal
cell
systems. For long-term production of recombinant proteins in mammalian
systems,
2() stable
expression in cell lines may be effected. For example, NSEQ may be
transformed into cell lines using expression vectors that may contain viral
origins of
replication and/or endogenous expression elements and a selectable or visible
marker
gene on the same or on a separate vector. The invention is not to be limited
by the
vector or host cell employed.
Alternatively, RNA and/or polypeptide may be expressed from a vector
comprising NSEQ using an in vitro transcription system or a coupled in vitro
transcription/translation system respectively.
In general, host cells that contain NSEQ and/or that express a polypeptide
encoded by the NSEQ, or a portion thereof, may be identified by a variety of
procedures known to those of skill in the art. These procedures include, but
are not
limited to, DNA/DNA or DNA/RNA hybridizations, PCR amplification, and protein
bioassay or immunoassay techniques that include membrane, solution, or chip
based
technologies for the detection and/or quantification of nucleic acid or amino
acid
sequences. Immunological methods for detecting and measuring the expression of
polypeptides using either specific polyclonal or monoclonal antibodies are
known in the
art. Examples of such techniques include enzyme-linked immunosorbent assays
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(ELISAs), radioimmunoassays (RIAs), and fluorescence activated cell sorting
(FACS).
Those of skill in the art may readily adapt these methodologies to the present
invention.
Host cells comprising NSEQ may thus be cultured under conditions for the
transcription of the corresponding RNA (mRNA, siRNA, shRNA etc.) and/or the
expression of the polypeptide from cell culture. The polypeptide produced by a
cell
may be secreted or may be retained intracellularly depending on the sequence
and/or
the vector used. As will be understood by those of skill in the art,
expression vectors
containing NSEQ may be designed to contain signal sequences that direct
secretion of
the polypeptide through a prokaryotic or eukaryotic cell membrane. Due to the
inherent
degeneracy of the genetic code, other DNA sequences that encode the same,
substantially the same or a functionally equivalent amino acid sequence may be
produced and used, for example, to express a polypeptide encoded by NSEQ. The
nucleotide sequences of the present invention may be engineered using methods
generally known in the art in order to alter the nucleotide sequences for a
variety of
purposes including, but not limited to, modification of the cloning,
processing, and/or
expression of the gene product. DNA shuffling by random fragmentation and PCR
reassembly of gene fragments and synthetic oligonucleotides may be used to
engineer
the nucleotide sequences. For example, oligonucleotide-mediated site-directed
mutagenesis may be used to introduce mutations that create new restriction
sites, alter
glycosylation patterns, change codon preference, produce splice variants, and
so forth.
In addition, a host cell strain may be chosen for its ability to modulate
expression of the
inserted sequences or to process the expressed polypeptide in the desired
fashion.
Such modifications of the polypeptide include, but are not limited to,
acetylation,
carboxylation, glycosylation, phosphorylation, lipidation, and acylation. Post-
translational processing, which cleaves a "prepro" form of the polypeptide,
may also be
used to specify protein targeting, folding, and/or activity. Different host
cells that have
specific cellular machinery and characteristic mechanisms for post-
translational
activities (e.g., CHO, HeLa, MDCK, HEK293, and W138) are available
commercially
and from the American Type Culture Collection (ATCC) and may be chosen to
ensure
the correct modification and processing of the expressed polypeptide.
Those of skill in the art will readily appreciate that natural, modified, or
recombinant nucleic acid sequences may be ligated to a heterologous sequence
resulting in translation of a fusion polypeptide containing heterologous
polypeptide
moieties in any of the aforementioned host systems. Such heterologous
polypeptide
moieties may facilitate purification of fusion polypeptides using commercially
available
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affinity matrices. Such moieties include, but are not limited to, glutathione
S-
transferase (GST), maltose binding protein, thioredoxin, calmodulin binding
peptide, 6-
His (His), FLAG, c-myc, hemaglutinin (HA), and antibody epitopes such as
monoclonal
antibody epitopes.
In yet a further aspect, the present invention relates to a polynucleotide
which
may comprise a nucleotide sequence encoding a fusion protein, the fusion
protein may
comprise a fusion partner fused to a peptide fragment of a protein encoded by,
or a
naturally occurring allelic variant polypeptide encoded by, the po(ynucleotide
sequence
described herein.
io Those of skill
in the art will also readily recognize that the nucleic acid and
polypeptide sequences may be synthesized, in whole or in part, using chemical
or
enzymatic methods well known in the art. For example, peptide synthesis may be
performed using various solid-phase techniques and machines such as the ABI
431A
Peptide synthesizer (PE Biosystems) may be used to automate synthesis. If
desired,
the amino acid sequence may be altered during synthesis and/or combined with
sequences from other proteins to produce a variant protein.
The present invention additionally relates to a bioassay for evaluating
compounds as potential antagonists of the polypeptide described herein, the
bioassay
may comprise:
a) culturing test cells in culture medium containing increasing
concentrations of at least one compound whose ability to inhibit the action of
a
polypeptide described herein is sought to be determined, wherein the test
cells
may contain a polynucleotide sequence described herein (for examp(e, in a
form having improved trans-activation transcription activity, relative to wild-
type
polynucleotide, and comprising a response element operatively linked to a
reporter gene); and thereafter
b) monitoring in the cells the level of expression of the product of
the reporter gene (encoding a reporter mo(ecule) as a function of the
concentration of the potential antagonist compound in the culture medium,
thereby indicating the ability of the potential antagonist compound to inhibit
activation of the polypeptide encoded by, the polynucleotide sequence
described herein.
The present invention further relates to a bioassay for evaluating compounds
as potential agonists for a polypeptide encoded by the polynucleotide sequence
described herein, the bioassay may comprise:
a) culturing test cells in culture medium containing increasing
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concentrations of at least one compound whose ability to promote the action of
the po(ypeptide encoded by the polynucleotide sequence described herein is
sought to be determined, wherein the test cells may contain a polynucleotide
sequence described herein (for example, in a form having improved trans-
activation transcription activity, relative to wild-type polynucleotide, and
comprising a response element operatively linked to a reporter gene); and
thereafter
b) monitoring in the cells the level of expression of the product of
the reporter gene as a function of the concentration of the potential agonist
compound in the culture medium, thereby indicating the ability of the
potential
agonist compound to promote activation of a polypeptide encoded by the
polynucleotide sequence described herein.
Use of NSEQ as a Identification Tool or as a Diagnostic Screening Toot
The skilled artisan will readily recognize that NSEQ may be used to identify a
particular cell, cell type, tissue, disease and thus may be used for
diagnostic purposes
to determine the absence, presence, or altered expression (i.e. increased or
decreased compared to normal) of the expression product of a gene. Suitable
NSEQ
may be for example, between 10 and 20 or longer, i.e., at least 10 nucleotides
long or
at least 12 nucleotides long, or at least 15 nucleotides long up to any
desired length
and may comprise, for example, RNA, DNA, branched nucleic acids, andfor
peptide
nucleic acids (PNAs). In one alternative, the polynucleotides may be used to
detect
and quantify gene expression in samples in which expression of NSEQ is
correlated
with disease. In another alternative, NSEQ may be used to detect genetic
polymorphisms associated with a disease. These polymorphisms may be detected,
for
example, in the transcript, cDNA or genomic DNA.
The invention provides for the use of at least one of the NSEQ described
herein on an array and for the use of that array in a method of detection of a
particular
cell, cell type, tissue, disease for the prognosis or diagnosis of cancer. The
method
may comprise hybridizing the array with a patient sample (putatively
comprising or
comprising a target polynucleotide sequence substantially complementary to a
NSEQ)
under conditions to allow complex formation (between NSEQ and target
polynucleotide), detecting complex formation, wherein the complex formation is
indicative of the presence of the polynucleotide and wherein the absence of
complex
formation is indicative of the absence of the polynucleotide in the patient
sample. The
presence or absence of the polynucleotide may be indicative of cancer such as,
for
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example, ovarian cancer or other cancer as indicated herein.
The method may also comprise the step of quantitatively or qualitatively
comparing (e.g., with a computer system, apparatus) the level of complex
formation in
the patient sample to that of standards for normal cells or individual or
other type,
origin or grade of cancer.
The present invention provides one or more compartmentalized kits for
detection of a polynucleotide and/or polypeptide for the diagnosis or
prognosis of
ovarian cancer. A first kit may have a receptacle containing at least one
isolated
NSEQ or probe comprising NSEQ. Such a probe may bind to a nucleic acid
fragment
which is present/absent in normal cells but which is absent/present in
affected or
diseased cells. Such a probe may be specific for a nucleic acid site that is
normally
active/inactive but which may be inactive/active in certain cell types.
Similarly, such a
probe may be specific for a nucleic acid site that may be abnormally expressed
in
certain cell types. Finally, such a probe may identify a specific mutation The
probe
may be capable of hybridizing to the nucleic acid sequence which is mutated
(not
identical to the normal nucleic acid sequence). or may be capable of
hybridizing to
nucleic acid sequences adjacent to the mutated nucleic acid sequences. The
probes
provided in the present kits may have a covalently attached reporter molecule.
Probes
and reporter molecules may be readily prepared as described above by those of
skill in
the art.
Antibodies (e.g., isolated antibody) that may specifically bind to a protein
or
polypeptide described herein (a PSEQ) as well as nucleic acids encoding such
antibodies are also encompassed by the present invention.
As used herein the term "antibody" means a monoclonal antibody, a
polyclonal antibody, a single chain antibody, a chimeric antibody, a humanized
antibody, a deimmunized antibody, an antigen-binding fragment, an Fab
fragment: an
F(alci')2 fragment, and Fv fragment; CDRs, or a single-chain antibody
comprising an
antigen-binding fragment (e.g., a single chain Fv).
The antibody may originate for example, from a mouse, rat or any other
mammal or from other sources such as through recombinant DNA technologies.
The antibody may also be a human antibody which may be obtained, for
example, from a transgenic non-human mammal capable of expressing human lg
genes. The antibody may also be a humanised antibody which may comprise, for
example, one or more complementarity determining regions of non-human origin.
It
may also comprise a surface residue of a human antibody and/or framework
regions of
a human antibody. The antibody may also be a chimeric antibody which may
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comprise, for example, variable domains of a non-human antibody and constant
domains of a human antibody.
The antibody of the present invention may be mutated and selected based on
an increased affinity, solubility, stability, specificity and/or for one of a
polypeptide
described herein and/or based on a reduced immunogenicity in a desired host or
for
other desirable characteristics.
Suitable antibodies may bind to unique antigenic regions or epitopes in the
polypeptides, or a portion thereof. Epitopes and antigenic regions useful for
generating
antibodies may be found within the proteins, polypeptides or peptides by
procedures
li;) available to one of skill in the art. For example, short, unique
peptide sequences may
be identified in the proteins and polypeptides that have little or no homology
to known
amino acid sequences. Preferably the region of a protein selected to act as a
peptide
epitope or antigen is not entirely hydrophobic; hydrophilic regions are
preferred
because those regions likely constitute surface epitopes rather than internal
regions of
the proteins and polypeptides. These surface epitopes are more readily
detected in
samples tested for the presence of the proteins and polypeptides. Such
antibodies
may include, but are not limited to, polyclonal, monoclonal, chimeric, and
single chain
antibodies, Fab fragments, and fragments produced by a Fab expression library.
The
production of antibodies is well known to one of skill in the art and is not
intended to be
zo limited herein.
Peptides may be made by any procedure known to one of skill in the art, for
example, by using in vitro translation or chemical synthesis procedures or by
introducing a suitable expression vector into cells. Short peptides which
provide an
antigenic epitope but which by themselves are too small to induce an immune
response may be conjugated to a suitable carrier. Suitable carriers and
methods of
linkage are well known in the art. Suitable carriers are typically large
macromolecules
such as proteins, polysaccharides and polymeric amino acids. Examples include
serum albumins, keyhole limpet hemocyanin, ovalbumin, polylysine and the like.
One
of skill in the art may use available procedures and coupling reagents to link
the
desired peptide epitope to such a carrier. For example, coupling reagents may
be used
to form disulfide linkages or thioether linkages from the carrier to the
peptide of
interest. If the peptide lacks a disulfide group, one may be provided by the
addition of a
cysteine residue. Alternatively, coupling may be accomplished by activation of
carboxyl
groups.
The minimum size of peptides useful for obtaining antigen specific antibodies
may vary widely. The minimum size must be sufficient to provide an antigenic
epitope
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that is specific to the protein or polypeptide. The maximum size is not
critical unless it
is desired to obtain antibodies to one particular epitope. For example, a
large
polypeptide may comprise multiple epitopes, one epitope being particularly
useful and
a second epitope being immunodominant, etc. Typically, antigenic peptides
selected
from the present proteins and polypeptides will range without limitation. from
5 to about
100 amino acids in length. More typically, however, such an antigenic peptide
will be a
maximum of about 50 amino acids in length, and preferably a maximum of about
30
amino acids. It is usually desirable to select a sequence of about 6, 8, 10,
12 or 15
amino acids, up to about 20 or 25 amino acids (and any number therebetween).
Amino acid sequences comprising useful epitopes may be identified in a
number of ways. For example, preparing a series of short peptides that taken
together
span the entire protein sequence may be used to screen the entire protein
sequence.
One of skill in the art may routinely test a few large polypeptides for the
presence of an
epitope showing a desired reactivity and also test progressively smaller and
overlapping fragments to identify a preferred epitope with the desired
specificity and
reactivity.
As mentioned herein, antigenic polypeptides and peptides are useful for the
production of monoclonal and polyclonal antibodies. Antibodies to a
polypeptide
encoded by the polynucleotides of NSEQ, polypeptide analogs or portions
thereof,
may be generated using methods that are well known in the art. For example,
monoclonal antibodies may be prepared using any technique that provides for
the
production of antibody molecules by continuous cell lines in culture. These
include, but
are not limited to, the hybridoma, the human B-cell hybridoma, and the EBV-
hybridoma techniques. In addition, techniques developed for the production of
chimeric
antibodies may be used. Alternatively, techniques described for the production
of
single chain antibodies may be employed. Fabs that may contain specific
binding sites
for a polypeptide encoded by the polynucleotides of NSEQ, or a portion
thereof, may
also be generated. Various immunoassays may be used to identify antibodies
having
the desired specificity. Numerous protocols for competitive binding or
immunoradiometric assays using either polyclonal or monoclonal antibodies with
established specificities are well known in the art.
To obtain polyclonal antibodies, a selected animal may be immunized with a
protein or polypeptide. Serum from the animal may be collected and treated
according
to known procedures. Polyclonal antibodies to the protein or polypeptide of
interest
may then be purified by affinity chromatography. Techniques for producing
polyclonal
antisera are well known in the art.
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Monoclonal antibodies (MAbs) may be made by one of several procedures
available to one of skill in the art, for example, by fusing antibody
producing cells with
immortalized cells and thereby making a hybridoma. The general methodology for
fusion of antibody producing B cells to an immortal cell line is well within
the province
of one skilled in the art. Another example is the generation of MAbs from mRNA
extracted from bone marrow and spleen cells of immunized animals using
combinatorial antibody library technology.
One drawback of MAbs derived from animals or from derived cell lines is that
although they may be administered to a patient for diagnostic or therapeutic
purposes,
they are often recognized as foreign antigens by the immune system and are
unsuitable for continued use. Antibodies that are not recognized as foreign
antigens by
the human immune system have greater potential for both diagnosis and
treatment.
Methods for generating human and humanized antibodies are now well known in
the
art.
Chimeric antibodies may be constructed in which regions of a non-human
MAb are replaced by their human counterparts. A preferred chimeric antibody is
one
that has amino acid sequences that comprise one or more complementarity
determining regions (CDRs) of a non-human Mab that binds to a polypeptide
encoded
by the polynucleotides of NSEQ, or a portion thereof, grafted to human
framework
(FW) regions. Methods for producing such antibodies are well known in the art.
Amino
acid residues corresponding to CDRs and FWs are known to one of average skill
in
the art.
A variety of methods have been developed to preserve or to enhance affinity
for antigen of antibodies comprising grafted CDRs. One way is to include in
the
chimeric antibody the foreign framework residues that influence the
conformation of
the CDR regions. A second way is to graft the foreign CDRs onto human variable
domains with the closest homology to the foreign variable region. Thus,
grafting of one
or more non-human CDRs onto a human antibody may also involve the substitution
of
amino acid residues which are adjacent to a particular CDR sequence or which
are not
contiguous with the CDR sequence but which are packed against the CDR in the
overall antibody variable domain structure and which affect the conformation
of the
CDR. Humanized antibodies of the invention therefore include human antibodies
which
comprise one or more non-human CDRs as well as such antibodies in which
additional
substitutions or replacements have been made to preserve or enhance binding
characteristics.
Chimeric antibodies of the invention also include antibodies that have been
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humanized by replacing surface-exposed residues to make the MAb appear human.
Because the internal packing of amino acid residues in the vicinity of the
antigen-
binding site remains unchanged, affinity is preserved. Substitution of surface-
exposed
residues of a polypeptide encoded by the polynucleotides of NSEQ (or a portion
thereof)-antibody according to the invention for the purpose of humanization
does not
mean substitution of CDR residues or adjacent residues that influence affinity
for a
polypeptide encoded by the polynucleotides of NSEQ, or a portion thereof.
Chimeric antibodies may also include antibodies where some or all non-
human constant domains have been replaced with human counterparts. This
approach
has the advantage that the antigen-binding site remains unaffected. However,
significant amounts of non-human sequences may be present where variable
domains
are derived entirely from non-human antibodies.
Antibodies of the invention include human antibodies that are antibodies
consisting essentially of human sequences. Human antibodies may be obtained
from
5 phage display libraries wherein combinations of human heavy and light
chain variable
domains are displayed on the surface of filamentous phage. Combinations of
variable
domains are typically displayed on filamentous phage in the form of Fab's or
scFvs.
The library may be screened for phage bearing combinations of variable domains
having desired antigen-binding characteristics. Preferred variable domain
combinations are characterized by high affinity for a polypeptide encoded by
the
polynucleotides of NSEQ, or a portion thereof. Preferred variable domain
combinations
may also be characterized by high specificity for a polypeptide encoded by the
polynucleotides of NSEQ, or a portion thereof, and little cross-reactivity to
other related
antigens. By screening from very large repertoires of antibody fragments, (2-
10 x 101 )
a good diversity of high affinity Mabs may be isolated, with many expected to
have
sub-nanomolar affinities for a polypeptide encoded by the polynucleotides of
NSEQ, or
a portion thereof.
Alternatively, human antibodies may be obtained from transgenic animals into
which un-rearranged human Ig gene segments have been introduced and in which
the
endogenous mouse Ig genes have been inactivated. Preferred transgenic animals
contain very large contiguous Ig gene fragments that are over 1 Mb in size but
human
polypeptide-specific Mabs of moderate affinity may be raised from transgenic
animals
containing smaller gene loci. Transgenic animals capable of expressing only
human Ig
genes may also be used to raise polyclonal antiserum comprising antibodies
solely of
human origin.
Antibodies of the invention may include those for which binding
characteristics
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have been improved by direct mutation or by methods of affinity maturation.
Affinity
and specificity may be modified or improved by mutating CDRs and screening for
antigen binding sites having the desired characteristics. CDRs may be mutated
in a
variety of ways. One way is to randomize individual residues or combinations
of
residues so that in a population of otherwise identical antigen binding sites,
all twenty
amino acids may be found at particular positions. Alternatively, mutations may
be
induced over a range of CDR residues by error prone PCR methods. Phage display
vectors containing heavy and light chain variable region gene may be
propagated in
mutator strains of E. coli. These methods of mutagenesis are illustrative of
the many
methods known to one of skill in the art.
The antibody may further comprise a detectable label (reporter molecule)
attached thereto.
There is provided also methods of producing antibodies able to specifically
bind to one of a polypeptide, polypeptide fragments, or polypeptide analogs
described
is herein, the method may,comprise:
a) immunizing a mammal (e.g., mouse, a transgenic mammal
capable of producing human lg, etc.) with a suitable amount of a PSEQ
described herein including, for example, a polypeptide fragment
comprising at least 6 (e.g., 8, 10, 12 etc.) consecutive amino acids of a
PSEQ;
b) collecting the serum from the mammal; and
c) isolating the polypeptide-specific antibodies from the serum of
the mammal.
The method may further comprise the step of administering a second dose to
the mammal (e.g., animal).
Methods of producing a hybridoma which secretes an antibody that
specifically binds to a polypeptide are also encompassed herewith and are
known in
the art.
The method may comprise:
a) immunizing a mammal (e.g., mouse, a transgenic mammal
capable of producing human Ig, etc.) with a suitable amount of a PSEQ
thereof;
b) obtaining lymphoid cells from the immunized animal
obtained
from (a);
c) fusing the lymphoid cells with an immortalizing cell to produce
hybrid cells; and
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d) selecting hybrid cells which produce antibody that
specifically
binds to a PSEQ thereof.
Also encompassed by the present invention is a method of producing an
antibody that specifically binds to one of the polypeptide described herein,
the method
may comprise:
a) synthesizing a library of antibodies (e.g., antigen
binding
fragment) on phage or ribosomes;
b) panning the library against a sample by bringing the
phage or
ribosomes into contact with a composition comprising a polypeptide or
io polypeptide fragment described herein;
c) isolating phage which binds to the polypeptide or
polypeptide
fragment, and;
d) obtaining an antibody from the phage or ribosomes.
The antibody of the present invention may thus be obtained, for example,
from a library (e.g., bacteriophage library) which may be prepared, for
example, by
a) extracting cells which are responsible for production of
antibodies from a host mammal;
b) isolating RNA from the cells of (a);
c) reverse transcribing mRNA to produce cDNA;
d) amplifying the cDNA using a (antibody-specific) primer; and
e) inserting the cDNA of (d) into a phage display vector or
ribosome display cassette such that antibodies are expressed
on the phage or ribosomes.
In order to generate antibodies, the host animal may be immunized with
polypeptide and/or a polypeptide fragment and/or analog described herein to
induce
an immune response prior to extracting the cells which are responsible for
production
of antibodies.
The antibodies obtained by the means described herein may be useful for
detecting proteins, variant and derivative polypeptides in specific tissues or
in body
fluids. Moreover, detection of aberrantly expressed proteins or protein
fragments is
probative of a disease state. For example, expression of the present
polypeptides
encoded by the polynucleotides of NSEQ, or a portion thereof, may indicate
that the
protein is being expressed at an inappropriate rate or at an inappropriate
developmental stage. Hence, the present antibodies may be useful for detecting
diseases associated with protein expression from NSEQs disclosed herein.
For in vivo detection purposes, antibodies may be those which preferably
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recognize an epitope present at the surface of a tumor cell.
A variety of protocols for measuring polypeptides, including ELISAs, RIAs,
and FAGS, are well known in the art and provide a basis for diagnosing altered
or
abnormal levels of expression. Standard values for polypeptide expression are
established by combining samples taken from healthy subjects, preferably
human, with
antibody to the polypeptide under conditions for complex formation. The amount
of
complex formation may be quantified by various methods, such as photometric
means.
Quantities of polypeptide expressed in disease samples may be compared with
standard values. Deviation between standard and subject values may establish
the
parameters for diagnosing or monitoring disease.
Design of immunoassays is subject to a great deal of variation and a variety
of these are known in the art. Immunoassays may use a monoclonal or polyclonal
antibody reagent that is directed against one epitope of the antigen being
assayed.
Alternatively, a combination of monoclonal or polyclonal antibodies may be
used which
are directed against more than one epitope. Protocols may be based, for
example,
upon competition where one may use competitive drug screening assays in which
neutralizing antibodies capable of binding a polypeptide encoded by the
polynucleotides of NSEQ, or a portion thereof, specifically compete with a
test
compound for binding the polypeptide. Alternatively one may use, direct
antigen-
antibody reactions or sandwich type assays and protocols may, for example,
make use
of solid supports or immunoprecipitation. Furthermore, antibodies may be
labelled with
a reporter molecule for easy detection. Assays that amplify the signal from a
bound
reagent are also known. Examples include immunoassays that utilize avidin and
biotin,
or which utilize enzyme-labelled antibody or antigen conjugates, such as EL1SA
assays.
Kits suitable for immunodiagnosis and containing the appropriate labelled
reagents include antibodies directed against the polypeptide protein epitopes
or
antigenic regions, packaged appropriately with the remaining reagents and
materials
required for the conduct of the assay, as well as a suitable set of assay
instructions.
The present invention therefore provides a kit for specifically detecting a
polypeptide described herein, the kit may comprise, for example, an antibody
or
antibody fragment capable of binding specifically to the polypeptide described
herein.
In accordance with the present invention, the kit may be a diagnostic kit,
which may comprise:
a) one or more antibodies described herein; and
b) a detection reagent which may comprise a reporter group.
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In accordance with the present invention, the antibodies may be immobilized
on a solid support. The detection reagent may comprise, for example, an anti-
immunoglobulin, protein G, protein A or lectin etc. The reporter group may be
selected, without limitation, from the group consisting of radioisotopes,
fluorescent
groups, luminescent groups, enzymes, biotin and dye particles
Use of NSEQ, PSEQ as a Therapeutic or Therapeutic targets
One of skill in the art will readily appreciate that the NSEQ, PSEQ,
expression
systems, assays, kits and array discussed above may also be used to evaluate
the
to efficacy of a particular therapeutic treatment regimen, in animal
studies, in clinical
trials, or to monitor the treatment of an individual subject. Once the
presence of
disease is established and a treatment protocol is initiated, hybridization or
amplification assays may be repeated on a regular basis to determine if the
level of
mRNA or protein in the patient (patient's blood, tissue, cell etc.) begins to
approximate
the level observed in a healthy subject. The results obtained from successive
assays
may be used to show the efficacy of treatment over a period ranging from
several days
to many years.
In yet another aspect of the invention, NSEQ may be used therapeutically for
the purpose of expressing mRNA and polypeptide, or conversely to block
transcription
and/or translation of the mRNA. Expression vectors may be constructed using
elements from retroviruses, adenoviruses, herpes or vaccinia viruses, or
bacterial
plasmids, and the like. These vectors may be used for delivery of nucleotide
sequences to a particular target organ, tissue, or cell population. Methods
well known
to those skilled in the art may be used to construct vectors to express
nucleic acid
sequences or their complements.
Alternatively, NSEQ may be used for somatic cell or stem cell gene therapy.
Vectors may be introduced in vivo, in vitro, and ex vivo, For ex vivo therapy,
vectors
are introduced into stem cells taken from the subject, and the resulting
transgenic cells
are clonally propagated for autologous transplant back into that same subject.
Delivery
of NSEQ by transfection, liposome injections, or polycationic amino polymers
may be
achieved using methods that are well known in the art. Additionally,
endogenous
NSEQ expression may be inactivated using homologous recombination methods that
insert an inactive gene sequence into the coding region or other targeted
region of
NSEQ.
Depending on the specific goal to be achieved, vectors containing NSEQ may
be introduced into a cell or tissue to express a missing polypeptide or to
replace a non-
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functional polypeptide. Of course, when one wishes to express PSEQ in a cell
or
tissue, one may use a NSEQ able to encode such PSEQ for that purpose or may
directly administer PSEQ to that cell or tissue.
On the other hand, when one wishes to attenuate or inhibit the expression of
PSEQ, one may use a NSEQ (e.g., an inhibitory NSEQ) which is substantially
complementary to at least a portion of a NSEQ able to encode such PSEQ.
The expression of an inhibitory NSEQ may be done by cloning the inhibitory
NSEQ into a vector and introducing the vector into a cell to down-regulate the
expression of a polypeptide encoded by the target NSEQ. Complementary or anti-
sense sequences may also comprise an oligonucleotide derived from the
transcription
initiation site; nucleotides between about positions -10 and +10 from the ATG
may be
used. Therefore, inhibitory NSEQ may encompass a portion which is
substantially
complementary to a desired nucleic acid molecule to be inhibited and a portion
(sequence) which binds to an untranslated portion of the nucleic acid.
Similarly, inhibition may be achieved using triple helix base-pairing
methodology. Triple helix pairing is useful because it causes inhibition of
the ability of
the double helix to open sufficiently for the binding of polymerases,
transcription
factors, or regulatory molecules. Recent therapeutic advances using triplex
DNA have
been described in the literature. (See, e.g., Gee et al. 1994)
70 Ribozymes, enzymatic RNA molecules, may also be used to catalyze the
cleavage of mRNA and decrease the levels of particular mRNAs, such as those
comprising the polynucleotide sequences of the invention. Ribozymes may cleave
mRNA at specific cleavage sites. Alternatively, ribozymes may cleave mRNAs at
locations dictated by flanking regions that form complementary base pairs with
the
target mRNA. The construction and production of ribozymes is well known in the
art.
RNA molecules may be modified to increase intracellular stability and half-
life.
Possible modifications include, but are not limited to, the addition of
flanking
sequences at the 5' and/or 3' ends of the molecule, or the use of
phosphorothioate or
2 0-methyl rather than phosphodiester linkages within the backbone of the
molecule.
Alternatively, nontraditional bases such as inosine, queosine, and wybutosine,
as well
as acetyl-, methyl-, thio-, and similarly modified forms of adenine, cytidine,
guanine,
thymine, and uridine which are not as easily recognized by endogenous
endonucleases, may be included.
Pharmaceutical compositions are also encompassed by the present invention.
The pharmaceutical composition may comprise at least one NSEQ or PSEQ and a
pharmaceutically acceptable carrier.
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As it will be appreciated form those of skill in the art, the specificity of
expression NSEQ and/or PSEQ in tumor cells may advantageously be used for
inducing an immune response (through their administration) in an individual
having, or
suspected of having a tumor expressing such sequence. Administration of NSEQ
and/or PSEQ in individuals at risk of developping a tumor expressing such
sequence is
also encompassed herewith.
In addition to the active ingredients, a pharmaceutical composition may
contain pharmaceutically acceptable carriers comprising excipients and
auxiliaries that
facilitate processing of the active compounds into preparations that may be
used
pharmaceutically.
For any compound, the therapeutically effective dose may be estimated
initially either in cell culture assays or in animal models such as mice,
rats, rabbits,
dogs, or pigs. An animal model may also be used to determine the concentration
range and route of administration. Such information may then be used to
determine
useful doses and routes for administration in humans. These techniques are
well
known to one skilled in the art and a therapeutically effective dose refers to
that
amount of active ingredient that ameliorates the symptoms or condition.
Therapeutic
efficacy and toxicity may be determined by standard pharmaceutical procedures
in cell
cultures or with experimental animals, such as by calculating and contrasting
the ED50
(the dose therapeutically effective in 50% of the population) and LD50 (the
dose lethal
to 50% of the population) statistics. Any of the therapeutic compositions
described
above may be applied to any subject in need of such therapy, including, but
not limited
to, mammals such as dogs, cats, cows, horses, rabbits, monkeys, and most
preferably, humans.
/5 The pharmaceutical compositions utilized in this invention may be
administered by any number of routes including, but not limited to, oral,
intravenous,
intramuscular, intra-arterial, intramedullary, intrathecal, intraventricular,
transdermal,
subcutaneous, intraperitoneal, intranasal, enteral, topical, sublingual, or
rectal means.
The term "treatment" for purposes of this disclosure refers to both
therapeutic
treatment and prophylactic or preventative measures, wherein the object is to
prevent
or slow down (lessen) the targeted pathologic condition or disorder. Those in
need of
treatment include those already with the disorder as well as those prone to
have the
disorder or those in whom the disorder is to be prevented.
Use of NSEQ in General Research
The invention also provides products, compositions, processes and methods
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that utilize a NSEQ described herein, a polypeptide encoded by a NSEQ
described
herein, a PSEQ described herein for research, biological, clinical and
therapeutic
purposes. For example, to identify splice variants, mutations, and
polymorphisms and
to generate diagnostic and prognostic tools.
NSEQ may be extended utilizing a partial nucleotide sequence and employing
various PCR-based methods known in the art to detect upstream sequences such
as
promoters and other regulatory elements. Additionally, one may use an XL-PCR
kit
(PE Biosystems, Foster City Calif.), nested primers, and commercially
available cDNA
libraries (Life Technologies, Rockville Md.) or genomic libraries (Clontech,
Palo Alto
Calif.) to extend the sequence.
The polynucleotides (NSEQ) may also be used as targets in a microarray.
The microarray may be used to monitor the expression patterns of large numbers
of
genes simultaneously and to identify splice variants, mutations, and
polymorphisms.
Information derived from analyses of the expression patterns may be used to
determine gene function, to identify a particular cell, cell type or tissue,
to understand
the genetic basis of a disease, to diagnose a disease, and to develop and
monitor the
activities of therapeutic agents used to treat a disease. Microarrays may also
be used
to detect genetic diversity, single nucleotide polymorphisms which may
characterize a
particular population, at the genomic level.
The polynucleotides (NSEQ) may also be used to generate hybridization
probes useful in mapping the naturally occurring genomic sequence. Fluorescent
in
situ hybridization (FISH) may be correlated with other physical chromosome
mapping
techniques and genetic map data.
It is to be understood herein that a sequence which is upregulated in an
ovarian cancer cell (e.g., malignant ovarian cancer cell) may represent a
sequence
which is involved in or responsible for the growth, development, maligancy and
so on.
of the cancer cell (referred herein as a positive regulator of ovarian
cancer). It is also
to be understood that a sequence which is downregulated (unexpressed or
expressed
at low levels) in a malignant ovarian cancer cell may represent a sequence
which is
responsible for the maintenance of the normal status (untransformed) of an
ovarian
cell (referred herein as a negative regulator of ovarian cancer). Therefore,
both the
presence or absence of some sequences may be indicative of the disease or may
be
indicative of the disease, probability of having a disease, degree of severity
of the
disease (staging).
Therefore, the present invention relates in an aspect thereof to an isolated
polynucleotide (e.g., exogenous form of) which may comprise a member selected
from
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the group consisting of;
a) a polynucleotide which may comprise or consist of any one of SEQ ID
NO.:1 to SEQ ID NO.49 and SEQ ID NO.169,
b) a polynucleotide which may comprise the open reading frame of any
one of SEQ ID NO.:1 to SEQ ID NO.49 and SEQ ID NO.169,
c) a polynucleotide which may comprise a transcribed or transcribable
portion of any one of SEQ. ID. NOs:1 to 49 and 169, which may be, for
example, free of untranslated or untranslatable portion(s),
d) a polynucleotide which may comprise a translated or translatable
I 0 portion of any one of SEQ. ID. NOs:1 to 49 and 169 (e.g., coding
portion),
e) a polynucleotide which may comprise a sequence substantially identical
(e.g., from about 50 to 100%, or about 60 to 100% or about 70 to 100%
or about 80 to 100% or about 85, 90, 95 to 100% identical over the
entire sequence or portion of sequences) to a), b), c), or d);
f) a polynucleotide which may comprise a sequence substantially
complementary (e.g., from about 50 to 100%, or about 60 to 100% or
about 70 to 100% or about 80 to 100% or about 85, 90, 95 to 100%
complementarity over the entire sequence or portion of sequences) to
a), b), c), or d) and;
g) a fragment of any one of a) to f)
including polynucleotides which consist in the above.
More specifically, the present invention relates to expressed polynucleotides
which are selected from the group consisting of;
a) a polynucleotide which may comprise or consist of any one of SEQ ID
NO.: 1, SEQ ID NO.:14, SEQ ID NO.:16, SEQ ID NO.:19, SEQ ID
NO.:20, SEQ ID NO.:22, SEQ ID NO. :28, SEQ ID NO.:37, SEQ ID
NO.:41, SEQ ID NO.:45, SEQ ID NO.:46. SEQ ID NO.:47 and SEQ ID
NO. :49 and even more specifically those which are selected from the
group consisting of SEQ ID NO.: 14, SEQ ID NO.:19, SEQ ID NO.: 22,
SEQ ID NO.:37, SEQ ID NO.:41, SEQ ID NO.:45, SEQ ID NO.:46 and
SEQ ID NO. :49,
b) a polynucleotide which may comprise the open reading frame of any
one of SEQ ID NO.: 1, SEQ ID NO.:14, SEQ ID NO.:16, SEQ ID
NO.:19, SEQ ID NO.:20, SEQ ID NO.:22, SEQ ID NO.:28, SEQ ID
NO.:37, SEQ ID NO.:41, SEQ ID NO.:45, SEQ ID NO.:46, SEQ ID
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NO. :47 and SEQ ID NO.:49 and even more specifically those which are
selected from the group consisting of SEQ ID NO.: 14, SEQ ID NO.:19,
SEQ ID NO.: 22, SEQ ID NO.:37, SEQ ID NO.:41, SEQ ID NO.:45,
SEQ ID NO.:46 and SEQ ID NO.:49,
c) a polynucleotide which may comprise a transcribed or transcribable
portion of any one of SEQ ID NO.: 1, SEQ ID NO.:14, SEQ ID NO.:16,
SEQ ID NO.:19, SEQ ID NO.:20, SEQ ID NO.:22, SEQ ID NO.:28, SEQ
ID NO.:37, SEQ ID NO.:41, SEQ ID NO.:45, SEQ ID NO.:46, SEQ ID
NO. :47 and SEQ ID NO.:49 and even more specifically those which are
selected from the group consisting of SEQ ID NO.: 14, SEQ ID NO.:19,
SEQ ID NO.: 22, SEQ ID NO.:37, SEQ ID NO.:41, SEQ ID NO.:45,
SEQ ID NO.:46 and SEQ ID NO.:49, which may be, for example, free of
untranslated or untranslatable portion(s),
d) a polynucleotide which may comprise a translated or translatable
portion of any one of SEQ ID NO.: 1, SEQ ID NO.:14, SEQ ID NO.:16,
SEQ ID NO.:19, SEQ ID NO.:20, SEQ ID NO.:22, SEQ ID NO.:28, SEQ
ID NO.:37, SEQ ID NO.:41, SEQ ID NO.:45, SEQ ID NO.:46. SEQ ID
NO. :47 and SEQ ID NO.:49 and even more specifically those which are
selected from the group consisting of SEQ ID NO.: 14, SEQ ID NO.:19,
SEQ ID NO.: 22, SEQ ID NO.:37, SEQ ID NO.:41, SEQ ID NO.:45,
SEQ ID NO.:46 and SEQ ID NO.:49, (e.g., coding portion),
e) a polynucleotide which may comprise a sequence substantially identical
(e.g., from about 50 to 100%, or about 60 to 100% or about 70 to 100%
or about 80 to 100% or about 85, 90, 95 to 100% identical over the
entire sequence or portion of sequences) to a), b), c), or d);
f) a polynucleotide which may comprise a sequence substantially
complementary (e.g., from about 50 to 100%, or about 60 to 100% or
about 70 to 100% or about 80 to 100% or about 85, 90, 95 to 100%
complementarity over the entire sequence or portion of sequences) to
a), b), c), or d) and;
g) a fragment of any one of a) to f)
including polynucleotides which consist in the above.
Vectors (e.g., a viral vector, a mammalian vector, a plasmid, a cosmid, etc.)
which may comprise the polynucleotides described herein are also encompassed
by
the present invention. The vector may be, for example, an expression vector.
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The present invention also provides a library of polynucleotide comprising at
least one polynucleotide (e.g., at least two, etc.) described herein (may
include SEQ ID
NO. :50). The library may be, for example, an expression library. Some or all
of the
polynucleotides described herein may be contained within an expression vector.
The
present invention also relates to a polypeptide library which may comprise at
least one
(e.g., at least two, etc.) polypeptide as described herein.
In another aspect, the present invention provides arrays which may comprise
at least one polynucleotide (e.g., at least two, etc.) described herein.
The present invention also provides an isolated cell (e.g., an isolated live
cell such as
an isolated mammalian cell, a bacterial cell, a yeast cell, an insect cell,
etc.) which may
comprise the polynucleotide, the vector or the polypeptide described herein.
In yet a further aspect the present invention relates to a composition
comprising the polynucleotide and/or polypeptide described herein.
In accordance with the present invention, the composition may be, for
example, a pharmaceutical composition which may comprise a polynucleotide
and/or a
polypeptide described herein and a pharmaceutically acceptable carrier. More
specifically, the pharmaceutical composition may be used for the treatment of
ovarian
cancer and/or for inhibiting the growth of an ovarian cancer cell.
Polynucleotides fragments of those listed above includes polynucleotides
comprising at least 10 nucleic acids which may be identical to a corresponding
portion
of any one of a) to e) and more particularly a coding portion of any one of
SEQ ID
NO.11 to 49, 50 or 169.
Another examplary embodiment of polynucleotide fragments encompassed by
the present invention includes polynucleotides comprising at least 10 nucleic
acids
which may be substantially complementary to a corresponding portion of a
coding
portion of any one of SEQ ID NO.:1 to 49, 50 or 169 and encompasses, for
example,
fragments selected from the group consisting of any one of SEQ ID NO.: 103 to
150.
These above sequences may represent powerful markers of cancer and more
particularly of, ovarian cancer, breast cancer, prostate cancer, leukemia,
melanoma,
renal cancer, colon cancer, lung cancer, cancer of the central nervous system
and any
combination thereof.
Based on the results presented herein and upon reading the present
description, a person skilled in the art will understand that the appearance
of a positive
signal upon testing (hybridization, PCR amplification etc.) for the presence
of a given
sequence amongst those expressed in a cancer cell, indicates that such
sequence is
specifically expressed in that type of cancer cell. A person skilled in the
art will also
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understand that, sequences which are specifically expressed in a certain types
of
cancer cell may be used for developing tools for the detection of this
specific type of
cancer cell and may also be used as targets in the development of anticancer
drugs.
A positive signal may be in the form of a band in a gel following
electrophoresis, Northern blot or Western blot, a PCR fragment detected by
emission
of fluorescence, etc.
As it will be understood, sequences which are particularly useful for the
development of tools for the detection of cancer cell may preferably be
expressed at
lower levels in at least some normal cells (non-cancerous cells).
to For example, in Figure 57 and related description, the appearance of a
band
upon RT-PCR amplification of mRNAs obtained from ovarian cancer cells, renal
cancer cells, lung cancer cells, breast cancer cells and melanoma cells
indicates that
SEQ ID NO.:1 is expressed in such cancer cells and that SEQ ID NO.:1 may
therefore
represent a valid marker and target for these types of cancer cells. Similar
conclusions may be derived from the results obtained from other Figures and
related
description.
NSEQs chosen among those which are substantially complementary to those
listed in Table 2, or to fragments of those of Table 2, may be used for the
treatment of
cancer.
The present invention therefore relates to a method for identifying a cancer
cell. The method may comprise contacting a cell, a cell sample (cell lysate),
a body
fluid (blood, urine, plasma, saliva etc.) or a tissue with a reagent which may
be, for
example, capable of specifically binding at least one NSEQ or PSEQ described
herein.
The method may more particularly comprise contacting a sequence isolated or
derived
such cell, sample, fluid or tissue. The complex formed may be detected using
methods
known in the art.
In accordance with the present invention, the presence of the above
mentioned complex may be indicative (a positive indication of the presence) of
the
presence of a cancer cell.
The present invention also relates in an additional aspect thereof to a method
for the diagnosis or prognosis of cancer. The method may comprise, for
example,
detecting, in a cell, tissue, sample, body fluid, etc., at least one NSEQ or
PSEQ
described herein.
The cell, cell sample, body fluid or tissue may originate, for example, from
an
individual which has or is suspected of having a cancer and more particularly
ovarian
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cancer, breast cancer, prostate cancer, leukemia, melanoma, renal cancer,
colon
cancer, lung cancer and/or cancer of the central nervous system
Any of the above mentioned methods may further comprise comparing the level
obtained with at least one reference level or value.
Detection of NSEQ may require an amplification (e.g., PCR) step in order to
have sufficient material for detection purposes. ,
In accordance with the present invention, the polynucleotide described herein
may comprise, for example, a RNA molecule, a DNA molecule, including those
which
are partial or complete, single-stranded or double-stranded, hybrids, modified
by a
group etc.
Other aspects of the present invention which are encompassed herewith
comprises the use of at least one NSEQ or PSEQ described herein and derived
antibodies in the manufacture of a composition for identification or detection
of a
cancer cell (e.g., a tumor cell) or for inhibiting or lowering the growth of
cancer cell
(e.g., for treatment of ovarian cancer or other cancer).
As some NSEQ and PSEQ are expressed at higher levels in malignant
ovarian cancer than in LMP detection of such NSEQ or PSEQ in a sample from an
individual (or in vivo) one may rule-out a low malignant potential ovarian
cancer and
may therefore conclude in a diagnostic of a malignant ovarian cancer.
Furthermore,
detection of the NSEQ or PSEQ in a cell, tissue, sample or body fluid from an
individual may also be indicative of a late-stage malignant ovarian cancer. As
such,
therapies adapted for the treatment of a malignant ovarian cancer or a late-
stage
malignant ovarian cancer may be commenced.
In accordance with an embodiment of the present invention, the method may
also comprise a step of qualitatively or quantitatively comparing the level
(amount,
presence) of at least one complex present in the test cell, test sample, test
fluid or test
tissue with the level of complex in a normal cell, a normal cell sample, a
normal body
fluid, a normal tissue or a reference value (e.g., for a non-cancerous
condition).
The normal cell may be any cell which does not substantially express the
desired sequence to be detected. Examples of such normal cells are included
for
example, in the description of the drawings section. A normal cell sample or
tissue
thus include, for example, a normal (non-cancerous) ovarian cell, a normal
breast cell,
a normal prostate cell, a normal lymphocyte, a normal skin cell, a normal
renal cell, a
normal colon cell. a normal lung cell and/or a normal cell of the central
nervous
system. For comparison purposes , a normal cell may be chosen from those of
identical or similar cell type.
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Of course, the presence of more than one complex may be performed in
order to increase the precision of the diagnostic method. As such, at least
two
complexes (e.g., formed by a first reagent and a first polynucleotide and a
second
reagent or a second polynucleotide) or multiple complexes may be detected.
An exemplary embodiment of a reagent which may be used for detecting a
NSEQ described herein is a polynucleotide which may comprise a sequence
substantially complementary to the NSEQ.
A suitable reference level or value may be, for example, derived from the
level
of expression of a specified sequence in a low malignant potential ovarian
cancer
ci and/or from a normal cell.
It will be understood herein that a higher level of expression measured in a
cancer cell, tissue or sample in comparison with a reference value or sample
is a
indicative of the presence of cancer in the tested individual.
For example, the higher level measured in an ovarian cell, ovarian tissue or a
sample of ovarian origin compared to a reference level or value for a normal
cell
(normal ovarian cell or normal non-ovarian cell) may be indicative of an
ovarian
cancer. For comparison purpose, the presence or level of expression of
a
desired NSEQ or PSEQ to be detected or identified may be compared with the
presence, level of expression, found in a normal cell which has been shown
herein not
to express the desired sequence.
Therapeutic uses and methods are also encompassed herewith.
The invention therefore provides polynucleotides which may be able to lower
or inhibit the growth of an ovarian cancer cell (e.g., in a mammal or
mammalian cell
thereof).
75 The present invention therefore relates in a further aspect to the use
of a
polynucleotide sequence which may be selected from the group consisting of
a) a polynucleotide which may comprise a sequence substantially
complementary to any of SEQ ID NO.:1 to SEQ ID NO.49, 50 or 169
b) a polynucleotide which may comprise a sequence substantially
complementary to a transcribed or transcribable portion of any one of SEQ.
ID. NOs:1 to 49, 50 or 169,
c) a polynucleotide which may comprise a sequence substantially
complementary to a translated or translatable portion of any one of SEQ.
ID. NOs:1 to 49, 50 or 169, and;
d) a fragment of any one of a) to c)
for reducing, lowering or inhibiting the growth of a cancer cell.
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The polynucleotide may be selected, for example, from the group consisting
of polynucleotides which may comprise a sequence of at least 10 nucleotides
which is
complementary to the nucleic acid sequence of any one of SEQ ID NO.: 1 to 49,
50
and 169 (to a translated portion which may be free, for example, of
untranslated
portions).
Of course, the present invention encompasses immunizing an individual by
administering a NSEQ (e.g., in an expression vector) or a PSEQ.
The present invention also relates to a method of reducing or slowing the
growth of an ovarian cancer cell in an individual in need thereof. The method
may
comprise administering to the individual a polynucleotide sequence which may
be
selected from the group consisting of
a) a polynucleotide which may comprise a sequence substantially
complementary (also including 100% complementary over a portion, e.g., a
perfect match) to any of SEQ ID NO.:1 to SEQ ID NO.49 and 169 or 50,
b) a polynucleotide which may comprise a sequence substantially
complementary (also including 100% complementary over a portion, e.g., a
perfect match) to a transcribed or transcribable portion of any one of SEQ.
ID. NOs:1 to 49 and 169 or 50,
c) a polynucleotide which may comprise a sequence substantially
complementary (also including 100% complementary over a portion, e.g., a
perfect match) to a translated or translatable portion of any one of SEQ. ID.
NOs:1 to 49 and 169 or 50, and;
d) a fragment of any one of a) to c).
The present invention therefore provides in yet another aspect thereof, a
siRNA or shRNA molecule that is able to lower the expression of a nucleic acid
selected from the group consisting of
a) a polynucleotide which may comprise any one of SEQ ID NO. .1 to SEQ
ID NO.:49 and SEQ ID NO.:169, or SEQ ID NO.:50,
b) a polynucleotide which may comprise a transcribed or transcribable
portion of any one of SEQ. ID. NOs:1 to 49 and 169, or SEQ ID NO.:50,
c) a polynucleotide which may comprise a translated or translatable
portion of any one of SEQ. ID. NOs:1 to 49 and 169 or SEQ ID NO.:50, and;
d) a polynucleotide which may comprise a sequence substantially identical
to a), b), or c).
Exemplary embodiment of polynucleotides are those which, for example, may
be able to inhibit the growth of an ovarian cancer cell, such as, for example,
a
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polynucleotide having or comprising a sequence selected from the group
consisting of
any one of SEQ ID NO. 103 to 150. These specific sequences are provided as
guidance only and are not intended to limit the scope of the invention.
The present invention also provides a kit for the diagnosis of cancer. The kit
may comprise at least one polynucleotide as described herein and/or a reagent
capable of specifically binding at least one polynucleotide described herein.
In a further aspect, the present invention relates to an isolated polypeptide
encoded by the polynucleotide described herein.
The present invention more particularly provides an isolated polypeptide
which may be selected from the group consisting of
a) a polypeptide which may comprise any one of SEQ ID NO. :51 to 88 and
170
b) a polypeptide which may be encoded by any one of the polynucleotide
described herein,
c) a fragment of any one of a) or b),
d) a derivative of any one of a) or b) and;
e) an analog of any one of a) or b).
In accordance with the present invention, the analog may comprise, for
example, at least one amino acid substitution, deletion or insertion in its
amino acid
sequence.
The substitution may be conservative or non-conservative. The polypeptide
analog may be a biologically active analog or an immunogenic analog which may
comprise, for example, at least one amino acid substitution (conservative or
non
conservative), for example, 1 to 5, 1 to 10, 1 to 15, 1 to 20, 1 to 50 etc.
(including any
number there between) compared to the original sequence. An immunogenic analog
may comprise, for example, at least one amino acid substitution compared to
the
original sequence and may still be bound by an antibody specific for the
original
sequence.
In accordance with the present invention, a polypeptide fragment may
comprise, for example, at least 6 consecutive amino acids, at least 8
consecutive
amino acids or more of an amino acid sequence selected from the group
consisting of
polypeptides encoded by a polynucleotide selected from the group consisting of
SEQ
ID NO.: 1 to 49 and 169 or any one of SEQ. ID. NOs:51 to 88 and 170, including
variants and analogs thereof. The fragment may be immunogenic and may be used
for the purpose, for example, of generating antibodies.
Exemplary embodiments of polypeptide encompassed by the present
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invention are those which may be encoded by any one of SEQ ID NO. :1-49 and
169,
more particularly those encoded by any one of SEQ ID NO.:1, 14, 16, 19, 20,
22, 28,
37, 41, 45, 46, 47 or 49 and even more particularly those encoded by any one
of SEQ
ID NO.: 14, 19, 22, 37, 41, 45, 46 or 49.
In a further aspect the present invention relates to a polypeptide which may
be encoded by the isolated differentially expressed sequence of the present
invention.
The present invention as well relates to the polypeptide encoded by the non-
human
ortholog polynucleotide, analogs, derivatives and fragments thereof.
A person skilled in the art may easily determine the possible peptide
sequence encoded by a particular nucleic acid sequence as generally, a maximum
of
6 possible open-reading frames exist in a particular coding sequence. The
first
possible open-reading frame may start at the first nucleotide (5'-3') of the
sequence,
therefore using in a 5' to 3' direction nucleotides No. 1 to 3 as the first
codon, using
nucleotides 4 to 6 as the second codon, etc. The second possible open-reading
frame
may start at the second nucleotide (5'-3') of the sequence, therefore using in
a 5' to 3'
direction nucleotides No. 2 to 4 as the first codon, using nucleotides 5 to 7
as the
second codon, etc. Finally, the third possible open-reading frame may start at
the third
nucleotide (5'-3') of the sequence, therefore using in a 5' to 3' direction
nucleotides No.
3 to 5 as the first codon, using nucleotides 6 to 8 as the second codon, etc.
The fourth
possible open-reading frame may start at the first nucleotide of the sequence
in a 3' to
5' direction, therefore using in 3' to 5' direction, nucleotides No. 1 to 3 as
the first
codon, using nucleotides 4 to 6 as the second codon, etc. The fifth possible
open-
reading frame may start at the second nucleotide of the sequence in a 3' to 5'
direction, therefore using in a 3' to 5' direction, nucleotides No. 2 to 4 as
the first
codon, using nucleotides 5 to 7 as the second codon, etc. Finally, the sixth
possible
open-reading frame may start at the third nucleotide of the sequence in a 3'
to 5'
direction, therefore using in a 3' to 5' direction nucleotides No. 3 to 5 as
the first codon,
using nucleotides 6 to 8 as the second codon, etc.
In an additional aspect, the present invention relates to the use of at least
one
polypeptide in the manufacture of a composition for the identification or
detection of a
cancer cell (tumor cell). The polypeptide may be used, for example, as a
standard in
an assay and/or for detecting antibodies specific for the particular
polypeptide, etc.
In yet an additional aspect, the present invention relates to the use of at
least one
polypeptide described herein in the identification or detection of a cancer
cell, such as
for example, an ovarian cancer cell or any other cancer cell as described
herein.
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The present invention therefore relates in a further aspect, to the use of at
least one polypeptide described herein in the prognosis or diagnosis of
cancer, such
as, for example, a malignant ovarian cancer or a low malignant potential
ovarian
cancer.
As such and in accordance with the present invention, detection of the
polypeptide in a cell (e.g., ovarian cell), tissue (e.g., ovarian tissue),
sample or body
fluid from an individual may preferentially be indicative of a malignant
ovarian cancer
diagnosis over a low malignant potential ovarian cancer diagnosis and
therefore may
preferentially be indicative of a malignant ovarian cancer rather than a low
malignant
it) potential ovarian cancer.
Further in accordance with the present invention, the presence of the
polypeptide in a cell, tissue, sample or body fluid from an individual may
preferentially
be indicative of a late-stage malignant ovarian cancer.
There is also provided by the present invention, methods for identifying a
cancer cell, which may comprise, for example, contacting a test cell, a test
cell sample
(cell lysate), a test body fluid (blood, urine, plasma, saliva etc.) or a test
tissue with a
reagent which may be capable of specifically binding the polypeptide described
herein,
and detecting the complex formed by the polypeptide and reagent. The presence
of a
complex may be indicative (a positive indication of the presence) of a cancer
cell such
as for example, an ovarian cancer cell, a breast cancer cell, a prostate
cancer cell,
leukemia, melanoma, a renal cancer cell, a colon cancer cell, a lung cancer
cell, a
cancer cell of the central nervous system and any combination thereof.
The presence of a complex formed by the polypeptide and the specific
reagent may be indicative, for example, of ovarian cancer including, for
example, a low
malignant potential ovarian cancer or a malignant ovarian cancer.
However, the method is more particularly powerful for the detection of ovarian
cancer of the malignant type. Therefore, the presence of a complex may
preferentially
be indicative of a malignant ovarian cancer relative (rather than) to a low
malignant
potential ovarian cancer.
Detection of the complex may also be indicative of a late stage malignant
ovarian cancer.
In accordance with the present invention, the method may also comprise a
step of qualitatively or quantitatively comparing the level (amount, presence)
of at least
one complex present in a test cell, a test sample, a test fluid or a test
tissue with the
level of complex in a normal cell, a normal cell sample, a normal body fluid,
a normal
tissue or a reference value (e.g., for a non-cancerous condition).
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Of course, the presence of more than one polypeptide or complex (two
complexes or more (multiple complexes)) may be determined, e.g., one formed by
a
first specific reagent and a first polypeptide and another formed by a second
specific
reagent and a second polypeptide may be detected. Detection of more than one
polypeptide or complex may help in the determination of the tumorigenicity of
the cell.
An exemplary embodiment of a reagent, which may be used for the detection
of the polypeptide described herein, is an antibody and antibody fragment
thereof.
The present invention also relates to a kit which may comprise at least one of
the polypeptide described herein and/or a reagent capable of specifically
binding to at
least one of the polypeptide described herein.
As one skill in the art will understand, compositions which comprises a
polypeptide may be used, for example, for generating antibodies against the
particular
polypeptide, may be used as a reference for assays and kits, etc.
Additional aspects of the invention relates to isolated or purified antibodies
(including an antigen-binding fragment thereof) which may be capable of
specifically
binding to a polypeptide selected from the group consisting of;
a) a polypeptide comprising or consisting of any one of SEQ ID NO. :51 to
89 or 170, and;
b) a polypeptide comprising a polypeptide sequence encoded by any one of
the polynucleotide sequence described herein (e.g., a fragment of at least 6
amino acids of the polypeptide).
More particularly, exemplary embodiments of the present invention relates to
antibodies which may be capable of specifically binding a polypeptide
comprising a
polypeptide sequence encoded by any one of SEQ ID NO.: 1, 14, 16, 19, 20, 22,
28,
37, 41, 45, 46, 47 or 49, or a fragment of at least 6 amino acids of the
polypeptide.
Even more particular exemplary embodiments of the present invention relates
to antibodies which may be capable of specifically binding a polypeptide
comprising a
polypeptide sequence encoded by any one of SEQ ID NO.: 14, 19, 22, 37, 41, 45,
46
or 49, or a fragment of at least 6 amino acids of the polypeptide.
In yet an additional aspect, the present invention relates to a hybridoma cell
which is capable of producing an antibody which may specifically bind to a
polypeptide
selected from the group consisting of;
a) a polypeptide which may comprise any one of SEQ ID NO. :51 to 88, 89
and 170, and;
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b) a polypeptide which may comprise a polypeptide sequence encoded by
any one of the polynucleotide sequence described herein or a fragment of at
least 6 amino acids of the polypeptide.
Exemplary hybridoma which are more particularly encompassed by the
present invention are those which may produce an antibody which may be capable
of
specifically binding a polypeptide comprising a polypeptide sequence encoded
by any
one of SEQ ID NO.: 1, 14, 16, 19, 20, 22, 28, 37, 41, 45, 46, 47 or 49 or a
fragment of
at least 6 amino acids of the polypeptide.
Exemplary embodiments of hybridoma which are even more particularly
encompassed by the present invention are those which may produce an antibody
which is capable of specifically binding a polypeptide comprising a
polypeptide
sequence encoded by any one of SEQ ID NO.: 14, 19, 22, 37, 41, 45, 46 or 49 or
a
fragment of at least 6 amino acids of the polypeptide.
The present invention also relates to a composition which may comprise an
antibody described herein.
In a further aspect the present invention provides a method of making an
antibody
which may comprise immunizing a non-human animal with an immunogenic fragment
(at least 6 amino acids, at least 8 amino acids, etc.) of a polypeptide which
may be
selected, for example, from the group consisting of;
a) a polypeptide which may comprise or consist in any one of SEQ ID
NO.:51 to 88, 89 and 170 or a fragment thereof, and;
b) a polypeptide which may comprise a polypeptide sequence encoded by
any one of the polynucleotide sequence described herein or a portion
thereof.
Exemplary polypeptides which may, more particularly, be used for generating
antibodies are those which are encoded by any one of SEQ ID NO.: 1, 14, 16,
19, 20,
22, 28, 37, 41, 45, 46, 47 or 49 (and polypeptide comprising a polypeptide
fragment of
these particular PSEQ). Even more particular polypeptides encompassed by the
present invention are those which are encoded by any one of SEQ ID NO.: 14,
19, 22,
37, 41, 45, 46 or 49.
In a further aspect, the present invention relates to a method of identifying
a
compound which is capable of inhibiting the activity or function of a
polypeptide which
may be selected, for example from the group consisting of any one of SEQ ID
NO. :51
to 88 and 170 or a polypeptide comprising a polypeptide sequence encoded by
any
one of SEQ ID NO.:1 to 49 and 169 (e.g., a transcribed portion, a translated
portion, a
fragment, substantially identical and even substantially complementary
sequences).
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The method may comprise contacting the polypeptide with a putative compound an
isolating or identifying a compound which is capable of specifically binding
any one of
the above mentioned polypeptide. The compound may originate from a
combinatorial
library.
The method may also further comprise determining whether the activity or
function of the polypeptide (e.g., such as a function indicated at Table 2) is
affected by
the binding of the compound. Those compounds which capable of binding to the
polypeptide and which and/or which are capable of altering the function or
activity of
the polypeptide represents a desirable compound to be used in cancer therapy.
0 The method may also further comprise a step of determining the effect of
the
putative compound on the growth of a cancer cell such as an ovarian cancer
cell.
The present invention also relates to an assay and method for identifying a
nucleic acid sequence and/or protein involved in the growth or development of
ovarian
cancer. The assay and method may comprise silencing an endogenous gene of a
cancer cell such as an ovarian cancer cell and providing the cell with a
candidate
nucleic acid (or protein). A candidate gene (or protein) positively involved
in inducing
cancer cell death (e.g., apoptosis) (e.g., ovarian cancer cell ) may be
identified by its
ability to complement the silenced endogenous gene. For example, a candidate
nucleic acid involved in ovarian cancer provided to a cell for which an
endogenous
gene has been silenced, may enable the cell to undergo apoptosis more so in
the
presence of an inducer of apoptosis.
Alternatively, an assay or method may comprise silencing an endogenous
gene (gene expression) corresponding to the candidate nucleic acid or protein
sequence to be evaluated and determining the effect of the candidate nucleic
acid or
protein on cancer growth (e.g., ovarian cancer cell growth). A sequence
involved in
the promotion or inhibition of cancer growth, development or malignancy may
change
the viability of the cell, may change the ability of the cell to grow or to
form colonies,
etc. The activity of a polypeptide may be impaired by targeting such
polypeptide with
an antibody molecule or any other type of compound. Again, such compound may
be
identified by screening combinatorial libraries, phage libraries, etc.
The present invention also provides a method for identifying an inhibitory
compound (inhibitor, antagonist) able to impair the function (activity) or
expression of a
polypeptide described herein. The method may comprise, for example, contacting
the
(substantially purified or isolated) polypeptide or a cell expressing the
polypeptide with
a candidate compound and measuring the function (activity) or expression of
the
polypeptide. A reduction in the function or activity of the polypeptide
(compared to the
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absence of the candidate compound) may thus positively identify a suitable
inhibitory
compound.
In accordance with the present invention, the impaired function or activity
may
be associated, for example, with a reduced ability of the polypeptide to
reduce growth
of an ovarian cancer cell or a reduced enzymatic activity or function
identified for
example in Table 2.
The cell used to carry the screening test may not naturally (endogenously)
express the polypeptide or analogs, or alternatively the expression of a
naturally
expressed polypeptide analog may be repressed.
As used herein the term " sequence identity" relates to (consecutive)
nucleotides of a nucleotide sequence with reference to an original nucleotide
sequence which when compared are the same or have a specified percentage of
nucleotides which are the same.
The identity may be compared over a region or over the total sequence of a
nucleic acid sequence. Thus, "identity" may be compared, for example, over a
region
of 10, 19, 20 nucleotides or more (and any number therebetween) and more
preferably
over a longer region or over the entire region of a polynucleotide sequence
described
at Table 4 (e.g., any one of SEQ ID NO.:1 to 49 and 169). It is to be
understood
herein that gaps of non-identical nucleotides may be found between identical
nucleic
acids regions (identical nucleotides). For example, a polynucleotide may have
100%
identity with another polynucleotide over a portion thereof. However, when the
entire
sequence of both polynucleotides is compared, the two polynucleotides may have
50%
of their overall (total) sequence identity to one another.
Percent identity may be determined, for example, with n algorithm GAP,
BESTFIT, or FASTA in the Wisconsin Genetics Software Package Release 7.0,
using
default gap weights.
Polynucleotides of the present invention or portion thereof having from about
50 to about 100% and any range therebetween, or about 60 to about 100% or
about
70 to about 100% or about 80 to about 100% or about 85% to about 100%, about
90%
to about 100%, about 95% to about 100% sequence identity with an original
polynucleotide are encompassed herewith. It is known by those of skill in the
art, that
a polynucleotide having from about 50% to 100% identity may function (e.g.,
anneal to
a substantially complementary sequence) in a manner similar to an original
polynucleotide and therefore may be used in replacement of an original
polynucleotide.
For example a polynucleotide (a nucleic acid sequence) may comprise or have
from
about 50% to about 100% identity with an original polynucleotide over a
defined region
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and may still work as efficiently or sufficiently to achieve the present
invention.
The term "substantially identical" used to define the polynucleotides of the
present
invention refers to polynucleotides which have, for example, from 50% to '100%
sequence identity and any range therebetween but preferably at least 80%, at
least
85%, at least 90%, at least 95% sequence identity and also include 100%
identity with
that of an original sequence (including sequences 100% identical over the
entire
length of the polynucleotide sequence).
"Substantially identical" polynucleotide sequences may be identified by
providing a probe of about 10 to about 25, or more or about 10 to about 20
nucleotides
Ri long (or
longer) based on the sequence of any one of SEQ ID NOs.:1 to 49 and 169
(more particularly, a transcribed and/or translated portion of any one of SEQ
ID NOs.:
1 to 49 and 169) and complementary sequence thereof and hybridizing a library
of
polynucleotide (e.g., cDNA or else) originating from another species, tissue,
cell,
individual etc. A polynucleotide which hybridizes under highly stringent
conditions
(e.g., 6XSCC, 65 C) to the probe may be isolated and identified using methods
known
in the art. A sequence "substantially identical" includes for example, an
isolated allelic
variant, an isolated splice variant, an isolated non-human ortholog, a
modified NSEQ
etc.
As used herein the terms" sequence complementarity" refers to (consecutive)
nucleotides of a nucleotide sequence which are complementary to a reference
(original) nucleotide sequence. The complementarity may be compared over a
region
or over the total sequence of a nucleic acid sequence.
Polynucleotides of the present invention or portion thereof having from about
50 to about 100%, or about 60 to about 100% or about 70 to about 100% or about
80
to about 100% or about 85%, about 90%, about 95% to about 100% sequence
complementarity with an original polynucleotide are thus encompassed herewith.
It is
known by those of skill in the art, that a polynucleotide having from about
50% to 100%
complementarity with an original sequence may anneal to that sequence in a
manner
sufficient to carry out the present invention (e.g., inhibit expression of the
original
polynucleotide).
The term "substantially complementary" used to define the polynucleotides of
the present invention refers to polynucleotides which have, for example, from
50% to
100% sequence complementarity and any range therebetween but preferably at
least
80%, at least 85%, at least 90%, at least 95% sequence complementarity and
also
include 100% complementarity with that of an original sequence (including
sequences
100% complementarity over the entire length of the polynucleotide sequence).
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As used herein the term "polynucleotide" generally refers to any
polyribonucleotide or polydeoxyribo-nucleotide, which may be unmodified RNA or
DNA, or modified RNA or DNA. "Poiynucleotides" include, without limitation
single-
and double-stranded DNA, DNA that is a mixture of single- and double-stranded
regions, single- and double-stranded RNA, and RNA that is a mixture of single-
and
double-stranded regions, hybrid molecules comprising DNA and RNA that may be
single-stranded or, more typically, double-stranded or a mixture of single-
and double-
stranded regions. In
addition, "polynucleotide" refers to triple-stranded regions
comprising RNA or DNA or both RNA and DNA. The term polynucleotide also
io includes DNAs
or RNAs containing one or more modified bases and DNAs or RNAs
with backbones modified for stability or for other reasons. "Modified" bases
include, for
example, tritylated bases and unusual bases such as inosine. A variety of
modifications may be made to DNA and RNA; thus "polynucleotide" embraces
chemically, enzymatically or metabolically modified forms of polynucleotides
as
typically found or not in nature, as well as the chemical forms of DNA and RNA
characteristic of viruses and cells. "Polynucleotide" includes but is not
limited to linear
and end-closed molecules.
"Polynucleotide" also embraces relatively short
polynucleotides, often referred to as oligonucleotides.
"Polypeptides" refers to any peptide or protein comprising two or more amino
acids joined to each other by peptide bonds or modified peptide bonds (i.e.,
peptide
isosteres). "Polypeptide" refers to both short chains, commonly referred as
peptides,
oligopeptides or oligomers, and to longer chains generally referred to as
proteins. As
described above, polypeptides may contain amino acids other than the 20 gene-
encoded amino acids.
As used herein the term "polypeptide analog" or "analog" relates to mutants,
chimeras, fusions, a polypeptide comprising at least one amino acid deletion,
a
polypeptide comprising at least one amino acid insertion or addition, a
polypeptide
comprising at least one amino acid substitutions, and any other type of
modifications
made relative to a given polypeptide.
An "analog" is thus to be understood herein as a molecule having a biological
activity and/or chemical structure similar to that of a polypeptide described
herein. An
"analog" may have sequence similarity with that of an original sequence or a
portion of
an original sequence and may also have a modification of its structure as
discussed
herein. For example, an "analog" may have at least 80% or 85% or 90 % sequence
similarity with an original sequence or a portion of an original sequence. An
"analog"
may also have, for example; at least 70 % or even 50 % sequence similarity
with an
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original sequence or a portion of an original sequence and may function in a
suitable
manner.
A "derivative" is to be understood herein as a polypeptide originating from an
original sequence or from a portion of an original sequence and which may
comprise
one or more modification; for example, one or more modification in the amino
acid
sequence (e.g., an amino acid addition, deletion, insertion, substitution
etc.), one or
more modification in the backbone or side-chain of one or more amino acid, or
an
addition of a group or another molecule to one or more amino acids (side-
chains or
backbone).
Biologically active derivatives of the carrier described herein are
io encompassed by
the present invention. Also, an "derivative" may have, for example,
at least 50 %, 70%, 80%, 90% sequence similarity to an original sequence with
a
combination of one or more modification in a backbone or side-chain of an
amino acid,
or an addition of a group or another molecule, etc.
As used herein the term "biologically active" refers to an analog which
retains
i5 some or all of
the biological activity of the original polypeptide, i.e., to have some of the
activity or function associated with the polypeptide described at Table 2, or
to be able
to promote or inhibit the growth ovarian cancer.
Therefore, any polypeptide having a modification compared to an original
polypeptide which does not destroy significantly a desired activity, function
or
20 immunogenicity
is encompassed herein. It is well known in the art, that a number of
modifications may be made to the polypeptides of the present invention without
deleteriously affecting their biological activity. These modifications may, on
the other
hand, keep or increase the biological activity of the original polypeptide or
may
optimize one or more of the particularity (e.g. stability, bioavailability,
etc.) of the
25 polypeptides
of the present invention which, in some instance might be desirable.
Polypeptides of the present invention may comprise for example, those
containing
amino acid sequences modified either by natural processes, such as
posttranslational
processing, or by chemical modification techniques which are known in the art.
Modifications may occur anywhere in a polypeptide including the polypeptide
30 backbone, the
amino acid side-chains and the amino- or carboxy-terminus. It will be
appreciated that the same type of modification may be present in the same or
varying
degrees at several sites in a given polypeptide. Also, a given polypeptide may
contain
many types of modifications. lt is to be understood herein that more than one
modification to the polypeptides described herein are encompassed by the
present
35 invention to
the extent that the biological activity is similar to the original (parent)
polypeptide.
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As discussed above, polypeptide modification may comprise, for example,
amino acid insertion, deletion and substitution (i.e., replacement), either
conservative
or non-conservative (e.g., D-amino acids, desamino acids) in the polypeptide
sequence where such changes do not substantially alter the overall biological
activity
of the polypeptide.
Example of substitutions may be those, which are conservative (i.e., wherein
a residue is replaced by another of the same general type or group) or when
wanted,
non-conservative (i.e., wherein a residue is replaced by an amino acid of
another
type). In addition, a non-naturally occurring amino acid may substitute for a
naturally
occurring amino acid (i.e., non-naturally occurring conservative amino acid
substitution
or a non-naturally occurring non-conservative amino acid substitution).
As is understood, naturally occurring amino acids may be sub-classified as
acidic, basic, neutral and polar, or neutral and non-polar. Furthermore, three
of the
encoded amino acids are aromatic. It may be of use that encoded polypeptides
differing from the determined polypeptide of the present invention contain
substituted
codons for amino acids, which are from the same type or group as that of the
amino
acid to be replaced. Thus, in some cases, the basic amino acids Lys, Arg and
His may
be interchangeable; the acidic amino acids Asp and Glu may be interchangeable;
the
neutral polar amino acids Ser, Thr, Cys, Gln, and Asn may be interchangeable;
the
non-polar aliphatic amino acids Gly, Ala, Val, Ile, and Leu are
interchangeable but
because of size Gly and Ala are more closely related and Val, Ile and Leu are
more
closely related to each other, and the aromatic amino acids Phe, Trp and Tyr
may be
interchangeable.
It should be further noted that if the polypeptides are made synthetically,
substitutions by amino acids, which are not naturally encoded by DNA (non-
naturally
occurring or unnatural amino acid) may also be made.
A non-naturally occurring amino acid is to be understood herein as an amino
acid which is not naturally produced or found in a mammal. A non-naturally
occurring
amino acid comprises a D-amino acid, an amino acid having an acetylaminomethyl
group attached to a sulfur atom of a cysteine, a pegylated amino acid, etc.
The
inclusion of a non-naturally occurring amino acid in a defined polypeptide
sequence
will therefore generate a derivative of the original polypeptide. Non-
naturally occurring
amino acids (residues) include also the omega amino acids of the formula
NH2(CH2)nCOOH wherein n is 2-6, neutral nonpolar amino acids, such as
sarcosine, t-
butyl alanine, t-butyl glycine, N-methyl isoleucine, norleucine, etc.
Phenylglycine may
substitute for Trp, Tyr or Phe; citrulline and methionine sulfoxide are
neutral nonpolar,
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cysteic acid is acidic, and ornithine is basic. Proline may be substituted
with
hydroxyproline and retain the conformation conferring properties.
It is known in the art that analogs may be generated by substitutional
mutagenesis and retain the biological activity of the polypeptides of the
present
invention. These analogs have at least one amino acid residue in the protein
molecule
removed and a different residue inserted in its place. For example, one site
of interest
for substitutional mutagenesis may include but are not restricted to sites
identified as
the active site(s), or immunological site(s). Other sites of interest may be
those, for
example, in which particular residues obtained from various species are
identical.
These positions may be important for biological activity. Examples of
substitutions
identified as "conservative substitutions" are shown in Table A. If such
substitutions
result in a change not desired, then other type of substitutions, denominated
"exemplary substitutions" in Table A, or as further described herein in
reference to
amino acid classes, are introduced and the products screened.
In some cases it may be of interest to modify the biological activity of a
polypeptide by amino acid substitution, insertion, or deletion. For
example,
modification of a polypeptide may result in an increase in the polypeptide's
biological
activity, may modulate its toxicity, may result in changes in bioavailability
or in stability,
or may modulate its immunological activity or immunological identity.
Substantial
modifications in function or immunological identity are accomplished by
selecting
substitutions that differ significantly in their effect on maintaining (a) the
structure of the
polypeptide backbone in the area of the substitution, for example, as a sheet
or helical
conformation. (b) the charge or hydrophobicity of the molecule at the target
site, or (c)
the bulk of the side chain. Naturally occurring residues are divided into
groups based
on common side chain properties:
(1) hydrophobic: norleucine, methionine (Met), Alanine (Ala), Valine (Val),
Leucine (Leu), lsoleucine (11e)
(2) neutral hydrophilic: Cysteine (Cys), Serine (Ser), Threonine (Thr)
(3) acidic: Aspartic acid (Asp), Glutamic acid (Glu)
(4) basic: Asparagine (Asn), Glutamine (Gin), Histidine (His), Lysine
(Lys),
Arginine (Arg)
(5) residues that influence chain orientation: Glycine (Gly), Proline
(Pro); and
aromatic: Tryptophan (Trp), Tyrosine (Tyr), Phenylalanine (Phe)
Non-conservative substitutions will entail exchanging a member of one of
these classes for another.
TABLE A. Examplary amino acid substitution
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Original residue Exemplary substitution I Conservative
substitution
Ala (A) Val, Leu, Ile Val
Arg (R) Lys, Gln, Asn Lys
Asn (N) Gln, His, Lys, Arg Gln
Asp (D) Glu Glu
Cys (C) Ser Ser
Gln (Q) Asn Asn
Glu (E) Asp Asp
Gly (G) Pro Pro
His (H) Asn, Gln, Lys, Arg Arg
Ile (I) Leu, Val, Met, Ala, Phe, Leu
norleucine
Leu (L) Norleucine, Ile, Val, Met, îlle
Ala, Phe 1
Lys (K) Arg, Gln, Asn Arg
Met (M) Leu, Phe, Ile Leu
Phe (F) Leu, Val, Ile, Ala Leu
Pro (P) Gly Gly
Ser (S) Thr Thr
Thr (T) Ser Ser
Trp (W) Tyr Tyr
Tyr (Y) Trp, Phe, Thr, Ser Phe
Val (V) Ile, Leu, Met, Phe, Ala, Leu
norleucine
It is to be understood herein, that if a "range" or "group" of substances
(e.g.
amino acids), substituents" or the like is mentioned or if other types of a
particular
characteristic (e.g. temperature, pressure, chemical structure, time, etc.) is
mentioned,
the present invention relates to and explicitly incorporates herein each and
every
specific member and combination of sub-ranges or sub-groups therein
whatsoever.
Thus, any specified range or group is to be understood as a shorthand way of
referring
to each and every member of a range or group individually as well as each and
every
possible sub-ranges or sub-groups encompassed therein; and similarly with
respect to
any sub-ranges or sub-groups therein. Thus, for example, with respect to a
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percentage (%) of identity of from about 80 to 100%, it is to be understood as
specifically incorporating herein each and every individual A), as well as
sub-range,
such as for example 80%, 81%, 84.78%, 93%, 99% etc. with respect to a length
of
"about 10 to about 25" it is to be understood as specifically incorporating
each and
every individual numebr such as for example 10, 11, 12, 13, 14, 15 up to and
including
25; and similarly with respect to other parameters such as, concentrations,
elements,
etc...
Other objects, features, advantages, and aspects of the present invention will
become apparent to those skilled in the art from the following description. It
should be
understood, however, that the following description and the specific examples,
while
indicating preferred embodiments of the invention, are given by way of
illustration only.
Various changes and modifications within the spirit and scope of the disclosed
invention will become readily apparent to those skilled in the art from
reading the
following description and from reading the other parts of the present
disclosure.
BRIEF DESCRIPTION OF THE DRAWINGS
In the appended drawings:
Fig. 1 to Fig. 31, Fig. 33, Fig. 34, Fig. 36, Fig. 37, Fig. 39, Fig. 40, Fig.
42, Fig.
43, Fig. 46, Fig. 47, Fig. 49, Fig. 50 and Fig. 56 are pictures of macroarray
hybridization results showing the differential expression data for STAR
selected
ovarian cancer-related human sequences. Macroarrays were prepared using RAMP
amplified RNA from six human LMP samples (A-F 1) and twenty malignant ovarian
tumor samples (Table B) (A-F 2 and A-G 3-4), and 30 different normal human
tissues
(adrenal (A7), breast (67), jejunum (C7), trachea (D7), liver (E7), placenta
(F7), aorta
(G7), brain (H7), lung (A8), adrenal cortex (B8), esophagus (C8), colon (08),
ovary
(E8), kidney (F8), prostate (G8), thymus (H8), skeletal muscle (A9), vena cava
(B9),
stomach (C9), small intestine (D9), heart (E9), fallopian tube (F9), spleen
(G9), bladder
(H9), cervix (A10), pancreas (810), ileum (C10), duodenum (D10), thyroid (E10)
and
testicle (F10)). Also included on the RNA macroarray were breast cancer cell
lines
(MDA (A5), MCF7 (B5) and MCF7+estradiol (C5)) and LCM microdissected prostate
normal epithelium (A-C 6) and prostate cancer (D-F 6), prostate cancer cell
line,
LNCap (G6) and LNCap+androgen (H6). In these figures, the probe labeling
reaction
was also spiked with a dsDNA sequence for Arabidopsis, which hybridizes to the
same
sequence spotted on the macroarray (M) in order to serve as a control for the
labeling
reaction.
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Fig. 32, Fig. 35, Fig. 38, Fig. 41, Fig. 44, Fig. 45 and Fig. 48 are pictures
of
RT-PCR results showing the differential expression data for STAR selected
ovarian
cancer-related human sequences. Complimentary DNAs were prepared using random
hexamers from RAMP amplified RNA from six human LMP samples and at least
twenty malignant ovarian tumor samples (Table B) as indicated in the figures.
The
cDNAs were quantified and used as templates for PCR with gene-specific primers
using standard methods known to those skilled in the art.
Fig. 57 to Fig. 105 are pictures of RT-PCR results showing the differential
expression data for STAR selected cancer-related human sequences in RNA
samples
derived from the NCI-60 panel of cancer cell lines. These 59 cell lines are
derived from
tumors that encompass 9 human cancer types that include leukemia, the central
nervous system, breast, colon, lung, melanoma, ovarian, prostate, and renal.
Complimentary DNAs were prepared using random hexamers from RAMP amplified
RNA from 59 human cancer cell lines (Table C). The cDNAs were quantified and
used
as templates for PCR with gene-specific primers using standard methods known
to
those skilled in the art. For each PCR result depicted in Fig. 57 to Fig. 105,
equal
amounts of template cDNA used in each PCR reaction was confirmed by
reamplifying
GAPDH with a specific primer pair, OGS 315
(TGAAGGTCGGAGTCAACGGATTTGGT; SEQ. ID. NO. 167) and OGS 316
(CATGTGGGCCATGAGGTCCACCAC; SEQ. ID. NO. 168) for this housekeeping
gene.
More particularly,
Fig. 1 is a picture of the macroarray hybridization results showing the
differential expression data for STAR selected ovarian cancer-related human
SEQ. ID.
NO. 1. The STAR dsDNA clone representing SEQ. ID. NO. 1 was labeled with 32P
and
hybridized to the macroarray. The hybridization results obtained confirm its
upregulation in a majority of malignant ovarian cancer samples (A-F 2 and A-G
3-4)
compared to LMP samples (A-F 1). Expression of this sequence was only observed
in
one (placenta (F7)) of the 30 normal tissues and the breast cancer cell line,
MCF7 (B-
C5);
Fig. 2 is a picture of the macroarray hybridization results showing the
differential expression data for STAR selected ovarian cancer-related human
SEQ. ID.
NO. 2. The STAR dsDNA clone representing SEQ. ID. NO. 2 was labeled with 32P
and
hybridized to the macroarray. The hybridization results obtained confirm its
upregulation in a majority of malignant ovarian cancer samples (A-F 2 and A-G
3-4)
compared to LMP samples (A-F 1). Expression of this sequence was also evident
in
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PCT/CA2007/001134
six (breast (B7), placenta (F7), aorta (G7), colon (D8), ovary (E8) and thymus
(H8)) of
the 30 normal tissues;
Fig. 3 is a picture of the macroarray hybridization results showing the
differential expression data for STAR selected ovarian cancer-related human
SEQ. ID.
NO. 3. The STAR dsDNA clone representing SEQ. ID. NO. 3 was labeled with 32P
and
hybridized to the macroarray. The hybridization results obtained confirm its
upregulation in a majority of malignant ovarian cancer samples (A-F 2 and A-G
3-4)
compared to LMP samples (A-F 1) but overall, only low levels of expression. No
significant expression was seen in any of the normal tissues;
Fig. 4 is a picture of the macroarray hybridization results showing the
differential expression data for STAR selected ovarian cancer-related human
SEQ. ID.
NO. 4. The STAR dsDNA clone representing SEQ. ID. NO. 4 was labeled with 32P
and
hybridized to the macroarray. The hybridization results obtained confirm its
upregulation in a majority of malignant ovarian cancer samples (A-F 2 and A-G
3-4)
compared to LMP samples (A-F 1). Expression of this sequence was also evident
in
two (esophagus (C8) and fallopian tube (F9)) of the 30 normal tissues;
Fig. 5 is a picture of the macroarray hybridization results showing the
differential expression data for STAR selected ovarian cancer-related human
SEQ. ID.
NO. 5. The STAR dsDNA clone representing SEQ. ID. NO. 5 was labeled with 32P
and
hybridized to the macroarray. The hybridization results obtained confirm its
upregulation in a majority of malignant ovarian cancer samples (A-F 2 and A-G
3-4)
compared to LMP samples (A-F 1). Weak expression of this sequence similar to
that of
LMPs was also observed in many of the normal tissues;
Fig. 6 is a picture of the macroarray hybridization results showing the
differential expression data for STAR selected ovarian cancer-related human
SEQ. ID.
NO. 6. The STAR dsDNA clone representing SEQ. ID. NO. 6 was labeled with 32P
and
hybridized to the macroarray. The hybridization results obtained confirm its
upregulation in a majority of malignant ovarian cancer samples (A-F 2 and A-G
3-4)
compared to LMP samples (A-F 1). Expression of this sequence was also evident
in
three (liver (E7), placenta (F7) and kidney (F8)) of the 30 normal tissues;
Fig. 7 is a picture of the macroarray hybridization results showing the
differential expression data for STAR selected ovarian cancer-related human
SEQ. ID.
NO. 7. The STAR dsDNA clone representing SEQ. ID. NO. 7 was labeled with 32P
and
hybridized to the macroarray. The hybridization results obtained confirm its
upregulation in several malignant ovarian cancer samples (A-F 2 and A-G 3-4)
48
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compared to LMP samples (A-F 1). Expression of this sequence was only evident
in
one (testicle (F10)) of the 30 normal tissues;
Fig. 8 is a picture of the macroarray hybridization results showing the
differential expression data for STAR selected ovarian cancer-related human
SEQ. ID.
NO. 8. The STAR dsDNA clone representing SEQ. ID. NO. 8 was labeled with 32P
and
hybridized to the macroarray. The hybridization results obtained confirm its
upregulation in a majority of malignant ovarian cancer samples (A-F 2 and A-G
3-4)
compared to LMP samples (A-F 1). Expression of this sequence was only evident
in
two (esophagus (C8) and stomach (C9)) of the 30 normal tissues and the breast
and
io prostate cancer cell lines, MDA (A5) and LNCap (G6 and H6),
respectively;
Fig. 9 is a picture of the macroarray hybridization results showing the
differential expression data for STAR selected ovarian cancer-related human
SEQ. ID.
NO. 9. The STAR dsDNA clone representing SEQ. ID. NO. 9 was labeled with 32P
and
hybridized to the macroarray. The hybridization results obtained confirm its
upregulation in a majority of malignant ovarian cancer samples (A-F 2 and A-G
3-4)
compared to LMP samples (A-F 1). Expression of this sequence was only evident
in
one (placenta (F7)) of the 30 normal tissues, the breast cancer cell line,
MCF7 (B-C 5)
and LCM microdissected prostate cancer samples (D6 and F6);
Fig. 10 is a picture of the macroarray hybridization results showing the
differential expression data for STAR selected ovarian cancer-related human
SEQ. ID.
NO. 10. The STAR dsDNA clone representing SEQ. ID. NO. 10 was labeled with 32P
and hybridized to the macroarray. The hybridization results obtained confirm
its
upregulation in a majority of malignant ovarian cancer samples (A-F 2 and A-G
3-4)
compared to LMP samples (A-F 1). Expression of this sequence was only evident
in
one (testicle (F10)) of the 30 normal tissues, the breast cancer cell lines,
MDA (A5)
and MCF7 (B-C 5) and prostate cancer cell line, LNCap (G-H 6);
Fig. 11 is a picture of the macroarray hybridization results showing the
differential expression data for STAR selected ovarian cancer-related human
SEQ. ID.
NO. 11. The STAR dsDNA clone representing SEQ. ID. NO. 11 was labeled with 32P
and hybridized to the macroarray. The hybridization results obtained confirm
its
upregulation in a majority of malignant ovarian cancer samples (A-F 2 and A-G
3-4)
compared to LMP samples (A-F 1). Significant expression of this sequence was
only
evident in the breast cancer cell lines, MDA (A5) and MCF7 (B-C 5);
Fig. 12 is a picture of the macroarray hybridization results showing the
differential expression data for STAR selected ovarian cancer-related human
SEQ. ID.
NO. 12. The STAR dsDNA clone representing SEQ. ID. NO. 12 was labeled with 32P
49
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and hybridized to the macroarray. The hybridization results obtained confirm
its
upregulation in the majority of malignant ovarian cancer samples (A-F 2 and A-
G 3-4)
compared to LMP samples (A-F 1). Significant expression of this sequence was
only
evident in one (testicle (F10)) of the 30 normal tissues and the prostate
cancer cell
line, LNCap (G-H 6). Weaker expression was also observed in normal ovary (E8);
Fig. 13 is a picture of the macroarray hybridization results showing the
differential expression data for STAR selected ovarian cancer-related human
SEQ. ID.
NO. 13. The STAR dsDNA clone representing SEQ. ID. NO. 13 was labeled with 32P
and hybridized to the macroarray. The hybridization results obtained confirm
its
upregulation in a majority of malignant ovarian cancer samples (A-F 2 and A-G
3-4)
compared to LMP samples (A-F 1). Significant expression of this sequence was
only
evident in the breast cancer cell lines, MDA (A5) and MCF7 (B-C 5). Weaker
expression was also observed in some normal tissues and the prostate cancer
cell
line, LNCap (G-H 6);
Fig. 14 is a picture of the macroarray hybridization results showing the
differential expression data for STAR selected ovarian cancer-related human
SEQ. ID.
NO. 14. The STAR dsDNA clone representing SEQ. ID. NO. 14 was labeled with 32P
and hybridized to the macroarray. The hybridization results obtained confirm
its
upregulation in a majority of malignant ovarian cancer samples (A-F 2 and A-G
3-4)
compared to LMP samples (A-F 1). Weaker expression of this sequence was only
observed in the normal kidney (F8) tissue;.
Fig. 15 is a picture of the macroarray hybridization results showing the
differential expression data for STAR selected ovarian cancer-related human
SEQ. ID.
NO. 15. The STAR dsDNA clone representing SEQ. ID. NO. 15 was labeled with 32P
and hybridized to the macroarray. The hybridization results obtained confirm
its
upregulation in the majority of malignant ovarian cancer samples (A-F 2 and A-
G 3-4)
compared to LMP samples (A-F 1). Weaker expression of this sequence similar to
that
of the LMPs was noted in many of the normal tissues as well;
Fig. 16 is a picture of the macroarray hybridization results showing the
differential expression data for STAR selected ovarian cancer-related human
SEQ. ID.
NO. 16. The STAR dsDNA clone representing SEQ. ID. NO. 16 was labeled with 32P
and hybridized to the macroarray. The hybridization results obtained confirm
its
upregulation in the majority of malignant ovarian cancer samples (A-F 2 and A-
G 3-4)
compared to LMP samples (A-F 1). Significant expression of this sequence was
also
evident in the breast cancer cell lines, MDA (A5) and MCF7 (B-C 5). Weaker
CA 02826738 2013-09-05
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expression similar to that of the LMPs was seen in prostate and some normal
tissue
samples;
Fig. 17 is a picture of the macroarray hybridization results showing the
differential expression data for STAR selected ovarian cancer-related human
SEQ. ID.
NO. 17. The STAR dsDNA clone representing SEQ. ID. NO. 17 was labeled with 32P
and hybridized to the macroarray. The hybridization results obtained confirm
its
upregulation in the majority of malignant ovarian cancer samples (A-F 2 and A-
G 3-4)
compared to LMP samples (A-F 1). Significant expression of this sequence was
only
evident in two (breast (B7) and bladder (H9)) of the 30 normal tissues;
Fig. 18 is a picture of the macroarray hybridization results showing the
differential expression data for STAR selected ovarian cancer-related human
SEQ. ID.
NO. 18, The STAR dsDNA clone representing SEQ. ID. NO. 18 was labeled with 32P
and hybridized to the macroarray. The hybridization results obtained confirm
its
upregulation in a majority of malignant ovarian cancer samples (A-F 2 and A-G
3-4)
Is compared to
LMP samples (A-F 1). Significant expression of this sequence was also
evident in the breast cancer cell lines, MDA (A5) and MCF7 (B-C 5), and
somewhat
lower expression in prostate cancer cell line, LNCap (G-H 6) and eight normal
tissues
(adrenal (A7), placenta (F7), lung (A8), adrenal cortex (B8), esophagus (C8),
colon
(D8), ovary (E8) and testicle (F10));
Fig. 19A is a picture of the macroarray hybridization results showing the
differential expression data for STAR selected ovarian cancer-related human
SEQ. ID.
NO. 19. The STAR dsDNA clone representing SEQ. ID. NO. 19 was labeled with 32P
and hybridized to the macroarray. The hybridization results obtained confirm
its
upregulation in several malignant ovarian cancer samples (A-F 2 and A-G 3-4)
compared to LMP samples (A-F 1). Significant expression of this sequence was
also
only evident in the breast cancer cell line, MCF7 (B-C 5);
Fig. 19B (panels A and B) is a picture of RT-PCR data showing the differential
expression data for STAR selected ovarian cancer-related human SEQ. ID. NO. 19
and KCNMB2 gene belonging to Unigene cluster, Hs.478368. Primer pairs specific
to
either the STAR clone sequence for SEQ. ID. NO. 19 or the KCNMB2 gene were
used
to perform RT-PCR on normal ovarian tissue, and benign and different
stages/grades
of ovarian cancer. As indicated by the expected PCR amplicon product (Fig.
19B,
panel A), compared to normal (Lane 1), benign (Lanes 2-3) and LMPs (Lanes 4-7)
samples, increased expression of SEQ. ID. NO. 19 mRNA was evident in clear
cell
carcinoma (Lanes 8-9), late stage endometrioid (Lane 12) and malignant serous
(Lanes 15-17). These results confirm the upregulation of the gene expression
for SEQ.
51
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ID. NO. 19 in malignant ovarian cancer. However, the expression of KCNMB2 was
markedly different from that of SEQ. ID. NO. 19 showing essentially no
difference in its
expression amongst the different ovarian samples (Fig 19B, panel B);
Fig. 20 is a picture of the macroarray hybridization results showing the
differential expression data for STAR selected ovarian cancer-related human
SEQ. ID.
NO. 20. The STAR dsDNA clone representing SEQ. ID. NO. 20 was labeled with 32P
and hybridized to the macroarray. The hybridization results obtained confirm
its
upregulation in several malignant ovarian cancer samples (A-F 2 and A-G 3-4)
compared to LMP samples (A-F 1). Significant expression of this sequence was
also
evident in the four (jejunum (C7), trachea (D7), colon (D8) and thymus (H8))
of the 30
normal tissues;
Fig. 21 is a picture of the macroarray hybridization results showing the
differential expression data for STAR selected ovarian cancer-related human
SEQ. ID.
NO. 21. The STAR dsDNA clone representing SEQ. ID. NO. 21 was labeled with 32P
and hybridized to the macroarray. The hybridization results obtained confirm
its
upregulation in a majority of malignant ovarian cancer samples (A-F 2 and A-G
3-4)
compared to LMP samples (A-F 1). Significant expression of this sequence was
also
evident in the three (adrenal (A7), breast (B7) and aorta (G7)) of the 30
normal tissues;
Fig. 22 is a picture of the macroarray hybridization results showing the
differential expression data for STAR selected ovarian cancer-related human
SEQ. ID.
NO. 22. The STAR dsDNA clone representing SEQ. ID. NO. 22 was labeled with 32P
and hybridized to the macroarray. The hybridization results obtained confirm
its
upregulation in a majority of malignant ovarian cancer samples (A-F 2 and A-G
3-4)
compared to LMP samples (A-F 1). Significant expression of this sequence was
also
evident in the breast cancer cell line, MCF7 (B-C 5). Weaker expression
similar to that
of the LMPs was seen in a majority of the normal tissues;
Fig. 23 is a picture of the macroarray hybridization results showing the
differential expression data for STAR selected ovarian cancer-related human
SEQ. ID.
NO. 23. The STAR dsDNA clone representing SEQ. ID. NO. 23 was labeled with 32P
and hybridized to the macroarray. The hybridization results obtained confirm
its
upregulation in several malignant ovarian cancer samples (A-F 2 and A-G 3-4)
compared to LMP samples (A-F 1). Significant expression of this sequence was
also
evident in the breast cancer cell lines, MDA (A5) and MCF7 (B-C 5) and
prostate
cancer cell line, LNCap (G-H 6);
Fig. 24 is a picture of the macroarray hybridization results showing the
differential expression data for STAR selected ovarian cancer-related human
SEQ. ID.
52
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NO. 24. The STAR dsDNA clone representing SEQ. ID. NO. 24 was labeled with 32P
and hybridized to the macroarray. The hybridization results obtained confirm
its
upregulation in several of the malignant ovarian cancer samples (A-F 2 and A-G
3-4)
compared to LMP samples (A-F 1). Significant expression of this sequence was
also
evident in the breast cancer cell line, MCF7 (B-C 5);
Fig. 25 is a picture of the macroarray hybridization results showing the
differential expression data for STAR selected ovarian cancer-related human
SEQ. ID.
NO. 25. The STAR dsDNA clone representing SEQ. ID. NO. 25 was labeled with 32P
and hybridized to the macroarray. The hybridization results obtained confirm
its
upregulation in a majority of malignant ovarian cancer samples (A-F 2 and A-G
3-4)
compared to LMP samples (A-F 1). Significant expression of this sequence was
also
evident in the prostate cancer cell line, LNCap (G-H 6);
Fig. 26 is a picture of the macroarray hybridization results showing the
differential expression data for STAR selected ovarian cancer-related human
SEQ. ID.
NO. 26. The STAR dsDNA clone representing SEQ. ID. NO. 26 was labeled with 32P
and hybridized to the macroarray. The hybridization results obtained confirm
its
upregulation in a majority of malignant ovarian cancer samples (A-F 2 and A-G
3-4)
compared to LMP samples (A-F 1). Significant expression of this sequence was
also
evident in the breast cancer cell lines, MDA (A5) and MCF7 (B-C 5), prostate
cancer
cell line, LNCap (G-H 6) and one normal tissue, testicle (F10). Weaker
expression
similar to that of the LMPs was seen in some normal tissues as well:
Fig. 27 is a picture of the macroarray hybridization results showing the
differential expression data for STAR selected ovarian cancer-related human
SEQ. ID.
NO. 27. The STAR dsDNA clone representing SEQ. ID. NO. 27 was labeled with 32P
and hybridized to the macroarray. The hybridization results obtained confirm
its
upregulation in a majority of malignant ovarian cancer samples (A-F 2 and A-G
3-4)
compared to LMP samples (A-F 1). Significant expression of this sequence was
also
evident in the breast cancer cell lines, MDA (A5) and MCF7 (B-C 5), prostate
cancer
cell line, LNCap (G-H 6). Weaker expression similar to that of the LMPs was
seen in
seven (adrenal (A7), placenta (F7), lung (A8), esophagus (C8), colon (D8),
ovary (E8)
and testicle (F10)) of the 30 normal tissues as well;
Fig. 28 is a picture of the macroarray hybridization results showing the
differential expression data for STAR selected ovarian cancer-related human
SEQ. ID.
NO. 28. The STAR dsDNA clone representing SEQ. ID. NO. 28 was labeled with 32P
and hybridized to the macroarray. The hybridization results obtained confirm
its
upregulation in a majority of malignant ovarian cancer samples (A-F 2 and A-G
3-4)
53
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compared to LMP samples (A-F 1). Significant expression of this sequence was
also
evident in the breast cancer cell lines, MDA (A5) and MCF7 (B-C 5). Weaker
expression similar to that of LMPs was seen for all other tissues;
Fig. 29 is a picture of the macroarray hybridization results showing the
differential expression data for STAR selected ovarian cancer-related human
SEQ. ID.
NO. 29. The STAR dsDNA clone representing SEQ. ID. NO. 29 was labeled with 32P
and hybridized to the macroarray. The hybridization results obtained confirm
its
upregulation in a majority of malignant ovarian cancer samples (A-F 2 and A-G
3-4)
compared to LMP samples (A-F 1). Significant expression of this sequence was
also
evident in the breast cancer cell line, MCF7 (B-C 5) and three (breast (B7),
esophagus
(C8) and fallopian tube (F9)) of the 30 normal tissues;
Fig. 30 is a picture of the macroarray hybridization results showing the
differential expression data for STAR selected ovarian cancer-related human
SEQ. ID.
NO. 30. The STAR dsDNA clone representing SEQ. ID. NO. 30 was labeled with 32P
and hybridized to the macroarray. The hybridization results obtained confirm
its
upregulation in a majority of malignant ovarian cancer samples (A-F 2 and A-G
3-4)
compared to LMP samples (A-F 1). Significant expression of this sequence was
also
evident in the breast cancer cell line, MCF7 (B-C 5), prostate cancer samples
(D-H 6).
Weaker expression similar to that of LMPs was seen in only very few normal
tissues;
Fig. 31 is a picture of the macroarray hybridization results showing the
differential expression data for STAR selected ovarian cancer-related human
SEQ. ID.
NO. 31. The STAR dsDNA clone representing SEQ. ID. NO. 31 was labeled with 32P
and hybridized to the macroarray. The hybridization results obtained confirm
its
upregulation in a majority of malignant ovarian cancer samples (A-F 2 and A-G
3-4)
compared to LMP samples (A-F 1). Significant expression of this sequence was
also
evident in the breast cancer cell line, MCF7 (B-C 5), prostate cancer samples
(D-H 6).
Weaker expression similar to that of LMPs was seen in only very few normal
tissues;
Fig. 32 is a picture of RT-PCR data showing the differential expression data
for STAR selected ovarian cancer-related human SEQ. ID. NO. 32. For this gene,
the
macroarray data was not available. A primer pair, OGS 1077
(GCGTCCGGGCCTGTCTTCAACCT; SEQ. ID. NO. 153) and OGS 1078
(GCCCCACCCTCTACCCCACCACTA; SEQ. ID. NO. 154) for SEQ. ID. NO. 32 was
used to perform RT-PCR on normal ovarian tissue, and benign and different
stages/grades of ovarian cancer. As indicated by the expected PCR amplicon
product,
compared to normal (Lane 1) and benign (Lanes 2-3), increased expression of
SEQ.
ID. NO. 32 mRNA was evident in LMPs (Lanes 4-7), clear cell carcinoma (Lanes 8-
9),
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late stage endometrioid (Lane 12) and malignant serous (Lanes 15-17). These
results
confirm the upregulation of the gene expression for SEQ. ID. NO. 32 in
malignant
ovarian cancer;
Fig. 33 is a picture of the macroarray hybridization results showing the
differential expression data for STAR selected ovarian cancer-related human
SEQ. ID.
NO. 33. The STAR dsDNA clone representing SEQ. ID. NO. 33 was labeled with 32P
and hybridized to the macroarray. The hybridization results obtained confirm
its
upregulation in a majority of malignant ovarian cancer samples (A-F 2 and A-G
3-4)
compared to LMP samples (A-F 1). Significant expression of this sequence was
also
evident in the prostate cancer samples (B-F 6). Weaker expression was seen in
many
normal tissues and strong expression was seen trachea (D7), colon (D8), small
intestine (D9), thymus (H8) and spleen (G9). These results confirm the
upregulation of
the gene expression for SEQ. ID. NO. 33 in malignant ovarian cancer;
Fig. 34 is a picture of the macroarray hybridization results showing the
differential expression data for STAR selected ovarian cancer-related human
SEQ. ID.
NO. 34. The STAR dsDNA clone representing SEQ. ID. NO. 34 was labeled with 32P
and hybridized to the macroarray. The hybridization results obtained confirm
its
upregulation in a majority of malignant ovarian cancer samples (A-F 2 and A-G
3-4)
compared to LMP samples (A-F 1). Significant expression of this sequence was
also
evident in the prostate cancer samples (B-F 6). Weaker expression was seen in
many
normal tissues and strong expression was seen trachea (D7), colon (D8), small
intestine (D9), thymus (H8) and spleen (G9). These results confirm the
upregulation of
the gene expression for SEQ. ID. NO. 34 in malignant ovarian cancer;
Fig. 35 is a picture of RT-PCR data showing the differential expression data
for STAR selected ovarian cancer-related human SEQ. ID. NO. 35. For this gene,
the
macroarray data was not available. A primer pair, OGS 1141
(GAGATCCTGATCAAGGTGCAGG; SEQ. ID. NO. 155) and OGS 1142
(TGCACGCTCACAGCAGTCAGG; SEQ. ID. NO. 156) for SEQ. ID. NO. 35 was used
to perform RT-PCR on LMP samples, different stages/grades of ovarian cancer
and
normal human tissue samples. As indicated by the expected PCR amplicon product
(indicated as AB-0201), increased expression of SEQ. ID. NO. 35 mRNA was
evident
in some ovarian cancer lanes (lanes 10, 11, 14, 18, 28 and 29) and the mRNA
was not
expressed in LMP samples. Expression was observed in only one normal tissue
sample, ileum (lane 27). Equal amounts of template cDNA used in each PCR
reaction
was confirmed by reamplifying GAPDH with a specific primer pair, OGS 315
(TGAAGGTCGGAGTCAACGGATTTGGT; SEQ. ID. NO. 167) and OGS 316
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(CATGTGGGCCATGAGGTCCACCAC; SEQ. ID. NO. 168) for this housekeeping
gene. These results confirm the upregulation of the gene expression for SEQ.
ID. NO.
35 in malignant ovarian cancer;
Fig. 36 is a picture of the macroarray hybridization results showing the
Fig. 37 is a picture of the macroarray hybridization results showing the
differential expression data for STAR selected ovarian cancer-related human
SEQ. ID.
NO. 37. The STAR dsDNA clone representing SEQ. ID. NO. 37 was labeled with 32P
Fig. 38 is a picture of RT-PCR data showing the differential expression data
for STAR selected ovarian cancer-related human SEQ. ID. NO. 38. For this gene,
the
macroarray data was not available. A primer pair, OGS 1202
(AACATGACTAAGATGCCCAACC; SEQ. ID NO. 157) and OGS 1203
GAPDH with a specific primer pair, OGS 315
(TGAAGGICGGAGICAACGGATTIGGT; SEQ. ID. NO. 167) and OGS 316
(CATGTGGGCCATGAGGTCCACCAC; SEQ. ID. NO. 168) for this housekeeping
56
CA 02826738 2013-09-05
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Fig. 39 is a picture of the macroarray hybridization results showing the
differential expression data for STAR selected ovarian cancer-related human
SEQ. ID.
NO. 39. The STAR dsDNA clone representing SEQ. ID. NO. 39 was labeled with 32P
and hybridized to the macroarray. The hybridization results obtained confirm
its
upregulation in a majority of malignant ovarian cancer samples (A-F 2 and A-G
3-4)
compared to LMP samples (A-F 1). Strong expression was also observed in breast
cancer samples (A-C 5) and weak expression in prostate cancer samples (A-H 6).
Weaker expression was seen in a few normal tissues with strong expression in
testes
(F 10). These results confirm the upregulation of the gene expression for SEQ.
ID. NO.
39 in malignant ovarian cancer;
Fig. 40 is a picture of the macroarray hybridization results showing the
differential expression data for STAR selected ovarian cancer-related human
SEQ. ID.
NO. 40. The STAR dsDNA clone representing SEQ. ID NO. 40 was labeled with 32P
and hybridized to the macroarray. The hybridization results obtained confirm
its
upregulation in a majority of malignant ovarian cancer samples (A-F 2 and A-G
3-4)
compared to LMP samples (A-F 1). Weak expression was seen in a few normal
tissues with strong expression in kidney (F 8). These results confirm the
upregulation
of the gene expression for SEQ. ID. NO. 40 in malignant ovarian cancer;
Fig. 41 is a picture of RT-PCR data showing the differential expression data
for STAR selected ovarian cancer-related human SEQ. ID. NO. 41. For this gene,
the
macroarray data was not available. A primer pair, OGS 1212
(AAGCATAGCCATAGGTGATTGG; SEQ. ID. NO. 159) and OGS 1213
(ACAGGTATCAGACAAGGGAGCAG; SEQ. ID. NO. 160) for SEQ. ID. NO. 41 was
used to perform RT-PCR on LMP samples, different stages/grades of ovarian
cancer
and normal human tissue samples. As indicated by the expected PCR amplicon
product (indicated as AB-0532), increased expression of SEQ. ID. NO. 41 mRNA
was
evident in a large majority of the ovarian cancer lanes and weaker expression
was
seen in LMP samples. Expression was observed in a few normal tissue samples
such
as kidney, thymus and spleen (lanes 14, 16 and 23, respectively). Equal
amounts of
template cDNA used in each PCR reaction was confirmed by reamplifying GAPDH
with a specific primer pair, OGS 315 (TGAAGGTCGGAGTCAACGGATTTGGT; SEQ.
ID. NO. 167) and OGS 316 (CATGTGGGCCATGAGGTCCACCAC; SEQ. ID. NO.
168) for this housekeeping gene. These results confirm the upregulation of the
gene
expression for SEQ. ID. NO. 41 in malignant ovarian cancer;
Fig. 42 is a picture of the macroarray hybridization results showing the
differential expression data for STAR selected ovarian cancer-related human
SEQ. ID.
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NO. 42. The STAR dsDNA clone representing SEQ. ID. NO. 42 was labeled with 32P
and hybridized to the macroarray. The hybridization results obtained showed
its
expression in both malignant ovarian cancer samples (A-F 2 and A-G 3-4) and
LMP
samples (A-F 1). Weak expression was also observed in breast cancer samples (A-
C
5). Weak expression was seen in a few normal tissues with moderate expression
in
placenta (F 7). These results confirm the expression for SEQ. ID. NO. 42 in
malignant
ovarian cancer;
Fig. 43 is a picture of the macroarray hybridization results showing the
differential expression data for STAR selected ovarian cancer-related human
SEQ. ID.
to NO. 43. The
STAR dsDNA clone representing SEQ. ID. NO. 43 was labeled with 32P
and hybridized to the macroarray. The hybridization results obtained confirm
its
upregulation in a majority of malignant ovarian cancer samples (A-F 2 and A-G
3-4)
compared to LMP samples (A-F 1). Strong expression was also observed in breast
cancer samples (A-C 5) and weak expression in prostate cancer samples (A-H 6).
5 Weaker
expression was seen in normal tissues with strong expression in testes (F 10).
These results confirm the upregulation of the gene expression for SEQ. ID. NO.
43 in
malignant ovarian cancer;
Fig. 44 is a picture of RT-PCR data showing the differential expression data
for STAR selected ovarian cancer-related human SEQ. ID. NO. 44. For this gene,
the
20 macroarray data was not available. A primer pair, OGS 1171
(TTACGACCTATTTCTCCGTGG; SEQ. ID. NO. 161) and OGS 1172
(AATGCAATAATTGGCCACTGC; SEQ. ID. NO. 162) for SEQ. ID. NO. 44 was used to
perform RT-PCR on LMP samples, different stages/grades of ovarian cancer and
normal human tissue samples. As indicated by the expected PCR amplicon product
25 (indicated as
AB-0795), increased expression of SEQ. ID. NO. 44 mRNA was evident
in a large majority of the ovarian cancer lanes and weaker expression was seen
in
LMP samples. Expression was observed in several normal tissue samples such as
aorta, skeletal muscle, small intestine and spleen (lanes 7, 17, 20 and 23,
respectively). Equal amounts of template cDNA used in each PCR reaction was
30 confirmed by reamplifying GAPDH with a specific primer pair. OGS 315
(TGAAGGTCGGAGTCAACGGATTTGGT, SEQ. ID. NO. 167) and OGS 316
(CATGTGGGCCATGAGGTCCACCAC; SEQ. ID. NO. 168) for this housekeeping
gene. These results confirm the upregulation of the gene expression for SEQ.
ID. NO.
44 in malignant ovarian cancer;
35 Fig. 45 is a
picture of RT-PCR data showing the differential expression data
for STAR selected ovarian cancer-related human SEQ. ID. NO. 45. For this gene,
the
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macroarray data was not available. A primer pair, OGS 1175
(ACACATCAAACTGCTTATCCAGG; SEQ. ID. NO. 163) and OGS 1176
(ACTGATGTGAAAATGCACATCC; SEQ. ID. NO. 164) for SEQ. ID. NO 45 was used
to perform RT-PCR on LMP samples, different stages/grades of ovarian cancer
and
normal human tissue samples. As indicated by the expected PCR amplicon product
(indicated as AB-0846), increased expression of SEQ. ID. NO. 45 mRNA was
evident
in half of the ovarian cancer lanes and weaker expression was seen in LMP
samples.
Expression was observed in only a few normal tissue samples such as kidney,
fallopian tube and testes (lanes 14, 22 and 30, respectively). Equal amounts
of
template cDNA used in each PCR reaction was confirmed by reamplifying GAPDH
with a specific primer pair, OGS 315 (TGAAGGTCGGAGTCAACGGATTTGGT; SEQ.
ID. NO. 167) and OGS 316 (CATGIGGGCCATGAGGICCACCAC; SEQ. ID. NO.
168) for this housekeeping gene. These results confirm the upregulation of the
gene
expression for SEQ. ID. NO. 45 in malignant ovarian cancer;
Fig. 46 is a picture of the macroarray hybridization results showing the
differential expression data for STAR selected ovarian cancer-related human
SEQ. ID.
NO. 46. The STAR dsDNA clone representing SEQ. ID. NO. 46 was labeled with 32P
and hybridized to the macroarray. The hybridization results obtained confirm
its
upregulation in a majority of malignant ovarian cancer samples (A-F 2 and A-G
3-4)
compared to LMP samples (A-F 1). Weak expression was also observed in prostate
cancer samples (A-H 6). Weaker expression was seen in a few normal tissues
with
moderate expression in breast (B 7) and ovary (E 8). These results confirm the
upregulation of the gene expression for SEQ. ID. NO. 46 in malignant ovarian
cancer;
Fig. 47 is a picture of the macroarray hybridization results showing the
differential expression data for STAR selected ovarian cancer-related human
SEQ. ID.
NO. 47. The STAR dsDNA clone representing SEQ. ID. NO. 47 was labeled with 32P
and hybridized to the macroarray. The hybridization results obtained confirm
its
upregulation in a majority of malignant ovarian cancer samples (A-F 2 and A-G
3-4)
compared to LMP samples (A-F 1). Significant expression of this sequence was
also
evident in the prostate cancer samples (B-F 6). Weaker expression was seen in
many
normal tissues and strong expression was seen trachea (D7), colon (D8), small
intestine (D9), thymus (H8) and spleen (G9). These results confirm the
upregulation of
the gene expression for SEQ. ID. NO. 47 in malignant ovarian cancer;
Fig. 48 is a picture of RT-PCR data showing the differential expression data
for STAR selected ovarian cancer-related human SEQ. ID. NO. 48. For this gene,
the
macroarray data was not available. A primer pair, OGS 1282
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(ATGGCTCATACAGCACTCAGG; SEQ. ID. NO. 165) and OGS 1283
(GAACTGTCACTCCGGAAAGCCT; SEQ. ID. NO. 166) for SEQ. ID. NO. 48 was used
to perform RT-PCR on LMP samples, different stages/grades of ovarian cancer
and
normal human tissue samples. As indicated by the expected PCR amplicon product
(indicated as AB-1120), increased expression of SEQ. ID. NO. 48 mRNA was
evident
in a majority of the ovarian cancer lanes and weaker expression was seen in
LMP
samples. Expression was eveident in virtually all normal tissues. Equal
amounts of
template cDNA used in each PCR reaction was confirmed by reamplifying GAPDH
with a specific primer pair, OGS 315 (TGAAGGTCGGAGTCAACGGATTTGGT; SEQ.
ID. NO. 167) and OGS 316 (CATGTGGGCCATGAGGTCCACCAC; SEQ. ID. NO.
168) for this housekeeping gene. These results confirm the upregulation of the
gene
expression for SEQ. ID. NO. 48 in malignant ovarian cancer;
Fig. 49 is a picture of the macroarray hybridization results showing the
differential expression data for STAR selected ovarian cancer-related human
SEQ. ID.
NO. 49. The STAR dsDNA clone representing SEQ. ID. NO. 49 was labeled with 32P
and hybridized to the macroarray. The hybridization results obtained confirm
its
upregulation in a majority of malignant ovarian cancer samples (A-F 2 and A-G
3-4)
compared to LMP samples (A-F 1). Strong expression was also observed in breast
cancer samples (A-C 5) and weak expression in prostate cancer samples (A-H 6).
Weaker expression was seen in normal tissues. These results confirm the
upregulation
of the gene expression for SEQ. ID. NO. 49 in malignant ovarian cancer;
Fig. 50 is a picture of the macroarray hybridization results showing the
differential expression data for STAR selected ovarian cancer-related human
SEQ. ID.
NO. 50. The STAR dsDNA clone representing SEQ. ID. NO. 50 was labeled with 32P
and hybridized to the macroarray. The hybridization results obtained confirm
its
upregulation in a majority of malignant ovarian cancer samples (A-F 2 and A-G
3-4)
compared to LMP samples (A-F 1). Significant expression of this sequence was
also
evident in the seven (adrenal (A7), breast (B7), trachea (07), placenta (F7),
lung (A8),
kidney (F8) and fallopian tube (F9)) of the 30 normal tissues;
Fig. 51 is a picture showing an example of STAR subtraction for the ovarian
cancer samples. The housekeeping genes, GAPDH (Panel A) and 11-actin (Panel B)
were nicely subtracted for both LMP minus Malignant (SL133 to SL137) and
Malignant
minus LMP (SL123 to SL127) whereas, a known differentially expressed
upregulated
gene, CCNE1 (Panel C) in malignant ovarian tumors was not subtracted in
Malignant
minus LMP STAR libraries but instead, enriched (Lanes SL123 to SL127 compared
to
Lanes 6 to 10);
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Fig. 52 is a picture showing the effect of shRNAs on the expression of
endogenous genes encoded by SEQ.ID Nos. 1 and 3 in transfected TOV-21G cells.
Two shRNAs per SEQ.ID. were transfected in TOV-21G ovarian cancer cell lines
and
monitored by RT-PCR using gene-specific primers. In each case, both shRNAs
attenuated the expression of the genes;
Fig. 53 is a picture showing the effect of SEQ.ID.-specific shRNAs on the
proliferation of TOV-21G cells. Decreased proliferation is indicative of a
gene that,
when attenuated, is required for normal growth of the cancer cells. The cells
were
stably transfected with two separate shRNA expression vectors and the
proliferation of
the cells was measured in an MTT assay. The positive control plasmid expresses
a
shRNA that has homology to no known gene in humans;
Fig. 54 is a picture showing SEQ.ID.-specific shRNAs on the survival of TOV-
21G cells. Less staining is indicative of a gene that, when attenuated, is
required for
survival of the cancer cells in this assay. The cells were transiently
transfected with
two separate shRNA expression vectors and the remaining colonies were stained
with
crystal violet and photographed. The positive control plasmid expresses a
shRNA that
has homology to no known gene in humans;
Fig. 55A and 55B are pictures of RT-PCR data showing the differential
expression data for STAR selected ovarian cancer-related human SEQ. ID. NO.
01,
09, 12, 15, 17, 19, 20 and 24. To further demonstrate that the STAR SEQ. ID.
NOs.
selected after macroarray analysis were upregulated in malignant ovarian
cancer
samples compared to LMPs and normal ovarian samples, semi-quantitative RT-PCR
was performed for 25 cycles using HotStarTaq polymerase according to the
supplier
instructions (Qiagen). Furthermore, these results serve to demonstrate the
utility of
these sequences as potential diagnostic, prognostic or theranostic markers for
ovarian
cancer. For SEQ. ID. NOs. 01, 09, 12, 15, 17, 19, 20 and 24, a specific primer
pair for
each was used. The differential expression results obtained for each SEQ. ID.
NO.
tested are shown in Figure 55A and 55B. As indicated by the expected PCR
amplicon
product for each SEQ. ID. NO., there is a clear tendency towards increased
expression of the mRNAs corresponding to SEQ. ID. NOs. 01, 09, 12, 15, 17, 19,
20
and 24 in clear cell carcinoma (Lanes 8-9), late stage endometrioid (Lane 12)
and
different stages of malignant serous (Lanes 15-17) compared to normal (Lane
1),
benign (Lanes 2-3) and LMPs (Lanes 4-7) ovarian samples. These results confirm
the
upregulation of the gene expression for SEQ. ID. NOs. 01, 09, 12, 15, 17, 19,
20 and
24 in the different stages of malignant ovarian cancer as was observed using
the
macroarrays;
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Fig. 56 is a picture of the macroarray hybridization results showing the
differential expression data for STAR selected ovarian cancer-related human
SEQ. ID.
NO. 169. The STAR dsDNA clone representing SEQ. ID. NO. 169 was labeled with
32P
and hybridized to the macroarray. The hybridization results obtained confirm
its
upregulation in malignant ovarian cancer samples (A-F 2 and A-G 3-4) compared
to
LMP samples (A-F 1). Weaker expression was seen in some normal tissues and
strong expression was seen liver (E7) and aorta (G7). These results confirm
the
upregulation of the gene expression for SEQ. ID. NO. 169 in malignant ovarian
cancer;
Fig. 57 is a picture of RT-PCR data showing the differential expression data
for the STAR selected ovarian cancer-related human SEQ. ID. NO. 1 in RNA
samples
derived from the NCI-60 panel of cancer cell lines. A primer pair, OGS 1136
(GCTTAAAAGAGTCCTCCTGTGGC; SEQ. ID. NO. 171) and OGS 1044
(TGGACATTGTTCTTAAAGTGTGG; SEQ. ID. NO. 172) for SEQ. ID. NO. 1 was used
to perform RT-PCR. As indicated by the expected PCR amplicon, increased
expression of SEQ. ID. NO. 1 mRNA was evident in ovarian, renal, lung, colon,
breast
cancers and weaker expression was seen in melanoma samples;
Fig. 58 is a picture of RT-PCR data showing the differential expression data
for the STAR selected ovarian cancer-related human SEQ. ID. NO. 2 in RNA
samples
derived from the NCI-60 panel of cancer cell lines. A primer pair, OGS 1250
(AGGTTTTATGGCCACCGTCAG; SEQ. ID. NO. 173) and OGS 1251
(ATCCTATACCGCTCGGTTATGC; SEQ. ID. NO. 174) for SEQ. ID. NO. 2 was used
to perform RT-PCR. As indicated by the expected PCR amplicon, increased
expression of SEQ. ID. NO. 2 mRNA was evident in all nine cancer types but
weaker
expression was seen in melanoma and leukemia samples;
Fig. 59 is a picture of RT-PCR data showing the differential expression data
for the STAR selected ovarian cancer-related human SEQ. ID. NO. 3 in RNA
samples
derived from the NCI-60 panel of cancer cell lines. A primer pair, OGS 1049
(GGGCGGCGGCTCTTTCCTCCTC; SEQ. ID. NO. 175) and OGS 1050
(GCTAGCGGCCCCATACTCG; SEQ. ID. NO. 176) for SEQ. ID. NO. 3 was used to
perform RT-PCR. As indicated by the expected PCR amplicon, increased
expression
of SEQ. ID. NO. 3 mRNA was evident in eight cancer types and absent in the
leukemia
samples;
Fig. 60 is a picture of RT-PCR data showing the differential expression data
for the STAR selected ovarian cancer-related human SEQ. ID. NO. 4 in RNA
samples
derived from the NCI-60 panel of cancer cell lines. A primer pair, OGS 1051
(ACACTGGATGCCCTGAATGACACA; SEQ. ID. NO. 177) and OGS 1052
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(GCTTTGGCCC _______________________________________________________ GCTAA;
SEQ. ID. NO. 178) for SEQ. ID. NO. 4 was used to
perform RT-PCR. As indicated by the expected PCR amplicon, increased
expression
of SEQ. ID. NO. 4 mRNA was evident in melanoma, ovarian, CNS, and lung cancers
and weakly expressd in the leukemia samples;
Fig. 61 is a picture of RT-PCR data showing the differential expression data
for the STAR selected ovarian cancer-related human SEQ. ID. NO. 5 in RNA
samples
derived from the NCI-60 panel of cancer cell lines. A primer pair, OGS 1252
(CCCACTTCTGTCTTACTGCATC; SEQ. ID. NO. 179) and OGS 1253
(CATAGTACTCCAGGGCTTATTC; SEQ. ID. NO. 180) for SEQ. ID. NO. 4 was used to
perform RT-PCR. As indicated by the expected PCR amplicon, increased
expression
of SEQ. ID. NO. 5 mRNA was evident all cancer types;
Fig. 62 is a picture of RT-PCR data showing the differential expression data
for the STAR selected ovarian cancer-related human SEQ. ID. NO. 6 in RNA
samples
derived from the NCI-60 panel of cancer cell lines. A primer pair, OGS 1083
(AACGATTGCCCGGATTGATGACA; SEQ. ID. NO. 181) and OGS 1084
(TACTTGAGGCTGGGGTGGGAGATG; SEQ. ID. NO. 182) for SEQ. ID. NO. 6 was used
to perform RT-PCR. As indicated by the expected PCR amplicon, increased
expression of SEQ. ID. NO. 6 mRNA was evident all cancer types;
Fig. 63 is a picture of RT-PCR data showing the differential expression data
zo for the STAR
selected ovarian cancer-related human SEQ. ID. NO. 7 in RNA samples
derived from the NCI-60 panel of cancer cell lines. A primer pair, OGS 1053
(CACTACGCCAGGCACCCCCAAAAC; SEQ. ID. NO. 183) and OGS 1054
(CGAGGCGCACGGCAGTCT; SEQ. ID. NO. 184) for SEQ. ID. NO. 7 was used to
perform RT-PCR. As indicated by the expected PCR amplicon, increased
expression
of SEQ. ID. NO. 7 mRNA was evident only in ovarian cancer samples;
Fig. 64 is a picture of RT-PCR data showing the differential expression data
for the STAR selected ovarian cancer-related human SEQ. ID. NO. 8 in RNA
samples
derived from the NC1-60 panel of cancer cell lines. A primer pair, OGS 1037
(ATCCGTTGCTGCAGCTCGTTCCTC; SEQ. ID. NO. 185) and OGS 1038
(ACCCTGCTGACCTTCTTCCATTCC; SEQ. ID. NO. 186) for SEQ. ID. NO. 8 was
used to perform RT-PCR. As indicated by the expected PCR amplicon, increased
expression of SEQ. ID. NO. 8 mRNA was evident in all cancer types;
Fig. 65 is a picture of RT-PCR data showing the differential expression data
for the STAR selected ovarian cancer-related human SEQ. ID. NO. 9 in RNA
samples
derived from the NCI-60 panel of cancer cell lines. A primer pair, OGS 1045
(TCGGAGGAGGGCTGGCTGGTGTTT; SEQ. ID. NO. 187) and OGS 1046
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(CTTGGGCGTCTTGGAGCGGTTCTG; SEQ. ID. NO. 188) for SEQ. ID. NO. 9 was
used to perform RT-PCR. As indicated by the expected PCR amplicon, (lower band
on
the gel; the top band is an artifact of the PCR reaction) increased expression
of SEQ.
ID. NO. 9 mRNA was evident in ovarian, lung, colon, breast cancer, and
melanoma
and weakly expressed in leukemia and CNS cancer;
Fig. 66 is a picture of RT-PCR data showing the differential expression data
for the STAR selected ovarian cancer-related human SEQ. ID. NO. 10 in RNA
samples derived from the NCI-60 panel of cancer cell lines. A primer pair, OGS
1240
(AGAGCCTATTGAAGATGAACAG; SEQ. ID. NO. 189) and OGS 1241
(TGATTGCCCCGGATCCTCTTAGG; SEQ. ID. NO. 190) for SEQ. ID. NO. 10 was
used to perform RT-PCR. As indicated by the expected PCR amplicon, increased
expression of SEQ. ID. NO. 10 mRNA was evident in all cancer types;
Fig. 67 is a picture of RT-PCR data showing the differential expression data
for the STAR selected ovarian cancer-related human SEQ. ID. NO. 11 in RNA
samples derived from the NCI-60 panel of cancer cell lines. A primer pair, OGS
1304
(GGACAAATACGACGACGAGG; SEQ. ID. NO. 191) and OGS 1305
(GGTTTCTTGGGTAGTGGGC; SEQ. ID. NO. 192) for SEQ. ID. NO. 11 was used to
perform RT-PCR. As indicated by the expected PCR amplicon, increased
expression
of SEQ. ID. NO. 11 mRNA was evident in all cancer types;
Fig. 68 is a picture of RT-PCR data showing the differential expression data
for the STAR selected ovarian cancer-related human SEQ. ID. NO. 12 in RNA
samples derived from the NCI-60 panel of cancer cell lines. A primer pair, OGS
1039
(CCCCGGAGAAGGAAGAGCAGTA; SEQ. ID. NO. 193) and OGS 1040
(CGAAAGCCGGCAGTTAGTTATTGA; SEQ. ID. NO. 194) for SEQ. ID. NO. 12 was
used to perform RT-PCR. As indicated by the expected PCR amplicon, increased
expression of SEQ. ID. NO. 12 mRNA was evident in all cancer types but weakly
in
CNS cancer and leukemia;
Fig. 69 is a picture of RT-PCR data showing the differential expression data
for the STAR selected ovarian cancer-related human SEQ. ID. NO. 13 in RNA
samples derived from the NCI-60 panel of cancer cell lines. A primer pair, OGS
1095
(GGCGGGCAACGAATTCCAGGTGTC; SEQ. ID. NO. 195) and OGS 1096
(TCAGAGGTTCGTCGCATTTGTCCA; SEQ. ID. NO. 196) for SEQ. ID. NO. 13 was
used to perform RT-PCR. As indicated by the expected PCR amplicon, increased
expression of SEQ. ID. NO. 13 mRNA was evident in all cancer types;
Fig. 70 is a picture of RT-PCR data showing the differential expression data
for the STAR selected ovarian cancer-related human SEQ. ID. NO. 15 in RNA
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samples derived from the NCI-60 panel of cancer cell lines. A primer pair, OGS
1284
(CAACAGTCATGATGTGTGGATG; SEQ. ID. NO. 197) and OGS 1285
(ACTGCACCTTGTCCGTGTTGAC; SEQ. ID. NO. 198) for SEQ. ID. NO. 15 was used
to perform RT-PCR. As indicated by the expected PCR amplicon, increased
expression of SEQ. ID. NO. 15 mRNA was evident in ovarian, prostate, lung,
colon,
and breast cancer;
Fig. 71 is a picture of RT-PCR data showing the differential expression data
for the STAR selected ovarian cancer-related human SEQ. ID. NO. 16 in RNA
samples derived from the NCI-60 panel of cancer cell lines. A primer pair, OGS
1063
(CCGGCTGGCTGCTTTGTTTA; SEQ. ID. NO. 199) and OGS 1064
(ATGATCAGCAGGTTCGTTGGTAGG; SEQ. ID. NO. 200) for SEQ. ID. NO. 16 was
used to perform RT-PCR. As indicated by the expected PCR amplicon, increased
expression of SEQ. ID. NO. 16 mRNA was evident in ovarian, lung, colon, and
breast
cancer;
Fig. 72 is a picture of RT-PCR data showing the differential expression data
for the STAR selected ovarian cancer-related human SEQ. ID. NO. 17 in RNA
samples derived from the NCI-60 panel of cancer cell lines. A primer pair, OGS
1031
(ATGCCGGAAGTGAATGTGG; SEQ. ID. NO. 201) and OGS 1032
(GGTGACTCCGCC 1111 GAT; SEQ. ID. NO. 202) for SEQ. ID. NO. 17 was used to
perform RT-PCR. As indicated by the expected PCR amplicon, increased
expression
of SEQ. ID. NO. 17 mRNA was evident in ovarian, renal, lung, colon, and breast
cancer but weakly in CNS cancer;
Fig. 73 is a picture of RT-PCR data showing the differential expression data
for the STAR selected ovarian cancer-related human SEQ. ID. NO. 18 in RNA
samples derived from the NCI-60 panel of cancer cell lines. A primer pair, OGS
1308
(ACATTCGCTTCTCCATCTGG; SEQ. ID. NO. 203) and OGS 1309
(TGTCACGGAAGGGAACCAGG; SEQ. ID. NO. 204) for SEQ. ID. NO. 18 was used to
perform RT-PCR. As indicated by the expected PCR amplicon, increased
expression
of SEQ. ID. NO. 18 mRNA was evident in all cancer types;
Fig. 74 is a picture of RT-PCR data showing the differential expression data
for the STAR selected ovarian cancer-related human SEQ. ID. NO. 19 in RNA
samples derived from the NCI-60 panel of cancer cell lines. A primer pair, OGS
1069
(ACGCTGCCTCTGGGTCACTT; SEQ. ID. NO. 205) and OGS 1070
(TTGGCAAATCAATGGCTTGTAAT; SEQ. ID. NO. 206) for SEQ. ID. NO. 19 was used
to perform RT-PCR. As indicated by the expected PCR amplicon, increased
expression of SEQ. ID. NO. 19 mRNA was evident in all cancer types;
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Fig. 75 is a picture of RT-PCR data showing the differential expression data
for the STAR selected ovarian cancer-related human SEQ. ID. NO. 20 in RNA
samples derived from the NCI-60 panel of cancer cell lines. A primer pair, OGS
1061
(ATGGCTTGGGTCATCAGGAC; SEQ. ID. NO. 207) and OGS 1062
(GTGTCACTGGGCGTAAGATACTG; SEQ. ID. NO. 208) for SEQ. ID. NO. 20 was
used to perform RT-PCR. As indicated by the expected PCR amplicon, increased
expression of SEQ. ID. NO. 20 mRNA was evident in all cancer types but weakly
in
breast and colon cancer;
Fig. 76 is a picture of RT-PCR data showing the differential expression data
I() for the STAR
selected ovarian cancer-related human SEQ. ID. NO. 21 in RNA
samples derived from the NCI-60 panel of cancer cell lines. A primer pair, OGS
1097
(CACCAAATCAGCTGCTACTACTCC; SEQ. ID. NO. 209) and OGS 1098
(GATAAACCCCAAAGCAGAAAGATT; SEQ. ID. NO. 210) for SEQ. ID. NO. 21 was
used to perform RT-PCR. As indicated by the expected PCR amplicon, increased
expression of SEQ. ID. NO. 21 mRNA was evident in all cancer types but weakly
in
leukemia;
Fig. 77 is a picture of RT-PCR data showing the differential expression data
for the STAR selected ovarian cancer-related human SEQ. ID. NO. 22 in RNA
samples derived from the NCI-60 panel of cancer cell lines. A primer pair, OGS
1075
(CGAGATTCCGTGGGCGTAGG; SEQ. ID. NO. 211) and OGS 1076
(TGAGTGGGAGCTTCGTAGG; SEQ. ID. NO. 212) for SEQ. ID. NO. 22 was used to
perform RT-PCR. As indicated by the expected PCR amplicon, increased
expression
of SEQ. ID. NO. 22 mRNA was evident in ovarian, lung, breast, and CNS cancer.
Another larger transcript was weakly expressed in colon and renal cancer ion
addition
to melanoma;
Fig. 78 is a picture of RT-PCR data showing the differential expression data
for the STAR selected ovarian cancer-related human SEQ. ID. NO. 23 in RNA
samples derived from the NCI-60 panel of cancer cell lines. A primer pair, OGS
1232
(TCAGAGTGGACGTTGGATTAC; SEQ. ID. NO. 213) and OGS 1233
(TGCTTGAAATGTAGGAGAACA; SEQ. ID. NO. 214) for SEQ. ID. NO. 23 was used to
perform RT-PCR. As indicated by the expected PCR amplicon, increased
expression
of SEQ. ID. NO. 23 mRNA was evident in all cancer types;
Fig. 79 is a picture of RT-PCR data showing the differential expression data
for the STAR selected ovarian cancer-related human SEQ ID. NO. 24 in RNA
samples derived from the NCI-60 panel of cancer cell lines. A primer pair, OGS
1067
(GAGGGGCATCAATCACACCGAGAA; SEQ. ID. NO. 215) and OGS 1068
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(CCCCACCGCCCACCCATTTAGG; SEQ. ID. NO. 216) for SEQ. ID. NO. 24 was used
to perform RT-PCR. As indicated by the expected PCR amplicon, increased
expression of SEQ. ID. NO. 24 mRNA was evident in ovarian, renal, lung, colon,
breast cancer, and melanoma but weakly in CNS cancer and leukemia;
Fig. 80 is a picture of RT-PCR data showing the differential expression data
for the STAR selected ovarian cancer-related human SEQ. ID. NO. 25 in RNA
samples derived from the NCI-60 panel of cancer cell lines. A primer pair, OGS
1099
(GGGGGCACCAGAGGCAGTAA; SEQ. ID. NO. 217) and OGS 1100
(GGTTGTGGCGGGGGCAGTTGTG; SEQ. ID. NO. 218) for SEQ. ID. NO. 25 was
i() used to
perform RT-PCR. As indicated by the expected PCR amplicon, increased
expression of SEQ. ID. NO. 25 mRNA was evident in all cancer types but weakly
in
leukemia;
Fig. 81 is a picture of RT-PCR data showing the differential expression data
for the STAR selected ovarian cancer-related human SEQ. ID. NO. 26 in RNA
samples derived from the NCI-60 panel of cancer cell lines. A primer pair, OGS
1246
(ACAGACTCCTGTACTGCAAACC; SEQ. ID. NO. 219) and OGS 1247
(TACCGGITCGTCCTCTTCCTC; SEQ. ID. NO. 220) for SEQ. ID. NO. 26 was used to
perform RT-PCR. As indicated by the expected PCR amplicon, increased
expression
of SEQ. ID. NO. 26 mRNA was evident in all cancer types;
Fig. 82 is a picture of RT-PCR data showing the differential expression data
for the STAR selected ovarian cancer-related human SEQ. ID. NO. 27 in RNA
samples derived from the NCI-60 panel of cancer cell lines. A primer pair, OGS
1093
(GAAGTTCCTCACGCCCTGCTATC: SEQ. ID. NO. 221) and OGS 1094
(CTGGCTGGTGACCTGCTTTGAGTA; SEQ. ID. NO. 222) for SEQ. ID. NO. 27 was
used to perform RT-PCR. As indicated by the expected PCR amplicon, increased
expression of SEQ. ID. NO. 27 mRNA was evident in all cancer types;
Fig. 83 is a picture of RT-PCR data showing the differential expression data
for the STAR selected ovarian cancer-related human SEQ. ID. NO. 28 in RNA
samples derived from the NCI-60 panel of cancer cell lines. A primer pair, OGS
1332
(TAGGCGCGCCTGACATACAGCAATGCCAGTT; SEQ. ID. NO. 223) and OGS 1333
(TAAGAATGCGGCCGCGCCACATCTTGAACACTTTGC ; SEQ. ID. NO. 224) for
SEQ. ID. NO. 28 was used to perform RT-PCR. As indicated by the expected PCR
amplicon, increased expression of SEQ. ID. NO. 28 mRNA was evident in ovarian,
prostate, and renal cancer but weakly in all other types;
Fig. 84 is a picture of RT-PCR data showing the differential expression data
for the STAR selected ovarian cancer-related human SEQ. ID. NO. 29 in RNA
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samples derived from the NCI-60 panel of cancer cell lines. A primer pair, OGS
1101
(TGGGGAGGAGTTTGAGGAGCAGAC; SEQ. ID. NO. 225) and OGS 1102
(GTGGGACGGAGGGGGCAGTGAAG; SEQ. ID. NO. 226) for SEQ. ID. NO. 29 was
used to perform RT-PCR. As indicated by the expected PCR amplicon, increased
expression of SEQ. ID. NO. 29 mRNA was evident in ovarian, renal, lung, colon,
and
breast cancer but weakly in CNS cancer and melanoma;
Fig. 85 is a picture of RT-PCR data showing the differential expression data
for the STAR selected ovarian cancer-related human SEQ. ID. NO. 30 in RNA
samples derived from the NCI-60 panel of cancer cell lines. A primer pair, OGS
1300
io (GCAACTATTCGGAGCGCGTG; SEQ. ID. NO. 227) and OGS 1301
(CCAGCAGCTTGTTGAGCTCC; SEQ. ID. NO. 228) for SEQ. ID. NO 30 was used to
perform RT-PCR. As indicated by the expected PCR amplicon, increased
expression
of SEQ. ID. NO. 30 mRNA was evident in all cancer types;
Fig. 86 is a picture of RT-PCR data showing the differential expression data
for the STAR selected ovarian cancer-related human SEQ. ID. NO. 31 in RNA
samples derived from the NCI-60 panel of cancer cell lines. A primer pair, OGS
1302
(GGAGGAGCTAAGCGTCATCGC; SEQ. ID. NO. 229) and OGS 1303
(TCGCTTCAGCGCGTAGACC; SEQ. ID. NO. 230) for SEQ. ID. NO. 31 was used to
perform RT-PCR. As indicated by the expected PCR amplicon, increased
expression
of SEQ. ID. NO. 31 mRNA was evident in all cancer types;
Fig. 87 is a picture of RT-PCR data showing the differential expression data
for the STAR selected ovarian cancer-related human SEQ. ID. NO. 32 in RNA
samples derived from the NCI-60 panel of cancer cell lines. A primer pair, OGS
1077
(GCGTCCGGGCCTGTCTTCAACCT; SEQ. ID. NO. 153) and OGS 1078
(GCCCCACCCTCTACCCCACCACTA; SEQ. ID. NO. 154) for SEQ. ID. NO. 32 was
used to perform RT-PCR. As indicated by the expected PCR amplicon, increased
expression of SEQ. ID. NO. 32 mRNA was evident in ovarian cancer and melanoma
but weaker expression was detectable in CNS, breast, colon, lung, and renal
cancer;
Fig. 88 is a picture of RT-PCR data showing the differential expression data
for the STAR selected ovarian cancer-related human SEQ. ID. NO. 33 in RNA
samples derived from the NCI-60 panel of cancer cell lines. A primer pair, OGS
1292
(TATTAGTTGGGATGGTGGTAGCAC; SEQ. ID. NO. 231) and OGS 1294
(GAGAATTCGAGTCGACGATGAC; SEQ. ID. NO. 232) for SEQ. ID. NO. 33 was used
to perform RT-PCR. As indicated by the expected PCR amplicon, increased
expression of SEQ. ID. NO. 33 mRNA was evident only in ovarian cancer samples;
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Fig. 89 is a picture of RT-PCR data showing the differential expression data
for the STAR selected ovarian cancer-related human SEQ. ID. NO. 34 in RNA
samples derived from the NCI-60 panel of cancer cell lines. A primer pair, OGS
1242
(GAAATMTGTTGACGCAGICTCC; SEQ. ID. NO. 233) and OGS 1243
(AGGCACACAACAGAGGCAGTTC; SEQ. ID. NO. 234) for SEQ. ID. NO. 34 was used
to perform RT-PCR. As indicated by the expected PCR amplicon, increased
expression of SEQ. ID. NO. 34 mRNA was evident only in ovarian cancer samples;
Fig. 90 is a picture of RT-PCR data showing the differential expression data
for the STAR selected ovarian cancer-related human SEQ. ID. NO. 35 in RNA
io samples
derived from the NCI-60 panel of cancer cell lines. A primer pair, OGS 1141
(GAGATCCTGATCAAGGTGCAGG; SEQ. ID. Na 155) and OGS 1142
(TGCACGCTCACAGCAGTCAGG; SEQ. ID. NO. 156) for SEQ. ID. NO. 35 was used
to perform RT-PCR. As indicated by the expected PCR amplicon, increased
expression of SEQ. ID. NO. 35 mRNA was evident in ovarian. lung and breast
cancer,
but weakly in colon and CNS cancer;
Fig. 91 is a picture of RT-PCR data showing the differential expression data
for the STAR selected ovarian cancer-related human SEQ. ID. NO. 36 in RNA
samples derived from the NCI-60 panel of cancer cell lines. A primer pair, OGS
1280
(GTACATCAACCTCCTGCTGTCC; SEQ. ID. NO. 235) and OGS '1281
(GACATCTCCAAGTCCCAGCATG; SEQ. ID. NO. 236) for SEQ. ID. NO. 36 was used
to perform RT-PCR. As indicated by the expected PCR amplicon, increased
expression of SEQ. ID. NO. 36 mRNA was evident in all cancer types;
Fig. 92 is a picture of RT-PCR data showing the differential expression data
for the STAR selected ovarian cancer-related human SEQ. ID. NO. 37 in RNA
samples derived from the NCI-60 panel of cancer cell lines. A primer pair, OGS
1159
(AGTCTCTCACTGTGCCTTATGCC; SEQ. ID. NO. 237) and OGS 1160
(AGTCCTAAGAACTGTAAACG; SEQ. ID. NO. 238) for SEQ. ID. NO. 37 was used to
perform RT-PCR. As indicated by the expected PCR amplicon, increased
expression
of SEQ. ID. NO. 37 mRNA was evident only in ovarian and renal cancer;
Fig. 93 is a picture of RT-PCR data showing the differential expression data
for the STAR selected ovarian cancer-related human SEQ. ID. NO. 38 in RNA
samples derived from the NCI-60 panel of cancer cell lines. A primer pair, OGS
1202
(AACATGACTAAGATGCCCAACC; SEQ. ID. NO. 157) and OGS 1203
(AATCTCCTTCACCTCCACTACTG; SEQ. ID. NO. 158) for SEQ. ID. NO. 38 was used
to perform RT-PCR. As indicated by the expected PCR amplicon, increased
expression of SEQ. ID. NO. 38 mRNA was evident in all cancer types;
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Fig. 94 is a picture of RT-PCR data showing the differential expression data
for the STAR selected ovarian cancer-related human SEQ. ID. NO. 39 in RNA
samples derived from the NCI-60 panel of cancer cell lines. A primer pair, OGS
1310
(CATCTATACGTGGATTGAGGA; SEQ. ID. NO. 239) and OGS 1311
(ATAGGTACCAGGTATGAGCTG; SEQ. ID. NO. 240) for SEQ. ID. NO. 39 was used
to perform RT-PCR. As indicated by the expected PCR amplicon, increased
expression of SEQ. ID. NO. 39 mRNA was evident in all cancer types;
Fig. 95 is a picture of RT-PCR data showing the differential expression data
for the STAR selected ovarian cancer-related human SEQ. ID. NO. 40 in RNA
samples derived from the NCI-60 panel of cancer cell lines. A primer pair, OGS
1155
(TGTCCACATCATCATCGTCATCC; SEQ. ID. NO. 241) and OGS 1156
(TGTCACTGGTCGGTCGCTGAGG; SEQ. ID. NO. 242) for SEQ. ID. NO. 39 was used
to perform RT-PCR. As indicated by the expected PCR amplicon, increased
expression of SEQ. ID. NO. 39 mRNA was evident in all cancer types;
Fig. 96 is a picture of RT-PCR data showing the differential expression data
for the STAR selected ovarian cancer-related human SEQ. ID. NO. 41 in RNA
samples derived from the NCI-60 panel of cancer cell lines. A primer pair, OGS
1212
(AAGCATAGCCATAGGTGATTGG; SEQ. ID. NO. 159) and OGS 1213
(ACAGGTATCAGACAAGGGAGCAG; SEQ. ID. NO. 160) for SEQ. ID. NO. 41 was
used to perform RT-PCR. As indicated by the expected PCR amplicon, increased
expression of SEQ. ID. NO. 41 mRNA was evident only in ovarian and renal
cancer
and leukemia;
Fig. 97 is a picture of RT-PCR data showing the differential expression data
for the STAR selected ovarian cancer-related human SEQ. ID. NO. 42 in RNA
samples derived from the NCI-60 panel of cancer cell lines. A primer pair, OGS
1316
(CATGGGGCTTAAGATGTC; SEQ. ID. NO. 243) and OGS 1317
(GTCGATTTCTCCATCATCTG; SEQ. ID. NO. 244) for SEQ. ID. NO. 42 was used to
perform RT-PCR. As indicated by the expected PCR amplicon, increased
expression
of SEQ. ID. NO. 42 mRNA was evident in all cancer types;
Fig. 98 is a picture of RT-PCR data showing the differential expression data
for the STAR selected ovarian cancer-related human SEQ. ID. NO. 43 in RNA
samples derived from the NCI-60 panel of cancer cell lines. A primer pair, OGS
1306
(AAGAGGCGCTCTACTAGCCG; SEQ. ID. NO. 245) and OGS 1307
(CTTTCCACATGGAACACAGG; SEQ. ID. NO. 246) for SEQ. ID. NO. 43 was used to
perform RT-PCR. As indicated by the expected PCR amplicon, increased
expression
of SEQ. ID. NO. 43 mRNA was evident in all cancer types;
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Fig. 99 is a picture of RT-PCR data showing the differential expression data
for the STAR selected ovarian cancer-related human SEQ. ID. NO. 44 in RNA
samples derived from the NCI-60 panel of cancer cell lines. A primer pair, OGS
1171
(TTACGACCTATTTCTCCGTGG; SEQ. ID. NO. 161) and OGS 1172
(AATGCAATAATTGGCCACTGC; SEQ. ID. NO. 162) for SEQ. ID. NO. 44 was used to
perform RT-PCR. As indicated by the expected PCR amplicon, increased
expression
of SEQ. ID. NO. 44 mRNA was evident in all cancer types;
Fig. 100 is a picture of RT-PCR data showing the differential expression data
for the STAR selected ovarian cancer-related human SEQ. ID. NO. 45 in RNA
samples derived from the NCI-60 panel of cancer cell lines. A primer pair, OGS
1175
(ACACATCAAACTGCTTATCCAGG; SEQ. ID. NO. 163) and OGS 1176
(ACTGATGTGAAAATGCACATCC; SEQ. ID. NO. 164) for SEQ. ID. NO. 45 was used
to perform RT-PCR. As indicated by the expected PCR amplicon, increased
expression of SEQ. ID. NO. 45 mRNA was evident only in ovarian cancer samples;
Fig. 101 is a picture of RT-PCR data showing the differential expression data
for the STAR selected ovarian cancer-related human SEQ. ID. NO. 46 in RNA
samples derived from the NCI-60 panel of cancer cell lines. A primer pair, OGS
1286
(CAI ______________________________________________________________ I
FICCIGGAATTTGATACAG; SEQ. ID. NO. 247) and OGS 1287
(GTAGAGAGTTTATTTGGGCCAAG; SEQ. ID. NO. 248) for SEQ. ID. NO. 46 was
used to perform RT-PCR. As indicated by the expected PCR amplicon, increased
expression of SEQ. ID. NO. 46 mRNA was evident in all cancer types;
Fig. 102 is a picture of RT-PCR data showing the differential expression data
for the STAR selected ovarian cancer-related human SEQ. ID. NO. 47 in RNA
samples derived from the NCI-60 panel of cancer cell lines. A primer pair, OGS
1244
(CATCTATGGTAACTACAATCG; SEQ. ID. NO. 249) and OGS 1245
(GTAGAAGTCACTGATCAGACAC; SEQ. ID. NO. 250) for SEQ. ID. NO. 47 was used
to perform RT-PCR. As indicated by the expected PCR amplicon, increased
expression of SEQ. ID. NO. 47 mRNA was evident only in ovarian cancer;
Fig. 103 is a picture of RT-PCR data showing the differential expression data
for the STAR selected ovarian cancer-related human SEQ. ID. NO. 48 in RNA
samples derived from the NCI-60 panel of cancer cell lines. A primer pair, OGS
1282
(ATGGCTCATACAGCACTCAGG; SEQ. ID. NO. 165) and OGS 1283
(GAACTGTCACTCCGGAAAGCCT; SEQ. ID. NO. 166) for SEQ. ID. NO. 48 was used
to perform RT-PCR. As indicated by the expected PCR amplicon, increased
expression of SEQ. ID. NO. 48 mRNA was evident in all cancer types;
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Fig. 104 is a picture of RT-PCR data showing the differential expression data
for the STAR selected ovarian cancer-related human SEQ. ID. NO. 50 in RNA
samples derived from the NCI-60 panel of cancer cell lines_ A primer pair, OGS
1035
(CTGCCTGCCAACCITTCCATTTCT, SEQ. ID. NO. 251) and OGS 1036
(TGAGCAGCCACAGCAGCATTAGG; SEQ. ID. NO. 252) for SEQ. ID. NO. 50 was
used to perform RT-PCR. As indicated by the expected PCR amplicon, increased
expression of SEQ. ID. NO. 50 mRNA was evident in all cancer types but weak in
CNS
cancer and leukemia, and;
Fig. 105 is a picture of RT-PCR data showing the differential expression data
io for the STAR selected ovarian cancer-related human SEQ. ID. NO. 169 in
RNA
samples derived from the NCI-60 panel of cancer cell lines. A primer pair, OGS
1248
(CACCTGATCAGGTGGATAAGG; SEQ. ID. NO. 253) and OGS 1249
(TCCCAGGTAGAAGGTGGAATCC; SEQ. ID. NO. 254) for SEQ. ID. NO. 169 was
used to perform RT-PCR. As indicated by the expected PCR amplicon, increased
expression of SEQ. ID. NO. 169 mRNA was evident in ovarian, renal, and lung
cancer
but weak in CNS cancer.
DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS
The applicant employed a carefully planned strategy to identify and isolate
genetic sequences involved in ovarian cancer. The process involved the
following
steps: 1) preparation of highly representative cDNA libraries using mRNA
isolated from
LMPs and malignant ovarian cancer samples of human origin; 2) isolation of
sequences upregulated in the malignant ovarian cancer samples; 3)
identification and
characterization of upregulated sequences; 4) selection of upregulated
sequences for
tissue specificity; 5) determination of knock-down effects on ovarian cancer
cell line
proliferation and migration; and 6) determination of the expression pattern of
each
upregulated sequence in samples derived from nine different cancer types. The
results
discussed in this disclosure demonstrate the advantage of targeting ovarian
cancer-
related genes that are highly specific to this differentiated cell type
compared to normal
tissues and provide a more efficient screening method when studying the
genetic basis
of diseases and disorders. Polynucleotide and/or polypeptide sequences that
are
known but have not had a role assigned to them until the present disclosure
have also
been isolated and shown to have a critical role in ovarian cancer cell line
proliferation
and migration. Finally, novel polynucleotide and/or polypeptide sequences have
been
identified that play a role as well.
The present invention is illustrated in further details below in a non-
limiting
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fashion.
A- Material and Methods
Commercially available reagents referred to in the present disclosure were
used according to supplier's instructions unless otherwise indicated.
Throughout the
present disclosure certain starting materials were prepared as follows:
B - Preparation of LMP and malignant ovarian cancer cells
LMP and malignant ovarian tumor samples were selected based on
histopathology to identify the respective stage and grade (Table B) . LMP was
choosen
instead of normal ovarian tissue to avoid genes that associated with
proliferation due
to ovulation. Also very few cells would have been recovered and stromal cells
would
have been a major contaminant. LMP and serous (most common) ovarian tumors
represent the extremes of tumorigenicity, differentiation and invasion. Once
the sample
were selected, total RNA was extracted with TrizolTm (InVitrogen, Grand
Island, NY)
after the tissues were homogenized. The quality of the RNA was assessed using
a
2100 Bioanalyzer (Agilent Technologies, Palo Alto, CA)
Table B: shows the pathologies including grade and stage of the different
ovarian
cancer samples used on the macroarrays.
Position
N
Code
Pathologies Symbol Stage Grade on
No. Macroarray
15 _Borderline serous B lb B Al
B11
, 16 Borderline serous B 2a B
t-
17 Borderline/carcinoma serous B/CS 3c 1 Fl '
------ .
18 Borderline serous B 3c B C1
19 Borderline __ serous Blb B D1
,- -4 __ -,--
Borderline serous B la B __ El
42 Carcinoma serous of the surface CSS 3a 3 A4
22 ,Carcinoma serous CS lb 3 A2
+--- ---4
Carcinoma serous CS 2c 3 __ E2
23 Carcinoma serous CS 3c 3 F2 ¨
25 Carcinoma serous CS 3c 3 B2
26 Carcinoma serous CS 3c 3 A3
27 Carcinoma serous CS 3c 3 C2
28 Carcinoma serous CS 3c 3 D2
43 Carcinoma serous CS 3c 3 84
45 Carcinoma serous CS 3c 3 1 04
49 Carcinoma serous CS 3c 2 F4
41 Carcinoma endometrioide CE 3b 3 . - d3--
Carcinoma endometrioide CE __ 3c 3
I
44 Carcinoma endometrioide CE 3c 3 C4
_
- - -
39 Carcinoma endometrioide 1 CE 3c i 2 E3
,
Carcinoma endometrioide CE lc '
, 1 G4 1
46 Carcinoma endometrioide CE la E 2 T E4 _ i
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Position
MF Code
Pathologies Symbol Stage Grade on
No. Macroarray
34 Clear cell carcinoma CCC 3c 2 B3
38 Clear cell carcinoma CCC 3c 3 D3
37 Clear cell carcinoma CCC lc 2 C3
C- Method of Isolating Differentially Expressed mRNA
Key to the discovery of differentially expressed sequences unique to
malignant ovarian cancer is the use of the applicant's patented STAR
technology
(Subtractive Transcription-based Amplification of mRNA; U.S. Patent No.
5,712,127
Malek et al., 1998). Based on this procedure, mRNA isolated from malignant
ovarian
tumor sample is used to prepare "tester RNA", which is hybridized to
complementary
single-stranded "driver DNA" prepared from mRNA from LMP sample and only the
un-
hybridized "tester RNA" is recovered, and used to create cloned cDNA
libraries,
termed "subtracted libraries". Thus, the "subtracted libraries" are enriched
for
differentially expressed sequences inclusive of rare and novel mRNAs often
missed by
micro-array hybridization analysis. These rare and novel mRNA are thought to
be
representative of important gene targets for the development of better
diagnostic and
therapeutic strategies.
The clones contained in the enriched "subtracted libraries" are identified by
DNA sequence analysis and their potential function assessed by acquiring
information
available in public databases (NCBI and GeneCard). The non-redundant clones
are
then used to prepare DNA micro-arrays, which are used to quantify their
relative
differential expression patterns by hybridization to fluorescent cDNA probes.
Two
classes of cDNA probes may be used, those which are generated from either RNA
transcripts prepared from the same subtracted libraries (subtracted probes) or
from
mRNA isolated from different ovarian LMP and malignant samples (standard
probes).
The use of subtracted probes provides increased sensitivity for detecting the
low
abundance mRNA sequences that are preserved and enriched by STAR. Furthermore,
the specificity of the differentially expressed sequences to malignant ovarian
cancer is
measured by hybridizing radio-labeled probes prepared from each selected
sequence
to macroarrays containing RNA from different LMP and malignant ovarian cancer
samples and different normal human tissues.
A major challenge in gene expression profiling is the limited quantities of
RNA
available for molecular analysis. The amount of RNA isolated from many human
specimens (needle aspiration, laser capture micro-dissection (LCM) samples and
transfected cultured cells) is often insufficient for preparing: 1)
conventional tester and
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driver materials for STAR; 2) standard cDNA probes for DNA micro-array
analysis; 3)
RNA macroarrays for testing the specificity of expression; 4) Northern blots
and; 5) full-
length cDNA clones for further biological validation and characterization etc.
Thus, the
applicant has developed a proprietary technology called RAMP (RNA
Amplification
Procedure) (U.S. Patent Application No. 11/000,958 published under No. US
2005/0153333A1 on July 14, 2005 and entitled "Selective Terminal Tagging of
Nucleic
Acids"), which linearly amplifies the mRNA contained in total RNA samples
yielding
microgram quantities of amplified RNA sufficient for the various analytical
applications.
The RAMP RNA produced is largely full-length mRNA-like sequences as a result
of the
proprietary method for adding a terminal sequence tag to the 3'-ends of single-
stranded cDNA molecules, for use in linear transcription amplification.
Greater than
99.5% of the sequences amplified in RAMP reactions show <2-fold variability
and thus,
RAMP provides unbiased RNA samples in quantities sufficient to enable the
discovery
of the unique mRNA sequences involved in ovarian cancer.
D- Preparation of Human Malignant Ovarian Cancer Subtracted Library
Total RNA from five human ovarian LMP samples (MF-15, -16, -18, -19 and ¨
20) (Table B) and five malignant ovarian cancer samples (MF-22, -25, -27, -28
and ¨
30) (Table B) (CHUM, Montreal, QC) were prepared as described above. Following
a
slight modification of the teachings of Malek et al., 1998 (U.S. Patent No.
5,712,127)
i.e., preparation of the cDNA libraries on the paramagnetic beads as described
below),
1 pg of total RNA from each sample were used to prepare highly representative
cDNA
libraries on streptavidin-coated paramagnetic beads (InVitrogen, Grand Island,
NY) for
preparing tester and driver materials. In each case, first-strand cDNA was
synthesized
using an oligo dTil primer with 3' locking nucleotides (e.g., A, G or C), a 5'-
biotin
moiety and containing a Not I recognition site (OGS 364: SEQ. ID NO. 90) Next,
second-strand cDNA synthesis was performed according to the manufacturer's
procedure for double-stranded cDNA synthesis (Invitrogen, Burlington, ON) and
the
resulting double-stranded cDNA ligated to linkers containing an Asc I
recognition site
(New England Biolabs, Pickering, ON). The double-stranded cDNAs were then
digested with Asc I and Not I restriction enzymes (New England Biolabs,
Pickering,
ON), purified from the excess linkers using the cDNA fractionation column from
Invitrogen (Burlington, ON) as specified by the manufacturer. Each sample was
equally divided and ligated separately to specialized oligonucleotide promoter
tags,
TAG1 (OGS 594 and 595: SEQ. ID. NO: 91 and SEQ. ID. NO:92) and TAG2 (0GS458
and 459: SEQ. ID. NO:93 and SEQ. ID. NO:94) used for preparing tester and
driver
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materials, respectively. Thereafter, each ligated cDNA was purified by
capturing on the
streptavidin beads as described by the supplier (InVitrogen, Grand Island,
NY), and
transcribed in vitro with T7 RNA polymerase (Ambion, Austin, TX).
Next, in order to prepare 3'-represented tester and driver libraries, a 10-pg
aliquot of each of the in vitro synthesized RNA was converted to double-
stranded
cDNA by performing first-strand cDNA synthesis as described above followed by
primer-directed (primer OGS 494 (SEQ. ID. NO:95) for TAG1 and primer OGS 302
(SEQ. ID. NO:96) for TAG2) second-strand DNA synthesis using Advantage-2 Taq
polymerase (BD Biosciences Clontech, Mississauga, ON). The double-stranded
cDNA
to was purified using Qiaquick columns and quantified at A260nrn.
Thereafter, 6x 1-pg
aliquots of each double-stranded cDNA was digested individually with one of
the
following 4-base recognition restriction enzymes Rsa 1, Sau3A1, Mse I, Msp I,
HinPI I
and Bsh 12361 (MBI Fermentas, Burlington, ON), yielding up to six possible 3'-
fragments for each RNA species contained in the cDNA library. Following
digestion,
the restriction enzymes were inactivated with phenol and the set of six
reactions
pooled. The restriction enzymes sites were then blunted with T4 DNA polymerase
and
ligated to linkers containing an Asc I recognition site. Each linker-adapted
pooled DNA
sample was digested with Asc I and Not I restriction enzymes, desalted and
ligated to
specialized oligonucleotide promoter tags, TAG1 (OGS 594 and 595) for the
original
TAG1-derived materials to generate tester RNA and TAG2-related OGS 621 and 622
(SEQ. ID. NO:97 and SEQ. ID. NO:98) with only the promoter sequence for the
original TAG2-derived materials for generating driver DNA. The promoter-
ligated
materials were purified using the streptavidin beads, which were then
transcribed in
vitro with either T7 RNA polymerase (Ambion, Austin, TX), purified and
quantified at
A260nm= The resulting TAG1 3'-represented RNA was used directly as "tester
RNA"
whereas, the TAG2 3'-represented RNA was used to synthesize first-strand cDNA,
which then served as single-stranded "driver DNA". Each "driver DNA" reaction
was
treated with RNase A and RNase H to remove the RNA, phenol extracted and
purified
before use. An equivalent amount of each driver RNA for the five IMP samples
were
pooled before synthesis of the single-stranded driver DNA.
The following 3'-represented libraries were prepared:
Tester 1 (MF-22) - human malignant ovarian cancer donor 1
Tester 2 (MF-25) - human malignant ovarian cancer donor 2
Tester 3 (MF-27) - human malignant ovarian cancer donor 3
Tester 4 (MF-28) - human malignant ovarian cancer donor 4
Tester 5 (MF-30) - human malignant ovarian cancer donor 5
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Driver 1 (MF-15) - human ovarian LMP donor 1
Driver 2 (MF-16) - human ovarian LMP donor 2
Driver 3 (MF-18) - human ovarian LMP donor 3
Driver 4 (MF-19) - human ovarian LMP donor 4
Driver 5 (MF-20) - human ovarian LMP donor 5
Each tester RNA sample was subtracted following the teachings of U.S.
patent No. 5,712,127 with the pooled driver DNA (MF-15, -16, -18, -19 and ¨20)
in a
ratio of 1:100 for 2-rounds following the teachings of Malek et al., 1998
(U.S. Patent
No. 5,712,127). Additionally, control reactions containing tester RNA and no
driver
io DNA, and tester RNA plus driver DNA but no RNase H were prepared. The
tester RNA
remaining in each reaction after subtraction was converted to double-stranded
DNA,
and a volume of 5% removed and amplified in a standard PCR reaction for 30-
cycles
for analytical purposes. The remaining 95% of only the tester-driver plus
RNase H
subtracted samples after 2-rounds were amplified for 4-cycles in PCR, digested
with
Asc I and Not I restriction enzymes, and one half ligated into the pCATRMAN
(SEQ.
ID. NO:99) plasmid vector and the other half, into the p20 (SEQ. ID NO:100)
plasmid
vector. The legated materials were transformed into E. coli DH1OB and
individual
clones contained in the pCATRMAN libraries were picked for further analysis
(DNA
sequencing and hybridization) whereas, clones contained in each p20 library
were
pooled for use as subtracted probes. Each 4-cycles amplified cloned subtracted
library
contained between 15,000 and 25,000 colonies. Additionally, in order to
prepare
subtracted cDNA probes, reciprocal subtraction for 2-rounds was performed
using
instead, the pooled driver RNA as "tester" and each of the malignant tester
RNA as
"driver". The materials remaining after subtraction for each were similarly
amplified for
4-cycles in PCR, digested with Asc I and Not I restriction enzymes, and one
half
legated into the p20 plasmid vector.
The following cloned subtracted libraries were prepared:
SL123 - Tester 1 (MF-22) minus Pooled Driver (MF-15, -16, -18, -19 and ¨20)
SL124 - Tester 2 (MF-25) minus Pooled Driver (MF-15, -16, -18, -19 and ¨20)
SL125 - Tester 3 (MF-27) minus Pooled Driver (MF-15, -16, -18, -19 and ¨20)
SL126 - Tester 4 (MF-28) minus Pooled Driver (MF-15, -16, -18, -19 and ¨20)
SL127 - Tester 5 (MF-30) minus Pooled Driver (MF-15, -16, -18. -19 and ¨20)
SL133 - Pooled Driver (MF-15, -16, -18, -19 and ¨20) minus Tester 1 (MF-22)
SL134 - Pooled Driver (MF-15, -16, -18, -19 and ¨20) minus Tester 2 (MF-25)
SL135 - Pooled Driver (MF-15, -16, -18, -19 and ¨20) minus Tester 3 (MF-27)
SL136 - Pooled Driver (MF-15, -16, -18, -19 and ¨20) minus Tester 4 (MF-28)
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SL137 - Pooled Driver (MF-15, -16, -18, -19 and ¨20) minus Tester 5 (MF-30)
A 5-pL aliquot of the 30-cycles PCR amplified subtracted and non-subtracted
materials were visualized on a 1.5% agarose gel containing ethidium bromide
and then
transferred to Hybond N+ (Amersham Biosciences, Piscataway, NJ) nylon membrane
for Southern blot analysis. Using radiolabeled probes specific for GAPDH
(glyceraldehyde-3-phosphate dehydrogenase; Accession #M32599.1) and 0-actin
(Accession #X00351), which are typically non-differentially expressed house-
keeping
genes, it was evident that there was subtraction of both GAPDH and I3-actin
(Fig. 51,
Panels A and B). Yet, at the same time, a probe specific for CCNE1 (Accession
#
NM_001238, a gene known to be upregulated in malignant ovarian cancer,
indicated
that it was not subtracted (Fig. 51, Panel C). Based on these results, it was
anticipated
that the subtracted libraries would be enriched for differentially expressed
upregulated
sequences.
E - Sequence identification and annotation of clones contained in the
subtracted
libraries:
Approximately ¨5300 individual colonies contained in the pCATRMAN
subtracted libraries (SL123 to SL127) described above were randomly picked
using a
Qbot (Genetix Inc., Boston, MA) into 60 pL of autoclaved water. Then, 42 pL of
each
was used in a 100-pL standard PCR reaction containing oligonucleotide primers,
OGS
1 and OGS 142 and amplified for 40-cycles (94 C for 10 minutes, 40x (94 C for
40
seconds, 55 C for 30 seconds and 72 C for 2 minutes) followed by 72 C for 7
minutes) in 96-wells microtitre plates using HotStartTM Taq polymerase
(Qiagen,
Mississauga, ON). The completed PCR reactions were desalted using the 96-well
filter
plates (Corning) and the amplicons recovered in 100 pL 10mM Tris (pH 8.0). A 5-
pL
aliquot of each PCR reaction was visualized on a 1.5% agarose gel containing
ethidium bromide and only those reactions containing a single amplified
product were
selected for DNA sequence analysis using standard DNA sequencing performed on
an
ABI 3100 instrument (Applied Biosystems, Foster City, CA). Each DNA sequence
obtained was given a Sequence Identification Number and entered into a
database for
subsequent tracking and annotation.
Each sequence was selected for BLAST analysis of public databases (e.g.
NCB!). Absent from these sequences were the standard housekeeping genes
(GAPDH, actin, most ribosomal proteins etc.), which was a good indication that
the
subtracted library was depleted of at least the relatively abundant non-
differentially
expressed sequences.
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Once sequencing and annotation of the selected clones were completed, the
next step involved identifying those sequences that were actually upregulated
in the
malignant ovarian cancer samples compared to the LMP samples.
F - Hybridization analysis for identifying upregulated sequences
The PCR amplicons representing the annotated sequences from the
pCATRMAN libraries described above were used to prepare DNA microarrays. The
purified PCR amplicons contained in 70 pL of the PCR reactions prepared in the
previous section was lyophilized and each reconstituted in 20 pL of spotting
solution
comprising 3xSSC and 0.1% sarkosyl. DNA micro-arrays of each amplicon in
triplicate
were then prepared using CMT-GAP2 slides (Corning, Corning, NY) and the GMS
417
spotter (Affymetrix, Santa Clara, CA).
The DNA micro-arrays were then hybridized with either standard or
subtracted cy3 and cy5 labelled cDNA probes as recommended by the supplier
(Amersham Biosciences, Piscataway, NJ). The standard cDNA probes were
synthesized using RAMP amplified RNA prepared from the different human ovarian
LMP and malignant samples. It is well known to the skilled artisan that
standard cDNA
probes only provide limited sensitivity of detection and consequently, low
abundance
sequences contained in the cDNA probes are usually missed. Thus, the
hybridization
analysis was also performed using cy3 and cy5 labelled subtracted cDNA probes
prepared from in vitro transcribed RNA generated from subtracted libraries
(SLP123 to
SLP127 and SLP133 to SLP137) cloned into the p20 plasmid vector and represent
the
different tester and driver materials. These subtracted libraries may be
enriched for low
abundance sequences as a result of following the teachings of Malek et al.,
1998 (U.S.
Patent No. 5,712,127), and therefore, may provide increased detection
sensitivity.
All hybridization reactions were performed using the dye-swap procedure as
recommended by the supplier (Amersham Biosciences, Piscataway, NJ) and
approximately 750 putatively differentially expressed upregulated (>2-fold)
sequences
were selected for further analysis.
G - Determining malignant ovarian cancer specificity of the differentially
expressed sequences identified:
The differentially expressed sequences identified in Section F for the
different
human malignant ovarian cancer subtracted libraries (SL123 to SL127) were
tested for
specificity by hybridization to nylon membrane-based macroarrays. The
macroarrays
were prepared using RAMP amplified RNA from 6 LMP and 20 malignant human
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ovarian samples, and 30 normal human tissues (adrenal, liver, lung, ovary,
skeletal
muscle, heart, cervix, thyroid, breast, placenta, adrenal cortex, kidney, vena
cava,
fallopian tube, pancreas, testicle, jejunum, aorta, esophagus, prostate,
stomach,
spleen, ileum, trachea, brain, colon, thymus, small intestine, bladder and
duodenum)
purchased commercially (Ambion, Austin, TX). In addition, RAMP RNA prepared
from
breast cancer cell lines, MDA and MCF7, prostate cancer cell line, LNCap, and
a
normal and prostate cancer LCM microdissected sample. Because of the limited
quantities of mRNA available for many of these samples, it was necessary to
first
amplify the mRNA using the RAMP methodology. Each amplified RNA sample was
reconstituted to a final concentration of 250 ng/pL in 3xSSC and 0.1% sarkosyl
in a
96-well microtitre plate and 1 pL spotted onto Hybond N+ nylon membranes using
the
specialized MULTI-PRINT-1-m apparatus (VP Scientific, San Diego, CA), air
dried and
UV-cross linked. Of the ¨750 different sequences selected from SL123 to SL127
for
macroarray analysis, only 250 sequences were individually radiolabeled with a-
32P-
dCTP using the random priming procedure recommended by the supplier (Amersham,
Piscataway, NJ) and used as probes on the macroarrays thus far. Hybridization
and
washing steps were performed following standard procedures well known to those
skilled in the art.
Occasionally, the results obtained from the macroarray methodology were
inconclusive. For example, probing the membranes with certain STAR clones
resulted
in patterns where all the RNA samples appeared to express equal levels of the
message or in patterns .where there was no signal. This suggested that not all
STAR
clones were useful tools to verify the expression of their respective genes.
To
circumvent this problem, RT-PCR was used to determine the specificity of
expression_
Using the same RAMP RNA samples that were spotted on the macroarrays, 500 pg
of
RNA was converted to single-stranded cDNA with Thermoscript RT (lnvitrogen,
Burlington, ON) as described by the manufacturer. The cDNA reaction was
diluted so
that 1/200 of the reaction was used for each PCR experiment. After trial PCR
reactions
with gene-specific primers designed against each SEQ. ID NOs. to be tested,
the
linear range of the reaction was determined and applied to all samples, PCR
was
conducted in 96-well plates using Hot-Start Taq Polymerase from Qiagen
(Mississauga, ON) in a DNA Engine Tetrad from MJ Research. Half of the
reaction
mixture was loaded on a 1.2% agarose/ethidium bromide gel and the amplicons
visualized with UV light.
Of the 250 sequences tested, approximately 55% were found to be
upregulated in many of the malignant samples compared to the LMPs. However,
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of these sequences were also readily detected in a majority of the different
normal
human tissues. Based on these results, those sequences that were detected in
many
of the other human tissues at significantly elevated levels were eliminated.
Consequently, only 49 sequences, which appeared to be upregulated and highly
malignant ovarian cancer-specific, were selected for biological validation
studies. This
subset of 49 sequences include some genes previously reported in the
literature to be
upregulated in ovarian cancer but without demonstration of their relative
expression in
normal tissues. The macroarray data for FOLR1 (SEQ. ID. NO.50) is included to
exemplify the hybridization pattern and specificity of a gene that is already
known to be
involved in the development of ovarian cancer.
Fig. 1-49 and 51 show the macroarray hybridization signal patterns and RT-
PCR amplification data for the malignant ovarian cancer and normal human
tissues
relative to LMPs for the 50 sequences isolated and selected for biological
validation.
Amongst the 50 selected sequences, 27 were associated with genes having
functional
annotation 15 were associated with genes with no functional annotation and 8
were
novel sequences (genomic hits). The identification of gene products involved
in
regulating the development of ovarian cancer has thus led to the discovery of
highly
specific, including novel targets, for the development of new therapeutic
strategies for
ovarian cancer management. Representative sequences summarized in Table 2 are
presented below and corresponding sequences are illustrated in Table 4.
The present invention thus relates in one aspect thereof to a method of
representatively identifying a differentially expressed sequence involved in
ovarian
cancer. The sequence may be, for example, differentially expressed in a
malignant
ovarian cancer cell compared to a LMP ovarian cancer cell or normal ovarian
cells.
The sequence may be, for example, differentially expressed in a malignant
ovarian
cancer cell and a LMP ovarian cancer cell compared to a normal ovarian cell
The method of the present invention may comprise the following steps or
some of the following steps;
a) separately providing total messenger RNA from malignant and LMP
ovarian cancer cells, and normal ovarian cells, the total messenger RNA
may comprise, for examp(e, at least one endogeneously differentially
expressed sequence,
b) generating (e.g., single copy) of a) single-stranded cDNA from each
messenger RNA of malignant ovarian cancer cell and (e.g., random(y)
tagging the 3'-end of the single-stranded cDNA with a RNA polymerase
promoter sequence and a first sequence tag;
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c) generating (e.g., single copy) of a) single-stranded cDNA from each
messenger RNA of LMP ovarian cancer cells or normal ovarian cell and
(e.g., randomly) tagging the 3'-end of the single-stranded cDNA with a
RNA polymerase promoter sequence and a second sequence tag;
d) separately generating partially or completely double-stranded 5'-
tagged-DNA from each of b) and c), the double-stranded 5'-tagged-DNA
may thus comprise in a 5' to 3' direction, a double-stranded RNA
polymerase promoter, a first or second sequence tag and an expressed
nucleic acid sequence,
e) separately linearly amplifying a first and second tagged sense RNA from
each of d) with a RNA polymerase enzyme (which may be selected based
on the promoter used for tagging),
f) generating single-stranded complementary first or second tagged DNA
from one of e),
g) hybridizing the single-stranded complementary first or second tagged
DNA of f) with the other linearly amplified sense RNA of e),
h) recovering unhybridized RNA with the help of the first or second
sequence tag (for example by PCR or hybridization), and;
i) identifying (determining) the nucleotide sequence of unhybridized RNA.
The method may further comprise the step of comparatively determining the
presence of the identified differentially expressed sequence in a cancer cell
relative to
a normal cell (e.g., a normal ovarian cell, a normal prostate cell, a normal
breast cell
etc.) or relative to a standard value.
The method may be used to preferentially identify a sequence which is
upregulated in malignant ovarian cancer cell compared to a cell from a low
malignancy
potential ovarian cancer and/or compared to a normal cell.
In accordance with the present invention, a sequence may be further selected
based on a reduced, lowered or substantially absent expression in a subset of
other
normal cell (e.g., a normal ovarian cell) or tissue, therefore representing a
candidate
sequence specifically involved in ovarian cancer.
The method may also further comprise a step of determining the complete
sequence of the nucleotide sequence and may also comprise determining the
coding
sequence of the nucleotide sequence.
A sequence may also be selected for its specificity to other types of tumor
cells, thus identifying a sequence having a more generalized involvement in
the
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development of cancer. These types of sequence may therefore represent
desirable
candidates having a more universal utility in the treatment and/or detection
of cancer.
The present invention also relates in a further aspect, to the isolated
differentially expressed sequence (polynucleotide and polypeptide) identified
by the
method of the present invention.
SEQ. ID. NO:1:
The candidate STAR sequence for SEQ. ID. NO:1 maps to a genomic hit and
est hits according to NCB1's nr and est databases (see Table 2). Although, the
matching ests are clustered into a new Unigene identifier number, Hs.555871,
the
STAR sequence does not map to any of the known mRNA sequences listed in this
cluster, which codes for guanine nucleotide binding protein (G protein), gamma
transducing activity polypeptide 1 (GNGT1). We have demonstrated that this
STAR
clone sequence is markedly upregulated in malignant ovarian cancer samples
i 5 compared to ovarian LMP samples and a majority of normal human tissues
(Figure 1),
which have not been previously reported. Thus, it is believed that the gene
comprising
this STAR sequence or a related gene member as is outlined in the Unigene
cluster
may be required for ovarian cancer tumorigenesis.
SEQ. ID. NO:2:
The candidate protein encoded by the isolated SEQ. ID. NO:2 is associated
with a previously identified gene that encodes a predicted polypeptide,
interferon-
induced protein 44-like (IF144L) with an unknown function (see Table 2). We
have
demonstrated that expression of this gene is markedly upregulated in malignant
ovarian cancer samples compared to ovarian LMP samples and a majority of
normal
human tissues (Figure 2), which have not been previously reported. Thus, it is
believed
that expression of this gene may be required for or involved for ovarian
cancer
tumorigenesis.
SEQ. ID. NO:3:
The candidate protein encoded by the isolated SEQ. ID. NO:3 is associated
with a previously identified gene that encodes a known polypeptide, HOX D1,
which
contains a homeobox DNA-binding domain. This gene is a member of the Antp
homeobox family and is nuclear sequence-specific transcription factor that is
previously known to be involved in differentiation and limb development (see
Table 2).
We have demonstrated that expression of this gene is markedly upregulated in
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malignant ovarian cancer samples compared to ovarian LMP samples and the
normal
human tissues (Figure 3), which have not been previously reported. Thus, it is
believed
that the gene may be required for, or involved in ovarian cancer tumorigenesis
as well.
SEQ. ID. NO:4:
The candidate protein encoded by the isolated SEQ. ID. NO:4 is associated
with a previously identified gene that encodes a hypothetical polypeptide,
L0C92196,
similar to death-associated protein with an unknown function (see Table 2). We
have
demonstrated that expression of this gene is markedly upregulated in malignant
lo ovarian cancer samples compared to ovarian LMP samples and a majority of
normal
human tissues (Figure 4), which have not been previously reported. Thus, it is
believed
that expression of this gene may be required for, or involved in ovarian
cancer
tumorigenesis.
SEQ. ID. NO:5
The candidate protein encoded by the isolated SEQ. ID. NO:5 is associated
with a previously identified gene that encodes a predicted polypeptide,
interferon-
induced protein with tetratricopeptide repeats 1 (IFIT1), with unknown
function (see
Table 2). We have demonstrated that expression of this gene is markedly
upregulated
in malignant ovarian cancer samples compared to ovarian LMP samples and a
majority of normal human tissues (Figure 5), which have not been previously
reported.
Thus, it is believed that expression of this gene may be required for, or
involved in
ovarian cancer tumorigenesis.
SEQ. ID. NO:6:
The candidate protein encoded by the isolated SEQ. ID. NO:6 is associated
with a previously identified gene that encodes a known protein, glycine
dehydrogenase
(GLDC) (decarboxylating; glycine decarboxylase, glycine cleavage system
protein P),
which is a mitochondrial enzyme that catalyzes the degradation of glycine (see
Table
2). We have demonstrated that expression of this gene is markedly upregulated
in
malignant ovarian cancer samples compared to ovarian LMP samples and a
majority
of normal human tissues (Figure 6), which have not been previously reported.
Thus, it
is believed that expression of this gene may be required for, or involved in
ovarian
cancer tumorigenesis. The GLDC activity may be detected, for example. by
measuring
the degradation of glycine into urea.
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SEQ. ID. NO:7:
The candidate protein encoded by the isolated SEQ. ID. NO:7 is associated
with a previously identified gene that encodes a protein, dipeptidase 3
(DPEP3), which
has membrane dipeptidase (proteolysis and peptidolysis) activity (see Table
2). We
__ have demonstrated that expression of this gene is markedly upregulated in
malignant
ovarian cancer samples compared to ovarian LMP samples and a majority of
normal
human tissues (Figure 7), which have not been previously reported. Thus, it is
believed
that expression of this gene may be required for, or involved in ovarian
cancer
tumorigenesis.
SEQ. ID. NO:8
The candidate protein encoded by the isolated SEQ. ID. NO:8 is associated
with a previously identified gene that encodes a protein, neuromedin U (NMU),
which
is a neuropeptide with potent activity on smooth muscle (see Table 2). We have
__ demonstrated that expression of this gene is markedly upregulated in
malignant
ovarian cancer samples compared to ovarian LMP samples and a majority of
normal
human tissues (Figure 8), which have not been previously reported. Thus, it is
believed
that expression of the gene may be required for, or involved in ovarian cancer
tumorigenesis.
SEQ. ID. NO:9
The candidate protein encoded by the isolated SEQ. ID. NO:9 is associated
with a previously identified gene that encodes a protein, bone morphogenetic
protein 7
(BMP7), which plays a role in calcium regulation and bone homeostasis (see
Table 2).
__ We have demonstrated that expression of this gene is markedly upregulated
in
malignant ovarian cancer samples compared to ovarian LMP samples and a
majority
of normal human tissues (Figure 9), which have not been previously reported.
Thus, it
is believed that expression of the gene may be required for, or involved in
ovarian
cancer tumorigenesis.
SEQ. ID. NO:10
The candidate protein encoded by the isolated SEQ. ID. NO:10 is associated
with a previously identified gene that encodes a protein, cyclin-dependent
kinase
inhibitor 3 (CDKN3) (CDK2-associated dual specificity phosphatase), which is
__ expressed at the G1 to S transition of the cell cycle (see Table 2). We
have
demonstrated that expression of this gene is markedly upregulated in malignant
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ovarian cancer samples compared to ovarian LMP samples and a majority of
normal
human tissues (Figure 10), which have not been previously reported. Thus, it
is
believed that expression of the gene may be required for, or involved in
ovarian cancer
tumorigenesis.
SEQ. ID. NO:11
The candidate protein encoded by the isolated SEQ. ID. NO:11 is associated
with a previously identified gene that encodes a protein, CDC28 protein kinase
regulatory subunit 1B (CKS1B), which has cyclin-dependent protein kinase
activity in
o cell cycle regulation (see Table 2). We have demonstrated that expression
of this gene
is markedly upregulated in malignant ovarian cancer samples compared to
ovarian
LMP samples and a majority of normal human tissues (Figure 11), which have not
been previously reported. Thus, it is believed that expression of the gene may
be
required for, or involved in ovarian cancer tumorigenesis.
SEQ. ID. NO:12
The candidate protein encoded by the isolated SEQ. ID. NO:12 is associated
with a previously identified gene that encodes a protein, preferentially
expressed
antigen in melanoma (PRAME), which has no known function (see Table 2). We
have
demonstrated that expression of this gene is markedly upregulated in malignant
ovarian cancer samples compared to ovarian LMP samples and a majority of
normal
human tissues (Figure 12), which have not been previously reported. Thus, it
is
believed that expression of the gene may be required for, or involved in
ovarian cancer
tumorigenesis.
SEQ. ID. NO:13
The candidate protein encoded by the isolated SEQ. ID. NO:13 is associated
with a previously identified gene that encodes a protein, ISG15 ubiquitin-like
modifier
(ISG15), which is associated with ubiquitin-dependent protein catabolism (see
Table
2). We have demonstrated that expression of this gene is markedly upregulated
in
malignant ovarian cancer samples compared to ovarian LMP samples and a
majority
of normal human tissues (Figure 13), which have not been previously reported.
Thus, it
is believed that expression of the gene may be required for, or involved in
ovarian
cancer tumorigenesis.
SEQ. ID. NO:14
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The candidate STAR sequence represented by the isolated SEQ. ID. NO:14
is associated with a previously identified partial gene sequence related to
Accession #
A1922121.1 (see Table 2), which codes for a yet unknown protein. We have
demonstrated that this STAR clone sequence is markedly upregulated in
malignant
ovarian cancer samples compared to ovarian LMP samples and a majority of
normal
human tissues (Figure 14), which have not been previously reported. Thus, it
is
believed that expression of the gene corresponding to this STAR sequence (and
polynucleotide sequences comprising the STAR sequence) may be required for, or
involved in ovarian cancer tumorigenesis.
SEQ. ID. NO:15
The candidate protein encoded by the isolated SEQ. ID. NO:15 is associated
with a previously identified gene that encodes a hypothetical protein,
FLJ33790, which
has no known function (see Table 2). We have demonstrated that expression of
this
gene is markedly upregulated in malignant ovarian cancer samples compared to
ovarian LMP samples and a majority of normal human tissues (Figure 15), which
have
not been previously reported. Thus, it is believed that expression of the gene
may be
required for, or involved in ovarian cancer tumorigenesis.
SEQ. ID. NO:16
The STAR sequence represented by the isolated SEQ. ID. NO:16 maps to a
previously identified est, BG213598 that is from a transcribed genomic locus
contained
in the Unigene cluster, Hs.334302, which encodes a yet unknown protein (see
Table
2). We have demonstrated that this STAR clone sequence is markedly upregulated
in
malignant ovarian cancer samples compared to ovarian LMP samples and a
majority
of normal human tissues (Figure 16), which have not been previously reported.
Thus, it
is believed that expression of the gene corresponding to this STAR sequence
(and
polynucleotides comprising this STAR sequence) or a related gene member as is
outlined in the Unigene cluster may be required for, or involved in ovarian
cancer
tumorigenesis.
SEQ. ID. NO:17
The candidate protein encoded by the isoalted SEQ. ID. NO:17 is associated
with a previously identified gene that encodes a protein, V-set domain
containing T cell
activation inhibitor 1 (VTCN1), which has no known function (see Table 2). We
have
demonstrated that expression of this gene is markedly upregulated in malignant
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ovarian cancer samples compared to ovarian LMP samples and a majority of
normal
human tissues (Figure 17), which have not been previously reported. Thus, it
is
believed that expression of the gene may be required for, or involved in
ovarian cancer
tumorigenesis.
SEQ. ID. NO:18
The candidate protein encoded by the isolated SEQ. ID. NO:18 is associated
with a previously identified gene that encodes a protein, kinesin family
member 20A
(KIF20A), which is involved in cell division in and membrane traffic within
the Golgi
apparatus (see Table 2). We have demonstrated that expression of this gene is
markedly upregulated in malignant ovarian cancer samples compared to ovarian
LMP
samples and a majority of normal human tissues (Figure 18), which have not
been
previously reported. Thus, it is believed that expression of the gene may be
required
for, or involved in ovarian cancer tumorigenesis.
SEQ. ID. NO:19
The STAR sequence represented by the isolated SEQ. ID. NO:19 maps to a
genomic hit, Accession #AY769439 and to a group of ests represented by
Accession #
AA744939. The ests are clustered into Unigene identifier, Hs.478368
representing the
protein, potassium large conductance calcium-activated channel, subfamily M,
beta
member 2 (KCNMB2). However, the STAR sequence does not overlap with any of the
mRNA sequences listed thus far in the Hs.478368 Unigene cluster (see Table 2).
We
have demonstrated that this STAR clone sequence is markedly upregulated in
malignant ovarian cancer samples compared to ovarian LMP samples and a
majority
of normal human tissues (Figure 19A), which have not been previously reported.
In
addition, performing RT-PCR using primers specific to either the STAR clone
sequence for SEQ. ID. NO. 19 or the KCNMB2 sequence represented by Accession
No. NM_005832, the amplification profiles were not the same across a number of
ovarian samples tested (Figure 19B). It was obvious that KCNMB2 was expressed
in
essentially all ovarian samples including the normal at similar levels
whereas, PCR
amplicons for SEQ. ID. NO. 19 was observed at higher levels in the malignant
ovarian
tumor samples compared to the LMPs and normal ovarian samples (Figure 198).
Thus, it is believed that the expression of the gene corresponding to this
STAR
sequence (and polynucleotide sequences comprising the STAR sequence) or a
related
gene member may be required for, or involved in ovarian cancer tumorigenesis.
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SEQ. ID. NO:20
The STAR sequence represented by the isolated SEQ. ID. NO:20 maps to a
previously identified est, BU595315 belonging to a group of ests that is from
a
transcribed genomic locus contained in the Unigene cluster, Hs.603908, which
encodes a yet unknown protein (see Table 2). We have demonstrated that this
STAR
clone sequence is markedly upregulated in malignant ovarian cancer samples
compared to ovarian LMP samples and a majority of normal human tissues (Figure
20), which have not been previously reported. Thus, it is believed that
expression of
the gene corresponding to this STAR sequence (and polynucleotide sequences
11:1 comprising this STAR sequence) or a related gene member as is outlined
in the
Unigene cluster may be required for, or involved in ovarian cancer
tumorigenesis.
SEQ. ID. NO:21
The candidate protein encoded by the isolated SEQ. ID. NO:21 is a previously
is identified gene that encodes a protein, chemokine (C-X-C motif) ligand
10 (CXCL10),
which has chemokine activity (see Table 2). We have demonstrated that
expression of
this gene is markedly upregulated in malignant ovarian cancer samples compared
to
ovarian LMP samples and a majority of normal human tissues (Figure 21), which
have
not been previously reported. Thus, it is believed that expression of the gene
may be
20 required for, or involved in ovarian cancer tumorigenesis.
SEQ. ID. NO:22
The STAR sequence represented by the isolated SEQ. ID. NO:22 maps to
chromosome 14, and may represent a portion of an unknown gene sequence (see
25 Table 2). We have demonstrated that this STAR clone sequence is markedly
upregulated in malignant ovarian cancer samples compared to ovarian LMP
samples
and a majority of normal human tissues (Figure 22), which have not been
previously
reported. Thus, it is believed that expression of the gene corresponding to
this STAR
sequence (and polynuclotides comprising this STAR sequence) may be required
for,
30 or involved in ovarian cancer tumorigenesis.
SEQ. ID. NO:23
The candidate protein encoded by the isolated SEQ. ID. NO:23 is a previously
identified gene that encodes a protein, asparagine-linked glycosylation 8
homolog
35 (yeast, alpha-1,3-glucosyltransferase) (ALG8), which catalyzes the
addition of the
second glucose residue to the lipid-linked oligosaccharide precursor for N-
linked
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glycosylation of proteins (see Table 2). We have demonstrated that expression
of this
gene is markedly upregulated in malignant ovarian cancer samples compared to
ovarian LMP samples and a majority of normal human tissues (Figure 23), which
have
not been previously reported. Thus, it is believed that expression of the gene
may be
required for, or involved in ovarian cancer tumorigenesis.
SEQ. ID. NO:24
The candidate protein encoded by the isolated SEQ. ID. NO:24 is a previously
identified gene that encodes a protein, kidney associated antigen 1 (KAAG1),
which
io has no known function (see Table 2). We have demonstrated that
expression of this
gene is markedly upregulated in malignant ovarian cancer samples compared to
ovarian LMP samples and a majority of normal human tissues (Figure 24), which
have
not been previously reported. Thus, it is believed that expression of the gene
may be
required for, or involved in ovarian cancer tumorigenesis.
SEQ. D. NO:25
The candidate protein encoded by the isolated SEQ. ID. NO:25 is a previously
identified gene that encodes a protein, cyclin-dependent kinase inhibitor 2A
(CDKN2A), which is involved in cell cycle control, G1/S Checkpoint (see Table
2). We
have demonstrated that expression of this gene is markedly upregulated in
malignant
ovarian cancer samples compared to ovarian LMP samples and a majority of
normal
human tissues (Figure 25), which have not been previously reported. Thus, it
is
believed that expression of the gene may be required for, or involved in
ovarian cancer
tumorigenesis.
/5
SEQ. ID. NO:26
The candidate protein encoded by the isolated SEQ. ID. NO:26 is a previously
identified gene that encodes a protein, microtubule-associated protein homolog
(Xenopus laevis) (TPX2), which is involved in cell proliferation from the
transition G1/S
until the end of cytokinesis (see Table 2). We have demonstrated that
expression of
this gene is markedly upregulated in malignant ovarian cancer samples compared
to
ovarian LMP samples and a majority of normal human tissues (Figure 26), which
have
not been previously reported. Thus, it is believed that expression of the gene
may be
required for, or involved in ovarian cancer tumorigenesis.
SEQ. ID. NO:27
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The candidate protein encoded by the isolated SEQ. ID. NO:27 is a previously
identified gene that encodes a protein, ubiquitin-conjugating enzyme E2C
(UBE2C),
which is required for the destruction of mitotic cyclins and for cell cycle
progression
(see Table 2). We have demonstrated that expression of this gene is markedly
upregulated in malignant ovarian cancer samples compared to ovarian LMP
samples
and a majority of normal human tissues (Figure 27), which have not been
previously
reported. Thus, it is believed that expression of the gene may be required
for, or
involved in ovarian cancer tumorigenesis.
to SEQ. ID. NO:28
The STAR sequence represented by the isolated SEQ. ID. NO:28 maps to
cDNA FLJ35538 fis, clone SPLEN2002463 of Unigene cluster, Hs.590469 and may
represent a portion of an unknown gene sequence (see Table 2). We have
demonstrated that this STAR clone sequence is markedly upregulated in
malignant
ovarian cancer samples compared to ovarian LMP samples and a majority of
normal
human tissues (Figure 28), which have not been previously reported. Thus, it
is
believed that expression of the gene correspondong to this STAR sequence (and
polynucleotides comprising this STAR sequence) or a related gene member as is
outlined in the Unigene cluster may be required for, or involved in ovarian
cancer
tumorigenesis.
SEQ. ID. NO:29
The candidate protein encoded by the isolated SEQ. ID. NO:29 is a previously
identified gene that encodes a protein, cellular retinoic acid binding protein
2
(CRABP2), whose function has not been precisely determined but this isoform is
important in retinoic acid-mediated regulation of human skin growth and
differentiation
(see Table 2). We have demonstrated that expression of this gene is markedly
upregulated in malignant ovarian cancer samples compared to ovarian LMP
samples
and a majority of normal human tissues (Figure 29), which have not been
previously
reported. Thus, it is believed that expression of the gene may be required
for, or
involved in ovarian cancer tumorigenesis.
SEQ. ID. NO:30
The candidate protein encoded by the isolated SEQ. ID. NO:30 is a previously
identified gene that encodes a protein. Histone 3, H2a (HIST3H2A), which is
involved
in nucleosome formation (see Table 2). We have demonstrated that expression of
this
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gene is markedly upregulated in malignant ovarian cancer samples compared to
ovarian LMP samples and a majority of normal human tissues (Figure 30), which
have
not been previously reported. Thus, it is believed that expression of the gene
may be
required for, or involved in ovarian cancer tumorigenesis.
SEQ. ID. NO:31
The candidate protein encoded by the isolated SEQ. ID. NO:31 is a previously
identified gene that encodes a protein, Histone 1, H4h (HIST1H4H), which is
involved
in nucleosome formation (see Table 2). We have demonstrated that expression of
this
gene is markedly upregulated in malignant ovarian cancer samples compared to
ovarian LMP samples and a majority of normal human tissues (Figure 30), which
have
not been previously reported. Thus, it is believed that expression of the gene
may be
required for, or involved in ovarian cancer tumorigenesis.
SEQ. ID. N0:32
The candidate protein encoded by the isolated SEQ. ID. NO:32 is a previously
identified gene that encodes a hypothetical protein, Homeo box D3 (HOXD3),
which is
a nuclear transcription factor involved in development and differentiation
(see Table 2).
We have demonstrated that expression of this gene is markedly upregulated in
malignant ovarian cancer samples compared to ovarian LMP samples and a
majority
of normal human tissues (Figure 32), which have not been previously reported.
Thus, it
is believed that expression of the gene may be required for ovarian cancer
tumorigenesis.
SEQ. ID. NO:33
The candidate protein encoded by the isolated SEQ. ID. NO:33 is a previously
identified gene that encodes a member of the immunoglobulin gene family,
immunoglobulin constant gamma 1 (IGHG1), which probably plays a role in immune
response and antigen binding (see Table 2). We have demonstrated that
expression of
this gene is markedly upregulated in malignant ovarian cancer samples compared
to
ovarian LMP samples and a majority of normal human tissues (Figure 33), which
have
not been previously reported. The expression pattern of this gene is similar
to two
other genes disclosed here, SEQ. ID. NO. 34 and SEQ. ID. NO. 47, which also
encode
immunoglobulins. This type of clustered immunoglobulin expression in ovarian
cancer
has not been previously described. Thus, it is believed that expression of the
gene
may be required for ovarian cancer tumorigenesis.
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SEQ. ID. NO:34
The candidate protein encoded by the isolated SEQ. ID. NO:34 is a previously
identified gene that encodes a member of the immunoglobulin gene family,
immunoglobulin kappa constant (IGKC), which probably plays a role in immune
response and antigen biinding (see Table 2). We have demonstrated that
expression
of this gene is markedly upregulated in malignant ovarian cancer samples
compared to
ovarian LMP samples and a majority of normal human tissues (Figure 34), which
have
not been previously reported. The expression pattern of this gene is similar
to two
io other genes disclosed here, SEQ. ID. NO. 33 and SEQ. ID. NO. 47, which
also encode
immunoglobulins. This type of clustered immunoglobulin expression in ovarian
cancer
has not been previously described. Thus, it is believed that expression of the
gene
may be required for ovarian cancer tumorigenesis.
SEQ. ID. NO:35
The candidate protein encoded by the isolated SEQ. ID. NO:35 is a gene
located on chromosome 10 that encodes an open reading frame of unknown
function.
(see Table 2). The gene may encode a protein termed astroprincin that was
found to
be expressed in a critical region in DiGeorge syndrome. We have demonstrated
that
expression of this gene is markedly upregulated in malignant ovarian cancer
samples
compared to ovarian LMP samples and a majority of normal human tissues (Figure
35), which have not been previously reported. Thus, it is believed that
expression of
the gene may be required for, or involved in ovarian cancer tumorigenesis.
SEQ. ID. NO:36
The candidate protein encoded by the isolated SEQ. ID. NO:36 is a previously
identified gene that encodes a protein, histocompatibility (minor) 13 (HM13),
which has
no known function (see Table 2). We have demonstrated that expression of this
gene
is markedly upregulated in malignant ovarian cancer samples compared to
ovarian
LMP samples and a majority of normal human tissues (Figure 36), which have not
been previously reported. Thus, it is believed that expression of the gene may
be
required for, or involved in ovarian cancer tumorigenesis.
SEQ. ID. NO:37
The STAR sequence represented by the isolated SEQ. ID. NO:37 maps to
chromosome 13, and may represent a portion of an unknown gene sequence (see
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Table 2). We have demonstrated that this STAR clone sequence is markedly
upregulated in malignant ovarian cancer samples compared to ovarian LMP
samples
and a majority of normal human tissues (Figure 37), which have not been
previously
reported. Thus, it is believed that expression of the gene corresponding to
this STAR
sequence (and polynuclotides comprising this STAR sequence) may be required
for,
or involved in ovarian cancer tumorigenesis.
SEQ. ID. NO:38
The candidate protein encoded by the isolated SEQ. ID. NO:38 is a previously
identified gene that encodes a protein, frizzled-related protein (FRZB), which
is
associated with symptomatic osteoarthritis and may play a role in skeletal
morphogenesis (see Table 2). We have demonstrated that expression of this gene
is
markedly upregulated in malignant ovarian cancer samples compared to ovarian
LMP
samples and a majority of normal human tissues (Figure 38), which have not
been
previously reported. Thus, it is believed that expression of the gene may be
required
for, or involved in ovarian cancer tumorigenesis.
SEQ. ID. NO:39
The candidate protein encoded by the isolated SEQ. ID. NO:39 is a previously
identified gene that encodes a protein, forkhead box M1 (FOXM1), which is a
transcription factor that regulates genes involved in cell proliferation (see
Table 2). We
have demonstrated that expression of this gene is markedly upregulated in
malignant
ovarian cancer samples compared to ovarian LMP samples and a majority of
normal
human tissues (Figure 39), which have not been previously reported. Thus, it
is
believed that expression of the gene may be required for, or involved in
ovarian cancer
tumorigenesis.
SEQ. ID. NO:40
The candidate protein encoded by the isolated SEQ. ID. NO:40 is a gene
located on chromosome 20 that encodes an open reading frame of unknown
function.
(see Table 2). The gene is predicted to encode a membrane protein. We have
demonstrated that expression of this gene is markedly upregulated in malignant
ovarian cancer samples compared to ovarian LMP samples and a majority of
normal
human tissues (Figure 40), which have not been previously reported. Thus, it
is
believed that expression of the gene may be required for, or involved in
ovarian cancer
tumorigenesis.
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SEQ. ID. NO:41
The STAR sequence represented by the isolated SEQ. ID. NO:41 maps to
chromosome 1, and may represent a portion of an unknown gene sequence (see
Table 2). Weak homology has been found between SEQ. ID. NO. 41 and the envelop
proteins present at the surface of human endogenous retroviruses. We have
demonstrated that this STAR clone sequence is markedly upregulated in
malignant
ovarian cancer samples compared to ovarian LMP samples and a majority of
normal
human tissues (Figure 41), which have not been previously reported. Thus, it
is
believed that expression of the gene corresponding to this STAR sequence (and
polynuclotides comprising this STAR sequence) may be required for, or involved
in
ovarian cancer tumorigenesis.
SEQ. ID. NO:42
The candidate protein encoded by the isolated SEQ. ID. NO:42 is a gene
located on chromosome 16 that encodes an open reading frame of unknown
function.
(see Table 2). The gene is predicted to encode a membrane protein. We have
demonstrated that expression of this gene is markedly upregulated in malignant
ovarian cancer samples compared to ovarian LMP samples and a majority of
normal
human tissues (Figure 42), which have not been previously reported. Thus, it
is
believed that expression of the gene may be required for, or involved in
ovarian cancer
tumorigenesis.
SEQ. ID. NO:43
The candidate protein encoded by the isolated SEQ. ID. NO:43 is a previously
identified gene that encodes a protein, Rac GTPase activating protein 1
(RACGAP1),
which is a GTPase that interacts with Rho GTPases to control many cellular
processes
(see Table 2). These types of proteins are important effector molecules for
the
downstream signaling of Rho GTPases. We have demonstrated that expression of
this
gene is markedly upregulated in malignant ovarian cancer samples compared to
ovarian LMP samples and a majority of normal human tissues (Figure 43), which
have
not been previously reported. Thus, it is believed that expression of the gene
may be
required for, or involved in ovarian cancer tumorigenesis.
SEQ. ID. NO:44
The candidate protein encoded by the isolated SEQ. ID. NO:44 is a gene that
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encodes transmembrane protein 19 (TMEM19) that has no known function. (see
Table
2). The gene is predicted to encode a membrane protein. We have demonstrated
that
expression of this gene is markedly upregulated in malignant ovarian cancer
samples
compared to ovarian LMP samples and a majority of normal human tissues (Figure
44), which have not been previously reported. Thus, it is believed that
expression of
the gene may be required for, or involved in ovarian cancer tumorigenesis.
SEQ. ID. NO:45
The STAR sequence represented by the isolated SEQ. ID. NO:45 maps to
to chromosome 4, and may represent a portion of an unknown gene sequence
(see
Table 2). We have demonstrated that this STAR clone sequence is markedly
upregulated in malignant ovarian cancer samples compared to ovarian LMP
samples
and a majority of normal human tissues (Figure 45), which have not been
previously
reported. Thus, it is believed that expression of the gene corresponding to
this STAR
sequence (and polynuclotides comprising this STAR sequence) may be required
for,
or involved in ovarian cancer tumorigenesis.
SEQ. ID. NO:46
The STAR sequence represented by the isolated SEQ. ID. NO:46 maps to
chromosome 1, and may represent a portion of an unknown gene sequence (see
Table 2). We have demonstrated that this STAR clone sequence is markedly
upregulated in malignant ovarian cancer samples compared to ovarian LMP
samples
and a majority of normal human tissues (Figure 46), which have not been
previously
reported. Thus, it is believed that expression of the gene corresponding to
this STAR
sequence (and polynuclotides comprising this STAR sequence) may be required
for,
or involved in ovarian cancer tumorigenesis.
SEQ. ID. NO:47
The candidate protein encoded by the isolated SEQ. ID. NO:47 is a previously
identified gene with the Unigene cluster, Hs.449585, and may represent a
portion
immunoglobulin lambda locus (IGLV@), which probably plays a role in immune
response and antigen blinding (see Table 2). We have demonstrated that
expression
of this gene is markedly upregulated in malignant ovarian cancer samples
compared to
ovarian LMP samples and a majority of normal human tissues (Figure 47), which
have
not been previously reported. The expression pattern of this gene is similar
to two
other genes disclosed here, SEQ. ID. NO. 33 and SEQ. ID. NO. 34, which also
encode
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immunoglobulins. This type of clustered immunoglobulin expression in ovarian
cancer
has not been previously described. Thus, it is believed that expression of the
gene
may be required for ovarian cancer tumorigenesis.
SEQ. ID. NO:48
The candidate protein encoded by the isolated SEQ. ID. NO:48 is a previously
identified gene that encodes a protein, secretory carrier membrane protein 3
(SCAMP3), which functions as a cell surface carrier protein during vesicular
transport
(see Table 2). We have demonstrated that expression of this gene is markedly
upregulated in malignant ovarian cancer samples compared to ovarian LMP
samples
but it is also expressed in a majority of normal human tissues (Figure 48),
which have
not been previously reported. Thus, it is believed that expression of the gene
may be
required for, or involved in ovarian cancer tumorigenesis.
SEQ. ID. NO:49
The STAR sequence represented by the isolated SEQ. ID. NO:49 maps to
chromosome 2, and may represent a portion of an unknown gene sequence (see
Table 2). We have demonstrated that this STAR clone sequence is markedly
upregulated in malignant ovarian cancer samples compared to ovarian LMP
samples
and a majority of normal human tissues (Figure 49), which have not been
previously
reported. Thus, it is believed that expression of the gene corresponding to
this STAR
sequence (and polynuclotides comprising this STAR sequence) may be required
for,
or involved in ovarian cancer tumorigenesis.
SEQ. ID. NO:50
The candidate protein encoded by the isolated SEQ. ID. NO:50 is a previously
identified gene that encodes a protein, Folate receptor 1 (adult) (FOLR1),
with
members of this gene family having a high affinity for folic acid and for
several reduced
folic acid derivatives, and mediate delivery of 5-methyltetrahydrofolate to
the interior of
cells (see Table 2). We have demonstrated that this gene is markedly
upregulated in
malignant ovarian cancer samples compared to ovarian LMP samples and a
majority
of normal human tissues (Figure 50). The potential role of FOLR1 in ovarian
cancer
therapeutics has been previously documented (Leamon and Low, 200'1 and Jhaveri
et
al., 2006, United States Patent 7,030,236). By way of example of the FOLR1
gene
target, similar genes described herein with upregulation in malignant ovarian
tumors
and limited or no expression in a majority of normal tissues may also serve as
potential
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therapeutic targets for ovarian cancer.
SEQ. ID. NO:169
The candidate protein encoded by the isolated SEQ. ID. NO:169 is a
previously identified gene that encodes a protein, ceruloplasmin (CP), that
binds most
of the copper in plasma and is involved in the peroxidation of
Fe(I1)transferrin. The
deficiency of this metalloprotein, termed aceruloplasminemia, leads to iron
accumulation and tissue damage, and is associated diabetes and neurologic
diseases
(see Table 2). We have demonstrated that this gene is markedly upregulated in
io malignant ovarian cancer samples compared to ovarian LMP samples and a
majority
of normal human tissues (Figure 56) which have not been previously reported.
Thus, it
is believed that expression of the gene corresponding to this STAR sequence
(and
polynuclotides comprising this STAR sequence) may be required for, or involved
in
ovarian cancer tumorigenesis.
H- RNA Interference Studies
RNA interference is a recently discovered gene regulation mechanism that
involves the sequence-specific decrease in a gene's expression by targeting
the
mRNA for degradation and although originally described in plants, it has been
discovered across many animal kingdoms from protozoans and invertebrates to
higher
eukaryotes (reviewed in Agrawal et al., 2003). In physiological settings, the
mechanism of RNA interference is triggered by the presence of double-stranded
RNA
molecules that are cleaved by an RNAse III-like protein active in cells,
called Dicer,
which releases the 21-23 bp siRNAs. The siRNA, in a homology-driven manner,
complexes into a RNA-protein amalgamation termed RISC (RNA-induced silencing
complex) in the presence of mRNA to cause degradation resulting in attenuation
of
that mRNA's expression (Agrawal et al., 2003).
Current approaches to studying the function of genes, such as gene knockout
mice and dominant negatives, are often inefficient, and generally expensive,
and time-
consuming. RNA interference is proving to be a method of choice for the
analysis of a
large number of genes in a quick and relatively inexpensive manner. Although
transfection of synthetic siRNAs is an efficient method, the effects are often
transient
at best (Hannon G.J., 2002). Delivery of plasmids expressing short hairpin
RNAs by
stable transfection has been successful in allowing for the analysis of RNA
interference in longer-term studies (Brummelkamp et al., 2002; Elbashir et
al., 2001).
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I- Determination of knockdown effects on the proliferation of ovarian
cancer cell lines
In order to determine which ovarian cancer-specific genes participate in the
proliferation of ovarian cancer cells, an assay was developed using stably
transfected
cell lines that contain attenuated (i.e., knocked down) levels of the specific
gene being
investigated. Two human ovarian cancer cell lines derived from chemotherapy-
naïve
patients were utilized that have been previously characterized in terms of
their
morphology, tumorigenicity, and global expression profiles. In addition, these
analyses
revealed that these cell lines were excellent models for in vivo behavior of
ovarian
Kt tumors in humans (Provencher et al., 2000 and Samouelian et al., 2004).
These cell
lines are designated TOV-21G and TOV-112D.
The design and subcloning of individual shRNA expression cassettes and the
procedure utilized for the characterisation of each nucleotide sequence is
described
below. Selection of polynucleotides were chosen based on their upregulation in
ovarian tumors and the selective nature of their expression in these tumors
compared
to other tissues as described above. The design of shRNA sequences was
performed
using web-based software that is freely available to those skilled in the art
(Qiagen for
example). These chosen sequences, usually 19-mers, were included in two
complementary oligonucleotides that form the template for the shRNAs, i.e. the
19-nt
sense sequence, a 9-nt linker region (loop), the 19-nt antisense sequence
followed by
a 5-6 poly-T tract for termination of the RNA polymerase III. Appropriate
restriction
sites were inserted at the ends of these oligonucleotides to facilitate proper
positioning
of the inserts so that the transcriptional start point is at a precise
location downstream
of the hU6 promoter. The plasmid utilized in all RNA interference studies,
pSilencer 2.0
(SEQ. ID. NO. 101), was purchase from a commercial supplier (Ambion, Austin,
TX).
For each sequence selected, at least two different shRNA expression vectors
were
constructed to increase the chance of observing RNA interference.
TOV-21G or TOV-112D cells were seeded in 6-well plates in OSE
(Samouelian et al., 2004) containing 10% fetal bovine serum at a density of
600 000
cells/well, allowed to plate overnight and transfected with 1 pg of pSil-shRNA
plasmid
(Figure 53, sh-1 and sh-2) using the Fugene 6 reagent (Roche, Laval, QC).
After 16h
of incubation, fresh medium was added containing 2 pg/ml puromycin (Sigma, St.
Louis, Mo) to select for stable transfectants. Control cells were transfected
with a
control pSil (sh-scr (SEQ. ID. NO. 102) that contains a scrambled shRNA
sequence
that displays homology to no known human gene. After approximately 4-5 days,
pools
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and/or individual clones of cells were isolated and expanded for further
analyses. The
effectiveness of attenuation was verified in all shRNA cells lines. Total RNA
was
prepared by standard methods using TrizolTm reagent from cells grown in 6-well
plates
and expression of the target gene was determined by RT-PCR using gene-specific
primers. First strand cDNA was generated using Thermoscript (Invitrogen,
Burlington,
ON) and semi-quantitative PCR was performed by standard methods (Qiagen,
Mississauga, ON). 100% expression levels for a given gene was assigned to
those
found in the cell lines transfected with the control pSil plasmid (sh-scr).
Figure 52
shows representative results from the attenuation of two candidate genes, SEQ.
ID.
NO. 1 and SEQ. ID. NO. 3. When RT-PCR was performed using total RNA from the
control cell lines (Figure 52, pSil-scr), a single band of expected size was
observed.
When the total RNA from the cell line containing shRNAs to SEQ. ID. NO. 1
(0094)
(sh-1: SEQ. ID. NO. 103 and sh-2: SEQ. ID. NO. 104) or SEQ. ID. NO. 3 (0671)
(sh-1:
SEQ. ID. NO. 107 and sh-2: SEQ. ID. NO. 108) was amplified under identical
conditions, significant reduction in the levels of expression of these genes
were
observed. These results indicate that the shRNAs that were expressed in the
TOV-
21G stable transfectants were successful in attenuating the expression of
their target
genes. As a control for equal quantities of RNA in all reactions, the
expression of
glyceraldehyde-3-phosphate dehydrogenase (Figure 52, GAPDH) was monitored and
found to be expressed at equal levels in all samples used.
The proliferative ability of each shRNA-expressing cell line was determined
and compared to cells expressing the scrambled shRNA (control). Cell number
was
determined spectrophotometrically by MTT assay at 570 nm (Mosmann, 1983).
After
selection of stably shRNA expressing pools and expansion of the lines, 5 000
cells/well
of each cell lines was plated in 48-well plates in triplicate and incubated
for 4 days
under standard growth conditions. Representative data from 2 experiments SEM
is
displayed and experiments were typically repeated at least three times to
confirm the
results observed. Figure 53 shows representative results that were obtained
when the
proliferation assay was applied to stable TOV-21G cells lines. The cell number
after 4
days in the control cell line expressing the scrambled shRNA (Figure 53, sh
scr) was
arbitrarily set to 100%. TOV-21G cell lines containing shRNA against SEQ. ID.
NO. 1
(sh-1: SEQ. ID. NO. 103 and sh-2: SEQ. ID. NO. 104), SEQ. ID. NO. 3 (sh-1:
SEQ. ID.
NO. 107 and sh-2: SEQ. ID. NO. 108) and SEQ. ID. NO. 8 (0065) (sh-1: SEQ. ID.
NO.
117 and sh-2: SEQ. ID. NO. 118) exhibited less than 50% proliferation for at
least one
shRNA compared to the control cell line (Figure 53, sh-1 and sh-2 for each).
The
proliferation of TOV-21G cell lines containing shRNA against SEQ. ID. NO. 2
(0478)
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(sh-1: SEQ. ID. NO. 105 and sh-21 SEQ. ID. NO. 106) and SEQ. ID. NO. 7 (1096)
(sh-
1: SEQ. ID. NO. 115 and sh-2: SEQ. ID. NO. 116) was not affected to the same
extent
but significant inhibition of growth was still observed nevertheless. These
results
indicate that attenuation of these genes causes retardation in the growth of
this
ovarian cancer cell line. Several of these shRNA expression vectors were also
transfected into the TOV-112D cell line and similar results were obtained
(data not
shown). This suggested that these genes are important for proliferation of
ovarian
cancer cells.
The gene encoding the folate receptor 1, SEQ. ID. NO. 50 (0967A) (Figure
53, 0967A), which has been documented as being an important marker for ovarian
cancer (Leamon and Low, 2001), was also attenuated in TOV-21G cells, and
marked
growth inhibition was observed in the presence of the shRNAs (sh-1 SEQ. ID.
NO.
151 and sh-2: SEQ. ID. NO. 152). This gives credibility to the approach used
to
validate the genes presented in this patent and substantiated their functional
importance in the proliferation of ovarian cancer cells.
Table 1 below lists all of the genes tested and the average growth inhibition
(n=3-4) that was observed in TOV-21G cells. Differences of less than 20% (see
Table
1, <20) compared to the control cell lines represent cells where statistically
significant
reduction in proliferation was measured within a range of 5 ¨ 20%.
Table 1 ¨ List of genes tested in cell proliferation assay and results
Gene SEQ. ID. shRNA SEQ. ID. NO.
Alethia's Average
Growth
NO.
Gene inhibition
in TOV-
Code 21G cells
(%) (n=3-4)
Control SEQ. ID. NO. 102 ______________________________ 0
SEQ. ID. NO. 1 ¨ 0094 SEQ. ID. NOs. 103 and 104 47.9
SEQ. ID. NO. 2 0478 SEQ. ID. NOs. 105 and 106 41.7
SEQ. ID. NO. 3 0671 SEQ. ID. NOs. 107 and 108 65.7
SEQ. ID. NO. 4 0851 __ SEQ. ID. NOs. 109 and 110 21.5
SEQ. ID. NO. 5 0713 SEQ. ID. NOs. 111 and 112 42.3
SEQ. ID. NO. 6 1064 SEQ. ID. NOs. 113 and 114 28.9
SEQ. ID. NO. 8 0065 SEQ. ID. NOs. 117 and 118 32_5
SEQ. ID. NO. 10 0059 SEQ. ID. NOs. 121 and 122 - 52.4
SEQ. ID. NO. 11 0239 SEQ. ID. NOs. 123 and 124 22.8 jj
SEQ. ID. NO. 12 0291 SEQ. ID. NOs. 125 and 126 <20
SEQ. ID. NO. 13 ______ 0972 SEQ. ID. NOs. 127 and 128 <20
SEQ. ID. NO. 14- 0875 SEQ. ID. NOs. 129 and 130 <20
SEQ. ID. NO. 15 0420 SEQ. ID, NOs. 131 and 132 <20
SEQ. ID. NO. 16 0125 SEQ. ID. NOs. 133 and 134 <20
SEQ. ID. NO. 17 0531 SEQ. ID. NOs. 135 and 136 0
SEQ. ID. NO. 18 0967B SEQ. ID. NOs. 137 and 138 ¨ 0
SEQ. ID. NO. 19 0889 SEQ. ID. NOs. 139 and 140 <20
SEQ. ID. NO. 20 _ 0313 SEQ. ID. NOs. 141 and 142 <20
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Alethia's Average Growth
Gene SEQ. ID. shRNA SEQ. ID. NO.
Gene inhibition in TOV-
NO.
Code 21G cells
(%) (n=3-4)
SEQ. ID. NO. 21 1134 SEQ. ID. NOs. 143 and 144 <20
SEQ. ID. NO. 22 ¨ 0488 SEQ. ID. NOs. 145 and 146 j 0
SEQ. ID. NO. 23 0216 SEQ. ID. NOs. 147 and 148 <20
SEQ. ID. NO. 24 _ 0447 SEQ. ID. NOs. 149 and 150 0
SEQ. ID. NO. 50 0967A SEQ. ID. NOs. 151 and 152 47.4
J- A method for determining the requirement for specifc genes in the survival
of
ovarian cancer cells
As a means of complementing the growth inhibition data that was generated
__ with the stable TOV-21G cell lines, a colony survival assay was used to
determine the
requirement of the selected genes in the survival of the cancer cells. The
'colony
formation assay' or 'clonogenic assay' is a classical test to evaluate cell
growth after
treatment. The assay is widespread in oncological research areas where it is
used to
test the proliferating power of cancer cell lines after radiation and/or
treatment with
to __ anticancer agents. It was expected that the results obtained when
analyzing the genes
that were functionally important in ovarian cancer would correlate between the
growth
inhibition study and the colony survival assay.
TOV-21G cells were seeded in 12-well plates at a density of 50 000 cells/well
and transfected 24h later with 1 ug of pSil-shRNA vector, the same plasmids
used in
__ the previous assay. The next day, fresh medium was applied containing 2
pg/ml
puromycin and the selection of the cells was carried out for 3 days. The cells
were
washed and fresh medium without puromycin was added and growth continued for
another 5 days. To visualize the remaining colonies, the cells were washed in
PBS and
fixed and stained simultaneously in 1% crystal violet/10% ethanol in PBS for
15
__ minutes at room temperature. Following extensive washing in PBS, the dried
plates
were scanned for photographic analysis.
As shown in Figure 37 and as exemplified by SEQ. ID. NO. 1 (0094), SEQ.
ID. NO. 3 (0671), and SEQ. ID. NO. 9 (1313), the amount of TOV-21G-derived
colonies that survived correlated with the growth inhibition data. For
example, the
__ growth inhibition in the proliferation assay (Figure 53) and cell death in
the colony
assay (Figure 54) was greater in TOV-21G cells containing shRNA-2 compared to
shRNA-1 for SEQ. ID. NO. 1 (0094) (0094-sh2 stronger than 0094-sh1) and SEQ.
ID.
NO. 3 (0671) (0671-sh2 stronger than 0671-sh1) whereas, for SEQ. ID. NO. 9
(1313),
the 1313-sh1 was stronger than 1313-sh2. Similar convergence was observed with
__ several other genes that were analyzed using these two assays (data not
shown).
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Therefore, these results implied that a phenotypic manifestation in both
assays was
indicative of important genes that are functionally required in ovarian cancer
cells and
suggest that inhibition of the proteins they encode could be serve as
important targets
to develop new anticancer drugs.
K- A method for broadening the scope of intervention to other oncology
indications
One skilled in the art will recognize that the sequences described in this
invention have utilities in not only ovarian cancer, but these applications
can also be
I() expanded to other oncology indications where the genes are expressed.
To address
this, a PCR-based method was adapted to determine the expression pattern of
all
sequences described above in cancer cell lines isolated from nine types of
cancer. The
cancer types represented by the cell lines are leukemia, central nervous
sytem, breast,
colon, lung, melanoma, ovarian, prostate, and renal cancer (see Table C).
These RNA
i5 samples were obtained from the Developmental Therapeutics Program at the
NCl/NIH.
Using the same RAMP RNA samples that amplified from the total RNA samples
obtained from the NCI, 500 mg of RNA was converted to single-stranded cDNA
with
Thermoscript RT (Invitrogen, Burlington, ON) as described by the manufacturer.
The
cDNA reaction was diluted so that 1/200 of the reaction was used for each PCR
20 experiment. After trial PCR reactions with gene-specific primers
designed against each
SEQ. ID NOs. to be tested, the linear range of the reaction was determined and
applied to all samples, PCR was conducted in 96-well plates using Hot-Start
Taq
Polymerase from Qiagen (Mississauga, ON) in a DNA Engine Tetrad from MJ
Research. Half of the reaction mixture was loaded on a 1.2% agarose/ethidium
25 bromide gel and the amplicons visualized with UV light. To verify that
equal quantities
of RNA was used in each reaction, the level of RNA was monitored with GAPDH
expression.
Table C ¨ List of cancer cell lines from the NCI-60 panel
Cell line Cancer type
K-562 leukemia
MOLT-4 leukemia
¨ CCRF-CEMleukemia
RPMI-8226 leukemia
HL-60(TB) leukemia
SR leukemia
SF-268 ____________________________ CNS
SF-295 ____________________________ CNS
SF-539 CNS
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Cell line Cancer type
SNB-19 CNS
SNB-75 CNS
U251 CNS
BT-549 breast
HS 578T breast __
MCF7 breast
¨NCl/AD¨R-RES breast
MDA-MB-231 breast
MDA-MB-435 breast
T-47D breast
COLO 205 colon
HCC-2998 colon
HCT-116 colon
HCT-15 colon
HT29 colon
KM12 colon
SW-620 colon
A549/ATCC ______________________ non-small cell lung
EKVX non-small cell lung
HOP-62 non-small cell lung
HOP-92 non-small cell lung
¨NCI-H322M = non-small cell lung
_NCI-H226 non-small cell lung
NCI-H23 non-small cell lung_ __________________
NCI-H460 non-small cell lung
NCI-H522 non-small cell lung
LOX IMVI melanoma
M14 ____________________________ melanoma
MALME-3M melanoma
SK-MEL-2 melanoma
SK-MEL-28 ______________________ melanoma
SK-MEL-5 _______________________ melanoma
UACC-257 melanoma
UACC-62 melanoma
IGROV-1 ovarian
OVCAR-3 ovarian
OVCAR-4 ovarian
OVCAR-5 ovarian
OVCAR-8 ________________________ ovarian
SK-OV-3 ovarian
DU-145 prostate
PC-3 prostate
786-0 renal
A498 renal
ACHN renal
---
CAKI-1 renal
RXF-393 renal
SN-12C _________________________ renal
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Cell line Cancer type
TK-10 renal
U0-31 renal
One of skill in the art will readily recognize that orthologues for all
mammals
maybe identified and verified using well-established techniques in the art,
and that this
disclosure is in no way limited to one mammal. The term "mammal(s)" for
purposes of
this disclosure refers to any animal classified as a mammal, including humans,
domestic and farm animals, and zoo, sports, or pet animals, such as dogs,
cats, cattle,
horses, sheep, pigs, goats, rabbits, etc. Preferably, the mammal is human.
The sequences in the experiments discussed above are representative of the
NSEQ being claimed and in no way limit the scope of the invention. The
disclosure of
the roles of the NSEQs in proliferation of ovarian cancer cells satisfies a
need in the art
to better understand ovarian cancer disease, providing new compositions that
are
useful for the diagnosis, prognosis, treatment, prevention and evaluation of
therapies
for ovarian cancer and other cancers where said genes are expressed as well.
The art of genetic manipulation, molecular biology and pharmaceutical target
development have advanced considerably in the last two decades. It will be
readily
apparent to those skilled in the art that newly identified functions for
genetic sequences
and corresponding protein sequences allows those sequences, variants and
derivatives to be used directly or indirectly in real world applications for
the
development of research tools, diagnostic tools, therapies and treatments for
disorders
or disease states in which the genetic sequences have been implicated.
Although the present invention has been described herein above by way of
preferred embodiments thereof, it maybe modified, without departing from the
spirit
and nature of the subject invention as defined in the appended claims.
TABLE 2 ¨ Differentially expressed sequences found in malignant ovarian
cancer.
Nucleotide NCI31 Accession ORF Function
Set' 1101(12 No. Lnienc Number Nucleotide
4,1;ene Positions/
m boll( :env Polypeptide
11) sequence No.
SEQ ID NO. I STAR clone BX094904 Unknown Transcribed locus
but possibly 149-373 for
belonging to NM_021955 Hs.555871 guanine nucleotide
binding
cluster for protein (G protein),
Hs.555871 Hs.55587I encoding SEQ gamma transducing
ID NO.: 51 activity polypeptide 1
SEQ ID NO. 2 Hs.389724 NM 006820 242-1483 interferon-induced
protein
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Nucleotide NCR! Accession ORF Function
Sequence No. Unigene Number Nucleotide
#/(;ene Positions/
mind/(;ene Polypeptide
II) sequence No.
/1F144L encoding SEQ 44-like ; function
unknown
/ 10964 ID NO.: 52
SEQ ID NO. 3 1-Is.83465 NM 024501 224-1210 homeobox DI;
/ HOXD1 sequence-specific
/ 3231 encoding SEQ transcription factor that
is
ID NO.: 53 involved in differentiation
and limb development
SEQ ID NO. 4 1-1s.59761 NM 0010179 45-368 hypothetical protein
/ 1,0(792196 /0 L0C92196
/ 92196 encoding SEQ function unknown
ID NO.: 54
SEQ ID NO. 5 Hs.20315 NM 0010018 93-1529 interferon-induced
protein
/ IFITI 87 with tetratricopeptide
/ 3434 encoding SEQ repeats I ; function
ID NO.: 55 unknown
SEQ ID NO. 6 Hs.584238 NM 000170 151-3213 glycine
dehydrogenase
/ GLDC (decarboxylating; glycine
/ 2731 decarboxylase, glycine
encoding SEQ cleavage system protein
ID NO.: 56 P); mitochondrial glycine
cleavage system catalyzes
the degradation of glycine
SEQ ID NO. 7 Hs.302028 NM 022357 9-1550 dipeptidase 3;
proteolysis
/ DPEP3 and peptidolysis
/ 64180 encoding SEQ
ID NO.: 57
SEQ ID NO. 8 Hs.418367 NM 006681 106..630 neuromedin U
(NMU);
/ NMU neuropeptide signaling
/ 10874 encoding SEQ pathway, regulation of
ID NO.: 58 smooth muscle contraction
SEQ ID NO. 9 Ils.473163 NM001719 123-1418 bone
morphogenetic
/ BMP7 protein 7; cell growth
/ 655 and/or maintenance,
encoding SEQ growth, skeletal
ID NO.: 59 development, cytokine
activity, growth factor
activity
SEQ ID NO. 10 Hs.84113 NM 005192 62-700 cyclin-dependent kinase
/ CDKN3 inhibitor 3; a cyclin-
/ 1033 dependent kinase inhibitor,
encoding SEQ as well as,
ID NO.: 60 dephosphorylate CDK2
kinase which prevent the
activation of CDK2 kinase
SEQ ID NO. I l Hs.374378 NM 001826 10-249 CDC28 protein kinase
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Nucleotide NCIU Accession ORF
Function
Sequence No. rtiocrie Number
Nucleotide
Positions/
m bur( ;ene Polypeptide
11) sequence No.
/ CKSIB regulatory subunit 18
/ 1163 ; cell cycle, cytokinesis,
encoding SEQ cyclin-dependent
ID NO.: 61 protein kinase activity
SEQ ID NO. 12 Hs.30743 NM 006115 250-1779
preferentially expressed
/ PRAME antigen in melanoma
/ 23532 encoding SEQ ; function unknown
ID NO.: 62
-4 =
SEQ ID NO. 13 Hs.458485 NM 005101 76-573
ISG15 ubiquitin-like
ISGI5 modifier
/ 9636 encoding SEQ ; protein binding
ID NO.: 63
SEQ ID NO. 14 STAR clone A1922121.1 Novel genomic hit
SEQ ID NO. 15 Hs.29245 I NM 9010395 220-1311 hypothetical protein
/ FLJ33790 48 L0C283212
/ 283212 encoding SEQ ; function unknown
ID NO.: 64
SEQ ID NO. 16 Hs.334302 BG2 13598 Transcribed locus;
function unknown
SEQ ID NO. 17 I4s.546434 NM 024626 71-919 V-set domain containing T
/ VTCNI encoding SEQ cell activation inhibitor
I
/ 79679 ID NO.: 65 ; function unknown
SEQ ID NO. 18 Hs.73625 NM005733 28..2700 kinesin family member
/ KIF20A 20A
/ 10112 ; kinesin family,
encoding SEQ interacts with guanosine
ID NO.: 66 triphosphate (GTP)-
bound forms of RAB6A
and RAB6B
SEQ ID NO. 19 STAR clone AC117457 300-1007 novel genomic hit
but a possibly
of it belonging NM_005832 encoding SEQ potassium large
to cluster for Hs.478368 ID NO.: 67 conductance calcium-
Hs,478368 activated channel,
according to subfamily M, beta member
NCB1 2 for Hs.478368
SEQ ID NO. 20 Hs.603908 BU595315 = Transcribed
locus;
function unknown
SEQ ID NO. 21 Hs.632586 NM _001565 67-363 chemokine (C-X-C motif)
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Nucleotide NCIti Accession ORF Function
Sequence No. Unigene Number Nucleotide
4/Gene Positions/
mboli( Polypeptide
11) sequence No.
/ CXCLIO encoding SEQ ligand 10 ; chemokine
/ 3627 ID NO.: 68
SEQ ID NO. 22 STAR clone AL583809 Novel genomic hit
SEQ ID NO. 23 Hs.503368 NM 0010070 66-1469 asparagine-linked
/ ALG8 27 glycosylation 8 hornolog
/ 79053 (S. cerevisiae, alpha-1,3-
encoding SEQ glucosyltransferase)
ID NO.: 69 ; catalyzes the addition of
the second glucose residue
to the lipid-linked
oligosaccharide precursor
for N-linked glycosylation
of proteins
SEQ ID NO. 24 Hs.591801 NM_ 181337 738-992 kidney associated antigen
/ KAAG1 encoding SEQ i fumction unknown
ID NO.: 70
SEQ ID NO. 25 Hs.512599 NM 000077 213-683 cyclin-dependent kinase
/ CDKN2A encoding SEQ inhibitor 2A
/ 1029 ID NO.: 71 : cell cycle G I control
SEQ ID NO. 26 Hs.244580 NM012112 699-2942 TPX2, microtubule-
/ TPX2 associated, homolog
/ 22974 encoding SEQ (Xenopus laevis)
ID NO.: 72 ; involve in cell
proliferation
SEQ ID NO. 27 1-ls.93002 NM 007019 81-620 ubiquitin-conjugating
/ UBE2C enzyme E2C ; required for
/ 11065 the destruction of mitotic
encoding SEQ cyclins and for cell cycle
ID NO.: 73 progression
SEQ ID NO. 28 Hs.590469 AK092857 cDNA FLJ35538 fis, clone
SPLEN2002463; function
unknown
SEQ ID NO. 29 Hs.405662 NM 001878 138-554 cellular retinoic acid
CRABP2 binding protein 2
/ 1382 ; function unknown but
encoding SEQ may be involved in
ID NO.: 74 human skin growth and
, differentiation
SEQ ID NO. 30 Hs.26331 NM 033445 43-435 histone 3, 112a
/ H1ST3H2A encoding SEQ ; nucieosome formation
/92815 ID NO.: 75
SEQ ID NO. 31 Hs.591790 NM 003543 1-312 histone I. H4h
/ uusT1H4H encoding SEQ ; nucleosome formation
/ 8365 ID NO.: 76
SEQ ID NO. 32 Hs.93574 NM 006898 177-1475 homeobox 03; may play a
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.Nuelcotidc NCBI Accession ORF Function
Sequence Nu. Unigene Number Nucleotide
14;cene Positions/
Symbol/(;ene Polypeptide
II) sequence No.
TIOXD3 role in the regulation of
' 3232 encoding SEQ ID cell adhesion
processes
NO.: 77
SEQ ID NO. 33 Hs.525641 BC092518 61-1470 Immunoglobulin heavy
/ IGEIG1 constant gamma 1; may
/ 3500 play a role in immune
encoding SEQ ID response and antigen
NO.: 78 binding
SEQ ID NO. 34 Hs.592988 BC073793 10-717 Immunoglobulin kappa
/ 1GKC constant; may play a role
/3514 in immune response and
encoding SEQ ID antigen binding
NO.: 79
SEQ ID NO. 35 Hs.66762 AY683003 55-2727 Chromosome 10 ORF 38;
encoding SEQ ID unknown function
NO.: 80
SEQ ID NO. 36 Hs.373741 NM 178580 115-1299 Histocompatibility (minor)
/ SPP encoding SEQ ID 13; unknown function
/81502 NO.: 81
SEQ ID NO. 37 STAR clone AL157931 Novel genomic hit
SEQ ID NO. 38 Hs.128453 NM_ 001463 219-1196 Frizzled-related protein:
/ FRZB Wnt receptor signaling
2487 pathway, development.
encoding SEQ 11) skeletal, transmembrane
NO.: 82 receptor activity
SEQ ID NO. 39 Hs.239 NM_ 202003 266-2512 Forkhead box MI;
FOXM I transcriptional regulation
/ 2305
encoding SEQ ID
NO.: 83
SEQ ID NO. 40 Hs.46627 NM 152864 89-715 Chromosome 20 ORF 58;
unknown function
encoding SEQ ID
NO.: 84
SEQ ID NO. 41 STAR clone AK092936 Novel genomic hit
SEQ ID NO. 42 Gene ID 404550 BC009078 552-746 Chromosome 16 ORF 74;
unknown function
encoding SEQ ID
NO.: 85
SEQ ID NO. 43 lls.645513 NM_013277 225-2123 Rac GTPase activating
/ RACGAP I protein 1; electron
/ 29127 transport, intracellular
encoding SEQ ID signaling cascade; iron ion
NO.: 86 binding
SEQ ID NO. 44 Hs.645522 NM 018279 584-1594 Transmembrane protein
TMEM19 encoding SEQ ID 19; unknown function
55266 NO.: 87
SEQ ID NO. 45 STAR clone ACI09350 Novel genomic hit
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Nucleotide CIl Accession ORF Function
Sequence No. tnigene Number Nucleotide
g/Gene Positions/
!-; wind Gene Polypeptide
11) sequence No.
SEQ ID NO. 46 STAR clone AC104837 Novel genomic hit
SEQ ID NO. 47 STAR clone AC002060 Immunoglobulin lambda
variable group @,; may
play a role in antigen
bindin
SEQ ID NO. 48 Hs.200600 NM 005698 254-1297 Secretory carrier
/ SCAMP3 membrane protein 3; post-
/ 10067 Golgi transport, protein
encoding SEQ ID transport
NO.: 88
SEQ ID NO. 49 STAR clone AC068288
SEQ ID NO. 50 Hs.73769 NM 000802 26-799
folate receptor 1 (adult)
/ FOLR1 ;mediate delivery of 5-
/ 2348 methyltetrahydrofolate to
encoding SEQ ID the interior of cells
NO.: 89
SEQ ID NO. Ils.558314 NM 000096 251-3448 Ceruloplasmin; secreted
169 / CP protein; copper ion binding
/ 1356 or transport
encoding SEQ ID
NO.: 170
TABLE 3 ¨ List of additional sequences identification of plasmids,
oligonucleotides and shRNA oligonucleotides
Sett tienee name Dese ription
Identification
SEQ. ID. NO. 90 OGS 364 Oligo dTi I +Not 1+biotin
SEQ. ID. NO. 91 OGS 594 Oligonucleotide promoter tag l
SEQ. ID. NO. 92 OGS 595 Oligonucleotide promoter tag I
SEQ. ID. NO. 93 OGS 458 Oligonucleotide promoter tag 2
SEQ. ID. NO. 94 OGS 459 Oligonucleotide promoter tag 2
SEQ. ID. NO. 95 OGS 494 Primer for second-strand synthesis
from tag I
SEQ. ID. NO. 96 OGS 302 Primer for second-strand synthesis
from tag 2
SEQ. ID. NO. 97 OGS 62 1 Oligonucleotide promoter
SEQ. ID. NO. 98 OGS 622 Oligonucleotide promoter
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Sequence name 1)cscription
Identification
SEQ. ID. NO. 99 pCATRMAN Vector for STAR
SEQ. ID. NO. 100 p20 Vector for STAR
SEQ. ID. NO: 101 pSilenccr2.0 vector Vector for shRNA
SEQ. ID. NO: 102 sh-scr ' Control shRNA (Ambion)
SEQ. ID. NO: 103 sh-1 0094 shRNA sequence for SEQ. ID. NO. 1
SEQ. ID. NO: 104 sh-2 0094 shRNA sequence for SEQ. II). NO. 1
SEQ. ID. NO: 105 sh-1 0478 shRNA sequence for SEQ. ID. NO. 2
SEQ. ID. NO: 106 sh-2 0478 shRNA sequence for SEQ ID NO. 2
SEQ. ID. NO: 107 sh-1 0671 shRNA sequence for SEQ. ID. NO. 3
SEQ. ID. NO: 108 sh-2 0671 shRNA sequence for SEQ. ID. NO. 3
SEQ. ID. NO: 109 sh-1 0851 shRNA sequence for SEQ. ID. NO. 4
SEQ. ID. NO: 110 sh-2 0851 shRNA sequence for SEQ ID NO. 4
SEQ. ID. NO: 111 . sh-1 0713 shRNA sequence for SEQ. ID. NO. 5
SEQ. ID. NO: 112 sh-2 0713 shRNA sequence for SEQ. ID. NO. 5
SEQ. ID. NO: 113 sh-1 1064 shRNA sequence for SEQ. ID. NO. 5
SEQ. ID. NO: 114 sh-2 1064 shRNA sequence for SEQ ID NO. 6
SEQ. ID. NO: 115 sh- I 1096 ' shRNA sequence for SEQ. ID. NO. 7
SEQ. ID. NO: 116 sh-2 1096 shRNA sequence for SEQ. 11). NO. 7
SEQ. ID. NO: 117 sh-1 0065 shRNA sequence For SEQ. 11). NO. 8
SEQ. ID. NO: 118 sh-2 0065 shRNA sequence for SEQ ID NO. 8
SEQ. ID. NO: 119 sh-1 1313 shRNA sequence for SEQ. ID. NO. 9
SEQ. ID. NO: 120 sh-2 1313 shRNA sequence for SEQ ID NO. 9
SEQ. ID. NO: 121 sh-1 0059 shRNA sequence for SEQ. ID. NO. 10
SEQ. ID. NO: 122 sh-2 0059 shRNA sequence for SEQ ID NO. 10
SEQ. ID. NO: 123 sh-1 0239 ' shRNA sequence for SEQ. ID. NO. 11
SEQ. ID. NO: 124 sh-2 0239 shRNA sequence for SEQ ID NO, 11
SEQ. ID. NO: 125 sh-1 0291 shRNA sequence for SEQ. ID. NO. 12
SEQ. ID. NO: 126 sh-2 0291 shRNA sequence for SEQ ID NO. 12
SEQ. ID. NO: 127 sh-1 0972 shRNA sequence for SEQ. ID. NO. 13
SEQ. ID. NO: 128 sh-2 0972 shRNA sequence for SEQ ID NO. 13 '
SEQ. ID. NO: 129 sh-1 0875 shRNA sequence for SEQ. ID. NO. 14
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Sequence name Description
Identification
SEQ. ID. NO: 130 sh-2 0875 shRNA sequence for SEQ ID NO. 14
SEQ. ID. NO: 131 sh-1 0420 shRNA sequence for SEQ. ID. NO. 15
SEQ. ID. NO: 132 sh-2 0420 shRNA sequence for SEQ ID NO. 15
SEQ. Ill. NO: 133 sh-1 0125 shRNA sequence for SEQ. ID. NO. 16
SEQ. ID. NO: 134 sh-2 0125 shRNA sequence for SEQ ID NO. 16
SEQ. ID. NO: 135 sh-1 0531 shRNA sequence for SEQ. ID. NO. 17
SEQ. ID. NO: 136 sh-2 0531 shRNA sequence for SEQ ID NO. 17
SEQ. ID. NO: 137 sh-I 0967B shRNA sequence for SEQ. ID. NO. 18
SEQ. ID. NO: 138 sh-2 0967B shRNA sequence for SEQ 11) NO. 18
SEQ. ID. NO: 139 sh-1 0889 shRNA sequence for SEQ. 11). NO. 19
SEQ. ID. NO: 140 sh-2 0889 shRNA sequence for SEQ ID NO. 19
SEQ. ID. NO: 141 sh-1 0313 shRNA sequence for SEQ. ID. NO. 20
SEQ. ID. NO: 142 . sh-2 0313 shRNA sequence for SEQ ID NO. 20
SEQ. ID. NO: 143 sh-1 1134 shRNA sequence for SEQ. ID. NO. 21
SEQ. ID. NO: 144 sh-2 1134 shRNA sequence for SEQ ID NO. 21
SEQ. ID. NO: 145 sh-1 0488 shRNA sequence for SEQ. ID. NO. 22
SEQ. ID. NO: 146 sh-2 0488 shRNA sequence for SEQ ID NO. 22
SEQ. ID. NO: 147 sh-1 0216 shRNA sequence for SEQ. ID. NO. 23
SEQ. ID. NO: 148 sh-2 0216 shRNA sequence for SEQ ID NO. 23
SEQ. ID. NO: 149 sh-1 0447 shRNA sequence for SEQ. ID. NO. 24
SEQ. ID. NO: 150 sh-2 0447 shRNA sequence for SEQ ID NO. 24
SEQ. ID. NO: 151 sh-I 0967 shRNA sequence for SEQ. ID. NO. 50
SEQ. ID. NO: 152 sh-2 0967 shRNA sequence for SEQ II) NO. 50
SEQ. ID. NO: 153 OGS 1077 Forward primer for SEQ ID NO. 32
SEQ. ID. NO: 154 OGS 1078 Reverse primer for SEQ ID NO. 32
SEQ. ID. NO: 155 ¨ OGS 1141 Forward primer for SEQ ID NO. 35
SEQ. ID. NO: 156 - OGS 1142 Reverse primer for SEQ ID NO. 35
SEQ. ID. NO: 157 OGS 1202 Forward primer for SEQ ID NO. 38
SEQ. ID. NO: 158 OGS 1203 Reverse primer for SEQ ID NO. 38
SEQ. ID. NO: 159 OGS 1212 Forward primer for SEQ ID NO. 41
SEQ. ID. NO: 160 OGS 1213 Reverse primer for SEQ ID NO. 41
SEQ. ID. NO: 161 OGS 1171 Forward primer for SEQ ID NO. 44
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Sequence name Description
Identification
SEQ. ID. NO: 162 OGS 1172 Reverse primer for SEQ ID NO. 44
SEQ. ID. NO: 163 OGS 1175 Forward primer for SEQ ID NO. 45
SEQ. ID. NO: 164 OGS 1176 Reverse primer for SEQ ID NO. 45
SEQ. ID. NO: 165 OGS 1282 Forward primer for SEQ ID NO. 48
SEQ. ID. NO: 166 OGS 1283 Reverse primer for SEQ ID NO. 48
SEQ. ID. NO: 167 OGS 315 Forward primer for human GAPDH
SEQ. ID. NO: 168 OGS 316 Reverse primer for human GAPD11
SEQ. ID NO. 171 OGS 1136 Forward primer for SEQ ID NO. 1
SEQ. ID NO. 172 OGS 1044 Reverse primer for SEQ 11) NO. I
SEQ. ID NO. 173 OGS 1250 Forward primer for SEQ ID NO. 2
SEQ. ID NO. 174 OGS 1251 Reverse primer for SEQ ID NO. 2
SEQ. ID NO. 175 OGS 1049 Forward primer for SEQ ID NO. 3
SEQ. ID NO. 176 OGS 1050 Reverse primer for SEQ ID NO. 3
SEQ. ID NO. 177 OGS 1051 Forward primer for SEQ ID NO. 4
SEQ. ID NO. 178 OGS 1052 Reverse primer for SEQ ID NO. 4
SEQ. ID NO. 179 OGS 1252 Forward primer for SEQ ID NO. 5
SEQ. ID NO. 180 OGS 1253 Reverse primer for SEQ ID NO. 5
SEQ. ID NO. 181 OGS 1083 Forward primer for SEQ ID NO. 6
SEQ. It) NO. 182 OGS 1084 Reverse primer for SEQ ID NO. 6
SEQ. ID NO. 183 OGS 1053 Forward primer for SEQ ID NO. 7
SEQ. ID NO. 184 OGS 1054 Reverse primer for SEQ ID NO. 7
7
SEQ. ID NO. 185 OGS 1037 Forward primer for SEQ ID NO. 8
SEQ. ID NO. 186 OGS 1038 Reverse primer for SEQ ID NO. 8
SEQ. ID NO. 187 ()GS 1045 Forward primer for SEQ II) NO. 9
'SEQ. ID NO. 188 OGS 1046 Reverse primer for SEQ II) NO. 9
SEQ. ID NO. 189 OGS 1240 Forward primer for SEQ ID NO. 10
SEQ. ID NO. 190 OGS 1241 Reverse primer for SEQ ID NO. 10
- SEQ. ID NO. 191 OGS 1304 Forward primer for SEQ ID NO. i 1
SEQ. ID NO. 192 OGS 1305 Reverse primer for SEQ ID NO. 11
SEQ. ID NO. 193 OGS 1039 Forward primer for SEQ ID NO. 12 -
_
SEQ. ID NO. 194 OGS 1040 Reverse primer for SEQ ID NO. 12
SEQ. ID NO. 195 OGS 1095 Forward primer for SEQ ID NO. 13
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Sequence name Description
Identification
SEQ. ID NO. 196 OGS 1096 Reverse primer for SEQ ID NO. 13
SEQ. ID NO. 197 OGS 1284 Forward primer for SEQ ID NO. 15
SEQ. ID NO. 198 OGS 1285 Reverse primer for SEQ ID NO. 15 -
SEQ. ID NO. 199 OGS 1063 Forward primer for SEQ ID NO. 16
SEQ. ID NO. 200 OGS 1064 Reverse primer for SEQ ID NO. 16
SEQ. ID NO. 201 OGS 1031 Forward primer for SEQ ID NO. 17
SEQ. 1D NO. 202 OGS 1032 Reverse primer for SEQ ID NO. 17
SEQ. ID NO. 203 OGS 1308 Forward primer for SEQ ID NO. 18
SEQ. ID NO. 204 OGS 1309 Reverse primer for SEQ ID NO. 18
SEQ. ID NO. 205 OGS 1069 Forward primer for SEQ ID NO. 19
SEQ. ID NO. 206 OGS 1070 Reverse primer for SEQ ID NO. 19
SEQ. ID NO. 207 OGS 1061 Forward primer for SEQ ID NO. 20
SEQ. ID NO. 208 OGS 1062 Reverse primer for SEQ ID NO. 20
SEQ. ID NO. 209 = OGS 1097 Forward primer for SEQ ID NO. 21
SEQ. ID NO. 210 O= GS 1098 Reverse primer for SEQ ID NO. 21
SEQ. ID NO. 211 OGS 1075 Forward primer for SEQ ID NO. 22
SEQ. ID NO. 212 OGS 1076 Reverse primer for SEQ ID NO. 22
SEQ. ID NO. 213 OGS 1232 Forward primer for SEQ ID NO. 23
SEQ. ID NO. 214 = OGS 1233 Reverse primer for SEQ ID NO. 23
SEQ. ID NO. 215 OGS 1067 Forward primer for SEQ ID NO. 24
SEQ. ID NO. 216 OGS 1068 Reverse primer for SEQ ID NO. 24
SEQ. ID NO. 217 OGS 1099 Forward primer for SEQ ID NO. 25
SEQ. ID NO. 218 O= GS 1100 Reverse primer for SEQ ID NO. 25
SEQ. ID NO. 219 OGS 1246 Forward primer for SEQ NO. 26
SEQ. ID NO. 220 O= GS 1247 Reverse primer for SEQ ID NO. 26
SEQ. ID NO. 221 OGS 1093 Forward primer for SEQ ID NO. 27
SEQ. ID NO. 222 - O= GS 1094 Reverse primer for SEQ 11) NO. 27
SEQ. ID NO. 223 ()GS 1332 Forward primer for SEQ ID NO, 28
SEQ. ID NO. 224 - O= GS 1333 Reverse primcr for SEQ ID NO. 28
SEQ. ID NO. 225 OGS 1101 Forward primer for SEQ ID NO. 29
SEQ. ID NO. 226 OGS 1102 Reverse primer for SEQ ID NO. 29
SEQ. ID NO. 227 - O= GS 1300 Forward primer for SEQ ID NO. 30
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WO 2007/147265
PCT/CA2007/001134
Sequence n a in c I) esc ri ptio ii
Identifik=ation
SEQ. ID NO. 228 OGS 1301 Reverse primer
for SEQ ID NO. 30
SEQ. ID NO. 229 OGS 1302 Forward primer
for SEQ ID NO. 31
SEQ. ID NO. 230 OGS 1303 Reverse primer
for SEQ ID NO. 31
SEQ. ID NO. 231 OGS 1292 Forward primer
for SEQ ID NO. 33
SEQ. ID NO. 232 OGS 1294 Reverse primer
for SEQ ID NO. 33
SEQ. ID NO. 233 OGS 1242 Forward primer
for SEQ ID NO. 34
SEQ. ID NO. 234 OGS 1243 Reverse primer
for SEQ ID NO. 34
SEQ. ID NO. 235 OGS 1280 Forward primer
for SEQ ID NO. 36
SEQ. ID NO. 236 OGS 1281 ' Reverse primer
for SEQ ID NO. 36 -
SEQ. ID NO. 237 OGS 1159 Forward primer
for SEQ ID NO. 37
SEQ. ID NO. 238 OGS 1160 Reverse primer
for SEQ ID NO. 37
SEQ. ID NO. 239 OGS 1310 Forward primer
for SEQ ID NO. 39
SEQ. ID NO. 240 OGS 1311 Reverse primer
for SEQ ID NO. 39
SEQ. ID NO. 241 OGS 1155 Forward primer
for SEQ ID NO. 40
SEQ. ID NO. 242 OGS 1156 Reverse primer
for SEQ II) NO. 40
SEQ. ID NO. 243 OGS 1316 Forward primer
for SEQ ID NO. 42
SEQ. ID NO. 244 OGS 1317 Reverse primer
for SEQ 1D NO. 42
SEQ. ID NO. 245 OGS 1306 Forward primer
for SEQ ID NO. 43
SEQ. ID NO. 246 OGS 1307 Reverse primer
for SEQ ID NO. 43
SEQ. ID NO. 247 OGS 1286 Forward primer
for SEQ ID NO. 46
SEQ. ID NO. 248 OGS 1287 Reverse primer
for SEQ ID NO. 46
SEQ. ID NO. 249 OGS 1244 Forward primer
for SEQ ID NO. 47
SEQ. ID NO. 250 OGS 1245 Reverse primer
for SEQ ID Na 47
SEQ. ID NO. 251 OGS 1035 Forward primer
for SEQ ID NO. 50
SEQ. ID NO. 252 OGS 1036 Reverse primer
for SEQ ID NO. 50
SEQ. ID NO. 253 OGS 1248 Forward primer
for SEQ ID NO. 51
SEQ. ID NO. 254 ()GS 1249 Reverse primer
for SEQ ID NO. 51
115
CA 02826738 2013-09-05
WO 2007/147265
PCT/CA2007/001134
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DO
CGGCTGAGAGGCAGCGAACTCATCTTTGCCAGTACAGGAGCTTGTGCCGTGGCCCACAGCCCACAGCCCACAGCCATGG
G MGWDLTVKMLAGNEFQVSLSSSMSVSELKAQ
CTGGGACCTGACGGTGAAGATGCTGGCGGGCAACGAATTCCAGGTGTCCCTGAGCAGCTCCATGTCGGTGTCAGAGCTG
A ITQKIGVHAFQQRLAVHPSGVALQDRVPLAS
AGGCGCAGATCACCCAGAAGATTGGCGTGCACGCCTTCCAGCAGCGTCTGGCTGTCCACCCGAGCGGTGTGGCGCTGCA
G QGLGPGSTVLLVVDKCDEPLSILVRNNKGRS
GACAGGGTCCCCCTTGCCAGCCAGGGCCTGGGCCCTGGCAGCACGGTCCTGCTGGTGGTGGACAAATGCGACGAACCTC
T STYEVRLTQTVAHLKQQVSGLEGVQDDLFWL
GAGCATCCTGGTGAGGAATAACAAGGGCCGCAGCAGCACCTACGAGGTCCGGCTGACGCAGACCGTGGCCCACCTGAAG
C TFEGKPLEDQLPLGEYGLKPLSTVFMNLRLR ;7.!
AGCAAGTGAGCGGGCTGGAGGGTGTGCAGGACGACCTGTTCTGGCTGACCTTCGAGGGGAAGCCCCTGGAGGACCAGCT
C GGGTEPGGRS a
r.11
CCGCTGGGGGAGTACGGCCTCAAGCCCCTGAGCACCGTGTTCATGAATCTGCGCCTGCGGGGAGGCGGCACAGAGCCTG
G
CGGGCGGAGCTAAGGGCCTCCACCAGCATCCGAGCAGGATCAAGGGCCGGAAATAAAGGCTGTTGTAAGAGAAT
SEQ.ID NO.14
STAR clone:
P
TGCCCACTTGGCCCCTCCTTCCAAGGTGTACTTTACTTCCTTTCATTCCTGCTCTAATACTGTTTAGTACATTTTCACT
C
CTGCTCTAAAACTTGCCTCAGTCTCTCACTGTGCCTTATGCCCCTCAGCTGAATTCTTTCTTCTGAGCAGGCAGGAATT
G rs,
AGGTTGCTGCAGACGTGTATGCATTTGCCACCAGTAACATACTTTGGTGCCACATGACTAGGATATGTTCTCTAGTGCT
A rs,
ACATGTTCGTTTACAGTTCTTAGGACTCCCTGATA
i7.ao SEQ.ID NO.15
SEQ.ID NO.64 rs,
4- 0
GGCCGCCTGCGCGCCGCCAACAGCCTAGCGCTGCGCCGCGTGGCCGCCGCCTTCTCGCTGGCCCCGCTGGCCGAGCGCT
G MRWVRHDAPARRGQLRRLLEHVRLPLLAPAY
CGGCCGCGTCCTGCGTCAGGCCTTCGCCGAGGTGGCGCGCCACGCCGACTTCCTGGAGCTGGCGCCTGACGAGGTGGTG
G FLEKVEADELLQACGECRPLLLEARACFILG
O
CGCTGCTGGCGGACCCCGCGCTGGGCGTGGCGCGCGAGGAGGCCGTGTTTGAAGCGGCCATGCGCTGGGTGCGCCACGA
C REAGALRTRPRRFMDLAEVIVVIGGCDRKGL
GCGCCGGCCCGCCGCGGCCAGCTGCGACGCCTGCTGGAGCACGTGCGCCTGCCGCTACTGGCGCCCGCTTACTTCCTGG
A LKLPFADAYHPESQRWTPLPSLPGYTRSEFA cri
GAAGGTGGAGGCGGACGAGCTGCTGCAGGCCTGCGGCGAGTGCCGCCCGCTGCTGCTCGAGGCTCGCGCCTGCTTCATC
C ACALRNDVYVSGGHINSHDVWMFSSHLHTWI
TGGGCCGCGAGGCCGGTGCGCTGCGGACCCGGCCGCGGAGATTCATGGACCTAGCTGAAGTGATCGTGGTCATCGGCGG
T KVASLHKGRWRHKMAVVQGQLFAVGGFDGLR
TGCGACCGCAAAGGTCTCCTGAAGCTGCCCTTCGCCGATGCCTACCATCCAGAGAGCCAGCGGTGGACCCCACTGCCCA
G RLHSVERYDPFSNTWAAAAPLPEAVSSAAVA
CCTGCCCGGCTACACTCGCTCAGAATTCGCCGCCTGTGCTCTCCGCAATGACGTCTACGTCTCCGGAGGCCACATCAAC
A SCAGKLFVIGGARQGGVNTDKVQCFDPKEDR
GTCATGATGTGTGGATGTTTAGCTCCCATCTGCACACCTGGATCAAGGTAGCCTCTCTGCACAAGGGCAGGTGGAGGCA
C WSLRSPAPFSQRCLEAVSLEDTIYVMGGLMS
AAGATGGCAGTTGTGCAGGGGCAGCTGTTCGCGGTGGGTGGCTTCGACGGCCTGAGGCGCCTGCACAGCGTGGAGCGCT
A KIFTYDPGTDVWGEAAVLPSPVESCGVTVCD
CGACCCCTTCTCCAACACCTGGGCGGCCGCCGCGCCCCTCCCGGAGGCCGTGAGCTCGGCGGCGGTGGCGTCCTGCGCG
G GKVHILGGRDDRGESTDKVFTFDPSSGQVEV
GCAAGCTCTTCGTGATTGGGGGCGCCAGGCAGGGCGGCGTCAACACGGACAAGGTGCAGTGCTTTGACCCCAAGGAGGA
C QPSLQRCTSSHGCVTIIQSLGR
CGGTGGAGCCTGCGGTCACCAGCACCCTTCTCACAGCGGTGTCTCGAGGCTGTCTCCCTTGAGGACACCATCTATGTCA
T
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0040440U = WE-.
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N 0
0 P 4 0 E. E. E. 0+ 4 < 0 P E.E. E. E. 4 U 0 4 El E.
4 ,IG E.
0 E.UHE.<0 41 EPlc [LI 4 KGUOUKCHOHU4 PH C.11 HE.
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u) ril H
128
AGAAAATGAAGTTGATGTTTCAGTGAGACACCTGTATGCCAGAGAGTAAAAGGGATTATTGTGGATTCCTGAGAATTTT
C 0
TACATATGAAATCATGTCATCTATGAACAGAGATGGGACTGTCTCGTTGGAGGAAAACAAGCTCAGGGCTCCCACTGAT
T
CCACATTATGTTGCAAGCTCCTACGAAGCTCCCACTCA
=
SEQ.ID NO.23
SEQ.ID NO.69
f4
a
TTTCTCCGCATGCGCGGGATCCCGGATGTGGATCAAGTTGGTGGGAAGCGTGCGGTGCCGCAGCAATGGCGGCGCTCAC
A MAALTIATGTGNWFSALALGVTLLKCLLIPT
ATTGCCACGGGTACTGGCAATTGGTTTTCGGCTTTGGCGCTCGGGGTGACTCTTCTCAAATGCCTTCTCATCCCCACAT
A YHSTDFEVHRNWLAITHSLPISQWYYEATSE
CCATTCCACAGATTTTGAAGTACACCGAAACTGGCTTGCTATCACTCACAGTTTGCCAATATCACAGTGGTATTATGAG
G WTLDYPPFFAWFEYILSHVAKYFDQEMLNVH
CAACTTCAGAGTGGACGTTGGATTACCCCCCTTTCTTTGCATGGTTTGAGTATATCCTGTCACATGTTGCCAAATATTT
T NLNYSSSRTLLFQRFSVIFMDVLFVYAVREC
GATCAAGAAATGCTGAATGTCCATAATTTGAATTACTCCAGCTCAAGGACCTTACTTTTCCAGAGATTTTCCGTCATCT
T CKCIDGKKVGKELTEKPKFILSVLLLWNFGL
TATGGATGTACTCTTTGTGTATGCTGTCCGTGAGTGCTGTAAATGCATTGATGGAAAAAAAGTGGGTAAAGAACTTACA
G LIVDHIHFQYNGFLFGLMLLSIARLFQKRHM 0
AAAAGCCAAAATTTATTCTGTCGGTATTACTTCTGTGGAACTTCGGGTTATTAATTGTGGACCATATTCATTTTCAGTA
C EGAFLFAVLLHFKHIYLYVAPAYGVYLLRSY
AATGGCTTTTTATTTGGATTAATGCTACTCTCCATTGCACGATTATTTCAGAAAAGGCATATGGAAGGAGCATTTCTCT
T CFTANKPDGSIRWKSFSFVRVISLGLVVFLV
TGCTGTTCTCCTACATTTCAAGCATATCTACCTCTATGTAGCACCAGCTTATGGTGTATATCTGCTGCGATCCTACTGT
T SALSLGPFLALNQLPQVFSRLFPFKRGLCHA
TCACTGCAAATAAACCAGATGGGTCTATTCGATGGAAGAGTTTCAGCTTTGTTCGTGTTATTTCCCTGGGACTGGTTGT
T YWAPNFWALYNALDKVLSVIGLKLKFLDPNN
TTCTTAGTTTCTGCTCTTTCATTGGGTCCTTTCCTGGCCTTGAATCAGCTGCCTCAAGTCTTTTCCCGACTCTTTCCTT
T IPKASMTSGLVQQFQHTVLPSVTPLATLICT
CAAGAGGGGCCTCTGTCATGCATATTGGGCTCCAAACTTCTGGGCTTTGTACAATGCTTTGGACAAAGTGCTGTCTGTC
A LIAILPSIFCLWFKPQGPRGFLRCLTLCALS
TCGGTTTGAAATTGAAATTTCTTGATCCCAACAATATTCCCAAGGCCTCAATGACAAGTGGTTTGGTTCAGCAGTTCCA
A SFMFGWHVHEKAILLAILPMSLLSVGKAGDA
CACACAGTCCTTCCCTCAGTGACTCCCTTGGCAACCCTCATCTGCACACTGATTGCCATATTGCCCTCTATTTTCTGTC
T SIFLILTTTGHYSLFPLLFTAPELPIKILLM
TTGGTTTAAACCCCAAGGGCCCAGAGGCTTTCTCCGATGTCTAACTCTTTGTGCCTTGAGCTCCTTTATGTTTGGGTGG
C LLFTIYSISSLKTLFRRSFTLVAQAGVQWHD
O
ATGTTCATGAAAAAGCCATACTTCTAGCAATTCTCCCAATGAGCCTTTTGTCTGTGGGAAAAGCAGGAGACGCTTCGAT
T LS
TTTCTGATTCTGACCACAACAGGACATTATTCCCTCTTTCCTCTGCTCTTCACTGCACCAGAACTTCCCATTAAAATCT
T
ACTCATGTTACTATTCACCATATATAGTATTTCGTCACTGAAGACTTTATTCAGACGGAGTTTCACCCTTGTTGCCCAG
G
CTGGAGTGCAATGGCACGATCTCAGCTAACTGAAACCTCCGCCTCCCAGAAAAGAAAAACCTCTTTTTAATTGGATGGA
A
ACTTTCTACCTGCTTGGCCTGGGGCCTCTGGAAGTCTGCTGTGAATTTGTATTCCCTTTCACCTCCTGGAAGGTGAAGT
A
CCCCTTCATCCCTTTGTTACTAACCTCAGTGTATTGTGCAGTAGGCATCACATATGCTTGGTTCAAACTGTATGTTTCA
G
TATTGATTGACTCTGCTATTGGCAAGACAAAGAAACAATGAATAAAGGAACTGCTTAGATATG
V
SEQ.ID NO.24
SEQ.ID NO.70
CATTATGCTAACAGCATAAACATGCAGGGGGTGGGAGCAGGGTCACAAAAGTGAGTGTTGTCAATTCTACTTGGAATGA
A MDDDAAPRVEGVPVAVHKHALHDGLRQVAGP
AGGTTGAAATAATTTAAACAGTACGGGAAATGCAGAGCAATTTTCTCCTCTGGTGACAATATAGTGTCCAACACTTGGA
A GAAAAHLPRWPPPQLAASRREAPPLSQRPHR
GTGATTTTTAAGAATGTTTATTTAAATTAAAAGGATGGATTTCCAAGGAAAAAAAATAAGGAAAAGGAAAGAAAAAACT
G TQGAGSPPETNEKLTNPQVKEK =
-4
AACAGAAAACGCAAAAGTATCAGTTTGGTCACTAACCTTTGCAAGGATACCTTTTTATTTTCTTTAAGATTCCTGTTGT
T
TATACACAGATTTTAAGTTTACTCCTACTGCTGACCCAAGTGAAATTCCTTCTCCAGTCACAGTGTCAACCTCTACCCC
C
CAACTGCAACGAGAGTTTTGAGGGGCATCAATCACACCGAGAAGTCACAGCCCCTCAACCACTGAGGTGTGGGGGGGTA
G c":
GGATCTGCATTTCTTCATATCAACCCCACACTATAGGGCACCTAAATGGGTGGGCGGTGGGGGAGACCGACTCACTTGA
G 0
TTTCTTGAAGGCTTCCTGGCCTCCAGCCACGTAATTGCCCCCGCTCTGGATCTGGTCTAGCTTCCGGATTCGGTGGCCA
G
=
TCCGCGGGGTGTAGATGTTCCTGACGGCCCCAAAGGGTGCCTGAACGCCGCCGGTCACCTCCTTCAGGAAGACTTCGAA
G =
-4
CTGGACACCTTCTTCTCATGGATGACGACGCGGCGCCCCGCGTAGAAGGGGTCCCCGTTGCGGTACACAAGCACGCTCT
T
CACGACGGGCTGAGACAGGTGGCTGGACCTGGCGCTGCTGCCGCTCATCTTCCCCGCTGGCCGCCGCCTCAGCTCGCTG
C -4
t4
TTCGCGTCGGGAGGCACCTCCGCTGTCCCAGCGGCCTCACCGCACCCAGGGCGCGGGATCGCCTCCTGAAACGAACGAG
A a
AACTGACGAATCCACAGGTGAAAGAGAAGTAACGGCCGTGCGCCTAGGCGTCCACCCAGAGGAGACACTAGGAGCTTGC
A
GGACTCGGAGTAGACGCTCAAGTTTTTCACCGTGGCGTGCACAGCCAATCAGGACCCGCAGTGCGCGCACCACACCAGG
T
TCACCTGCTACGGGCAGAATCAAGGTGGACAGCTTCTGAGCAGGAGCCGGAAACGCGCGGGGCCTTCAAACAGGCACGC
C
TAGTGAGGGCAGGAGAGAGGAGGACGCACACACACACACACACACAAATATGGTGAAACCCAATTTCTTACATCATATC
T
GTGCTACCCTTTCCAAACAGCCTAATTTTTCTTTTCTCTCTTCTTGCACCTTTACCCCTCAATCTCCTGCTTCCTCCCA
A 0
ATTAAAGCAATTAAGTTCCTGG
P
rs,
SEQ.ID NO.25
SEQ.ID NO.71
rs,
CTCCTCCGAGCACTCGCTCACGGCGTCCCCTTGCCTGGAAAGATACCGCGGTCCCTCCAGAGGATTTGAGGGACAGGGT
C MEPAAGSSMEPSADWLATAAARGRVEEVRAL
GGAGGGGGCTCTTCCGCCAGCACCGGAGGAAGAAAGAGGAGGGGCTGGCTGGTCACCAGAGGGTGGGGCGGACCGCGTG
C LEAGALPNAPNSYGRRPIQVMMMGSARVAEL
CO4
GCTCGGCGGCTGCGGAGAGGGGGAGAGCAGGCAGCGGGCGGCGGGGAGCAGCATGGAGCCGGCGGCGGGGAGCAGCATG
G LLLHGAEPNCADPATLTRPVHDAAREGFLDT rs,
AGCCTTCGGCTGACTGGCTGGCCACGGCCGCGGCCCGGGGTCGGGTAGAGGAGGTGCGGGCGCTGCTGGAGGCGGGGGC
G LVVLHRAGARLDVRDAWGRLPVDLAEELGHR
CTGCCCAACGCACCGAATAGTTACGGTCGGAGGCCGATCCAGGTCATGATGATGGGCAGCGCCCGAGTGGCGGAGCTGC
T DVARYLRAAAGGTRGS
GCTGCTCCACGGCGCGGAGCCCAACTGCGCCGACCCCGCCACTCTCACCCGACCCGTGCACGACGCTGCCCGGGAGGGC
T NHARIDAAEGPSDIPD
TCCTGGACACGCTGGTGGTGCTGCACCGGGCCGGGGCGCGGCTGGACGTGCGCGATGCCTGGGGCCGTCTGCCCGTGGA
C
CTGGCTGAGGAGCTGGGCCATCGCGATGTCGCACGGTACCTGCGCGCGGCTGCGGGGGGCACCAGAGGCAGTAACCATG
C
CCGCATAGATGCCGCGGAAGGTCCCTCAGACATCCCCGATTGAAAGAACCAGAGAGGCTCTGAGAAACCTCGGGAAACT
T
AGATCATCAGTCACCGAAGGTCCTACAGGGCCACAACTGCCCCCGCCACAACCCACCCCGCTTTCGTAGTTTTCATTTA
G
AAAATAGAGCTTTTAAAAATGTCCTGCCTTTTAACGTAGATATATGCCTTCCCCCACTACCGTAAATGTCCATTTATAT
C
ATTTTTTATATATTCTTATAAAAATGTAAAAAAGAAAAACACCGCTTCTGCCTTTTCACTGTGTTGGAGTTTTCTGGAG
T
GAGCACTCACGCCCTAAGCGCACATTCATGTGGGCATTTCTTGCGAGCCTCGCAGCCTCCGGAAGCTGTCGACTTCATG
A
CAAGCATTTTGTGAACTAGGGAAGCTCAGGGGGGTTACTGGCTTCTCTTGAGTCACACTGCTAGCAAATGGCAGAACCA
A
AGCTCAAATAAAAATAAAATAATTTTCATTCATTCACTCAAAA
"0
¨3
SEQ.ID NO.26
SEQ.ID NO.72
=
AGTGGACTCACGCAGGCGCAGGAGACTACACTTCCCAGGAACTCCGGGCCGCGTTGTTCGCTGGTACCTCCTTCTGACT
T MSQVKSSYSYDAPSDFINFSSLDDEGDTQNI 9
CCGGTATTGCTGCGGTCTGTAGGGCCAATCGGGAGCCTGGAATTGCTTTCCCGGCGCTCTGATTGGTGCATTCGACTAG
G DSWFEEKANLENKLLGKNGTGGLFQGKTPLR
CTGCCTGGGTTCAAAATTTCAACGATACTGAATGAGTCCCGCGGCGGGTTGGCTCGCGCTTCGTTGTCAGATCTGAGGC
G KANLQQAIVTPLKPVDNTYYKEAEKENLVEQ =
AGGCTAGGTGAGCCGTGGGAAGAAAAGAGGGAGCAGCTAGGGCGCGGGTCTCCCTCCTCCCGGAGTTTGGAACGGCTGA
A SIPSNACSSLEVEAAISRKTPAQPQRRSLRL
-r
IVIDOILLVIDIDIVVIDIDVDVDDVDIDIDIDIDIDVIIIVVOVIDVOIDIDVOILIDIODOIVIVIIIIODOODDOV
DIV
DOVVVIII DDIIDVILDVIOIVD DIDVDVIIVDDIDIOIVVOIVD OVVISIVOVOIWIVVID
DeVVIDVIIVDVIIDI
III0IIVVOVO IIDIIIIDVD DIDVOSIOIDVELDVOILDVIVDOVVVOVOID3OIDD DDVIDDVIIIIDVDVD
DI DILLY
DDDWOVOVOODIDOODIIVDIDIIIDDIIDIODDVOOVIDDVVVDIDDVVIIDIDDIDDVOODIVVDDODDOODDVIVO
ODDIDOVOIDI DOVDIDWVIDDIDVD DIIVDD IDVDDIDIIVWD D DI DIVIOIDD
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C.)
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V
VVVVVDVDOVOVVDDVDOVDVIDVDVDDOOVODVDDII0VDOVODDMINDVDDDOVVOVIODVaIDDOIVVOVOVVOVO
C.;
SID DVS DVDOOD OVOVVVDDOVDVDWDVOIDVIDODI
OVaLLII3 DVVDOVDIIDVIDIDIIDDIDIIID DOOM/SID
a.
DLL DVDIVVVOVVOVOVVVOVVODDIIDIIIDODOVDOVaL
IS3DVDVVVD DIDDIDOOVVDISIDIIDOVDOV
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DVDVSIIIIVDID DaLID DDLIDVD
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VOL
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DOVILLI D DDIDDeSIVIIVDVDDOIOID D
VVDIDOVVVVIVOISVIODDDVVODVDDVOVVDDVaLVDVVDVVVDDVDDDSIVVODIIWOVDVVDVVOIIVDOIIIDD
DVD DVD IOVVDDDDIDDDVDIVVD DILL DVIDOVVOVVVVOID
DLLDIODDIDIIDIVOVVODIIIIVOVVIDVIDDDDII
0
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CA 02826738 2013-09-05
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GCCAAGTGCCATTTGGGGTCAGCATCCTCGTTTCAACACAGTGTGCTCTCTAGTTATCATGTGTAACGTGGGTTCTGTT
T 0
AGCGAAGATAGACTAGAGGACACGTTAGAGATGCCCTTCCCTGCTCCATCCCTGTGGCACCATTATGGTTTTTTGGCTG
T
=
TTGTATATACGGTTACGTATTAACTCTGGAATCCTATGGGCTCATCTTGCTCACCCAATGTGGGAGTCTGGTTTGAGCA
A =
GCGAGCTGAATGTGACTATTAAAAAAAATTTAAAAAAAAAAAAGAAAATCTTATGTACTATCCAAAAGTGCCAGAATGA
C 7.1
TCTTCTGTGCATTCTTCTTAAAGAGCTGCTTGGTTATCCAAAAATGAAAATTCAAAATAAACTCTGAAAAAAAAAAAAA
A
r.a
AAAAAA
SEQ.ID NO.36
SEQ.ID NO.81
CGTCACTTCCTGTTGCCTTAGGGGAACGTGGCTTTCCCTGCAGAGCCGGTGTCTCCGCCTGCGTCCCTGCTGCAGCAAC
C MDSALSDPHNGSAEAGGPTNSTTRPPSTPEG
GGAGCTGGAGTCGGATCCCGAACGCACCCTCGCCATGGACTCGGCCCTCAGCGATCCGCATAACGGCAGTGCCGAGGCA
G IALAYGSLLLMALLPIFFGALRSVRCARGKN
GCGGCCCCACCAACAGCACTACGCGGCCGCCTTCCACGCCCGAGGGCATCGCGCTGGCCTACGGCAGCCTCCTGCTCAT
G ASDMPETITSRDAARFPIIASCTLLGLYLFF 0
GCGCTGCTGCCCATCTTCTTCGGCGCCCTGCGCTCCGTACGCTGCGCCCGCGGCAAGAATGCTTCAGACATGCCTGAAA
C KIFSQEYINLLLSMYFFVLGILALSHTISPF
AATCACCAGCCGGGATGCCGCCCGCTTCCCCATCATCGCCAGCTGCACACTCTTGGGGCTCTACCTCTTTTTCAAAATA
T MNKFFPASFPNRQYQLLFTQGSGENKEEIIN
TCTCCCAGGAGTACATCAACCTCCTGCTGTCCATGTATTTCTTCGTGCTGGGAATCCTGGCCCTGTCCCACACCATCAG
C YEFDTKDLVCLGLSSIVGVWYLLRKHWIANN
CCCTTCATGAATAAGTTTTTTCCAGCCAGCTTTCCAAATCGACAGTACCAGCTGCTCTTCACACAGGGTTCTGGGGAAA
A LFGLAFSLNGVELLHLNNVSTGCILLGGLFI
CAAGGAAGAGATCATCAATTATGAATTTGACACCAAGGACCTGGTGTGCCTGGGCCTGAGCAGCATCGTTGGCGTCTGG
T YDVFWVFGTNVMVTVAKSFEAPIKLVFPQDL
ACCTGCTGAGGAAGCACTGGATTGCCAACAACCTTTTTGGCCTGGCCTTCTCCCTTAATGGAGTAGAGCTCCTGCACCT
C LEKGLEANNFAMLGLGDVVIPGIFIALLLRF
AACAATGTCAGCACTGGCTGCATCCTGCTGGGCGGACTCTTCATCTACGATGTCTTCTGGGTATTTGGCACCAATGTGA
T DISLKKNTHTYFYTSFAAYIFGLGLTIFIMH
GGTGACAGTGGCCAAGTCCTTCGAGGCACCAATAAAATTGGTGTTTCCCCAGGATCTGCTGGAGAAAGGCCTCGAAGCA
A IFKHAQPALLYLVPACIGFPVLVALAKGEVT
ACAACTTTGCCATGCTGGGACTTGGAGATGTCGTCATTCCAGGGATCTTCATTGCCTTGCTGCTGCGCTTTGACATCAG
C EMFSYESSAEILPHTPRLTHFPTVSGSPASL 0
TTGAAGAAGAATACCCACACCTACTTCTACACCAGCTTTGCAGCCTACATCTTCGGCCTGGGCCTTACCATCTTCATCA
T ADSMQQKLAGPRRRRPQNPSAM
GCACATCTTCAAGCATGCTCAGCCTGCCCTCCTATACCTGGTCCCCGCCTGCATCGGTTTTCCTGTCCTGGTGGCGCTG
G
CCAAGGGAGAAGTGACAGAGATGTTCAGCTACGAGTCCTCGGCGGAAATCCTGCCTCATACCCCGAGGCTCACCCACTT
C
CCCACAGTCTCGGGCTCCCCAGCCAGCCTGGCCGACTCCATGCAGCAGAAGCTAGCTGGCCCTCGCCGCCGGCGCCCGC
A
GAATCCCAGCGCCATGTAATGCCCAGCGGGTGCCCACCTGCCCGCTTCCCCCTACTGCCCCGGGGCCCAAGTTATGAGG
A
GTCAAATCCTAAGGATCCAGCGGCAGTGACAGAATCCAAAGAGGGAACAGAGGCATCAGCATCGAAGGGGCTGGAGAAG
A
AAGAGAAATGATGCAGCTGGTGCCCGAGCCTCTCAGGGCCAGACCAGACAGATGGGGGCTGGGCCCACACAGGCGTGCA
C
CGGTAGAGGGCACAGGAGGCCAAGGGCAGCTCCAGGACAGGGCAGGGGGCAGCAGGATACCTCCAGCCAGGCCTCTGTG
G
CCTCTGTTTCCTTCTCCCTTTCTTGGCCCTCCTCTGCTCCTCCCCACACCCTGCAGGCAAAAGAAACCCCCAGCTTCCC
C
CCTCCCCGGGAGCCAGGTGGGAAAAGTGGGTGTGATTTTTAGATTTTGTATTGTGGACTGATTTTGCCTCACATTAAAA
A
CTCATCCCATGGCCAGGGCGGGCCACTGTGCTCCTGGAAAAAAAAAA
=
SEQ.ID NO.37
=
=
STAR clone:
TGCCTCAGTCTCTCACTGTGCCTTATGCCCCTCAGCTGAATTCTTTCTTCTGAGCAGGCAGGAATTGAGGTTGCTGCAG
A
CGTGTATGCATTTGCCACCAGTAACATACTTTGGTGCCACATGACTAGGATATGTTCTCTAGTGCTAACATGTTCGTTT
A 0
CAGTTCTTAGGACTCCCTGATAGAAAAAAACACAAAAAAAAACACAAAAAAACCCAACCA
=
=
SEQ.ID NO.38
SEQ.ID NO.82
GTTGGGAAAGAGCAGCCTGGGCGGCAGGGGCGGTGGCTGGAGCTCGGTAAAGCTCGTGGGACCCCATTGGGGGAATTTG
A MVCGSPGGMLLLRAGLLALAALCLLRVPGAR
TCCAAGGAAGCGGTGATTGCCGGGGGAGGAGAAGCTCCCAGATCCTTGTGTCCACTTGCAGCGGGGGAGGCGGAGACGG
C AAACEPVRIPLCKSLPWNMTKMPNHLHHSTQ
GGAGCGGGCCTTTTGGCGTCCACTGCGCGGCTGCACCCTGCCCCATCCTGCCGGGATCATGGTCTGCGGCAGCCCGGGA
G ANAILAIEQFEGLLGTHCSPDLLFFLCAMYA
GGATGCTGCTGCTGCGGGCCGGGCTGCTTGCCCTGGCTGCTCTCTGCCTGCTCCGGGTGCCCGGGGCTCGGGCTGCAGC
C PICTIDFQHEPIKPCKSVCERARQGCEPILI
TGTGAGCCCGTCCGCATCCCCCTGTGCAAGTCCCTGCCCTGGAACATGACTAAGATGCCCAACCACCTGCACCACAGCA
C KYRHSWPENLACEELPVYDRGVCISPEAIVT
TCAGGCCAACGCCATCCTGGCCATCGAGCAGTTCGAAGGTCTGCTGGGCACCCACTGCAGCCCCGATCTGCTCTTCTTC
C ADGADFPMDSSNGNCRGASSERCKCKPIRAT
TCTGTGCCATGTACGCGCCCATCTGCACCATTGACTTCCAGCACGAGCCCATCAAGCCCTGTAAGTCTGTGTGCGAGCG
G QKTYFRNNYNYVIRAKVKEIKTKCHDVTAVV 0
GCCCGGCAGGGCTGTGAGCCCATACTCATCAAGTACCGCCACTCGTGGCCGGAGAACCTGGCCTGCGAGGAGCTGCCAG
T EVKEILKSSLVNIPRDTVNLYTSSGCLCPPL
GTACGACAGGGGCGTGTGCATCTCTCCCGAGGCCATCGTTACTGCGGACGGAGCTGATTTTCCTATGGATTCTAGTAAC
G NVNEEYIIMGYEDEERSRLLLVEGSIAEKWK
GAAACTGTAGAGGGGCAAGCAGTGAACGCTGTAAATGTAAGCCTATTAGAGCTACACAGAAGACCTATTTCCGGAACAA
T DRLGKKVKRWDMKLRHLGLSKSDSSNSDSTQ
TACAACTATGTCATTCGGGCTAAAGTTAAAGAGATAAAGACTAAGTGCCATGATGTGACTGCAGTAGTGGAGGTGAAGG
A SQKSGRNSNPRQARN
GATTCTAAAGTCCTCTCTGGTAAACATTCCACGGGACACTGTCAACCTCTATACCAGCTCTGGCTGCCTCTGCCCTCCA
C
C:
TTAATGTTAATGAGGAATATATCATCATGGGCTATGAAGATGAGGAACGTTCCAGATTACTCTTGGTGGAAGGCTCTAT
A
s.z
GCTGAGAAGTGGAAGGATCGACTCGGTAAAAAAGTTAAGCGCTGGGATATGAAGCTTCGTCATCTTGGACTCAGTAAAA
G
TGATTCTAGCAATAGTGATTCCACTCAGAGTCAGAAGTCTGGCAGGAACTCGAACCCCCGGCAAGCACGCAACTAAATC
C
CGAAATACAAAAAGTAACACAGTGGACTTCCTATTAAGACTTACTTGCATTGCTGGACTAGCAAAGGAAAATTGCACTA
T 0
TGCACATCATATTCTATTGTTTACTATAAAAATCATGTGATAACTGATTATTACTTCTGTTTCTCTTTTGGTTTCTGCT
T
O
CTCTCTTCTCTCAACCCCTTTGTAATGGTTTGGGGGCAGACTCTTAAGTATATTGTGAGTTTTCTATTTCACTAATCAT
G
AGAAAAACTGTTCTTTTGCAATAATAATAAATTAAACATGCTGTTACCAGAGCCTCTTTGCTGGAGTCTCCAGATGTTA
A
TTTACTTTCTGCACCCCAATTGGGAATGCAATATTGGATGAAAAGAGAGGTTTCTGGTATTCACAGAAAGCTAGATATG
C
CTTAAAACATACTCTGCCGATCTAATTACAGCCTTATTTTTGTATGCCTTTTGGGCATTCTCCTCATGCTTAGAAAGTT
C
CAAATGTTTATAAAGGTAAAATGGCAGTTTGAAGTCAAATGTCACATAGGCAAAGCAATCAAGCACCAGGAAGTGTTTA
T
GAGGAAACAACACCCAAGATGAATTATTTTTGAGACTGTCAGGAAGTAAAATAAATAGGAGCTTAAGAAAGAACATTTT
G
CCTGATTGAGAAGCACAACTGAAACCAGTAGCCGCTGGGGTGTTAATGGTAGCATTCTTCTTTTGGCAATACATTTGAT
T
TGTTCATGAATATATTAATCAGCATTAGAGAAATGAATTATAACTAGACATCTGCTGTTATCACCATAGTTTTGTTTAA
T V
TTGCTTCCTTTTAAATAAACCCATTGGTGAAAGTCCC
SEQ.ID NO.39
SEQ,ID NO.83 =
=
ACTGAAAGCTCCGGTGCCAGACCCCACCCCCGGCCCCGGCCCGGGACCCCCTCCCCTCCCGGGATCCCCCGGGGTTCCC
A MKTSPRRPLILKRRRLPLPVQNAPSETSEEE
CCCCGCCCGCACCGCCGGGGACCCGGCCGGTCCGGCGCGAGCCCCCGTCCGGGGCCCTGGCTCGGCCCCCAGGTTGGAG
G PKRSPAQQESNQAEASKEVAESNSCKFPAGI =
AGCCCGGAGCCCGCCTTCGGAGCTACGGCCTAACGGCGGCGGCGACTGCAGTCTGGAGGGTCCACACTTGTGATTCTCA
A KIINHPTMPNTQVVAIPNNANIHSIITALTA
-r
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SEQ.ID NO.95
0
GGGAGACGAAGACAGTAGA
SEQ.ID NO.96
t=J
a
GCCTGCACCAACAGTTAACA
SEQ.ID NO.97
GGAATTCTAATACGACTCACTATAGGGA
SEQ.ID NO.98
0
CGCGTCCCTATAGTGAGTCGTATTAGAATTC
SEQ.ID NO.99
TTTTCCCAGTCACGACGTTGTAAAACGACGGCCAGTGAATTCTAATACGACTCACTATAGGGAGATGGAGAAAAAAATC
A
CTGGACGCGTGGCGCGCCATTAATTAATGCGGCCGCTAGCTCGAGTGATAATAAGCGGATGAATGGCTGCAGGCATGCA
A
GCTTGGCGTAATCATGGTCATAGCTGTTTCCTGTGTGAAATTGTTATCCGCTCACAATTCCACACAACATACGAGCCGG
A
AGCATAAAGTGTAAAGCCTGGGGTGCCTAATGAGTGAGCTAACTCACATTAATTGCGTTGCGCTCACTGCCCGCTTTCC
A
GTCGGGAAACCTGTCGTGCCAGCTGCATTAATGAATCGGCCAACGCGCGGGGAGAGGCGGTTTGCGTATTGGGCGCTCT
T
CCGCTTCCTCGCTCACTGACTCGCTGCGCTCGGTCGTTCGGCTGCGGCGAGCGGTATCAGCTCACTCAAAGGCGGTAAT
A
CGGTTATCCACAGAATCAGGGGATAACGCAGGAAAGAACATGTGAGCAAAAGGCCAGCAAAAGGCCAGGAACCGTAAAA
A
GGCCGCGTTGCTGGCGTTTTTCCATAGGCTCCGCCCCCCTGACGAGCATCACAAAAATCGACGCTCAAGTCAGAGGTGG
C
GAAACCCGACAGGACTATAAAGATACCAGGCGTTTCCCCCTGGAAGCTCCCTCGTGCGCTCTCCTGTTCCGACCCTGCC
G
CTTACCGGATACCTGTCCGCCTTTCTCCCTTCGGGAAGCGTGGCGCTTTCTCAATGCTCACGCTGTAGGTATCTCAGTT
C
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T
ATCGTCTTGAGTCCAACCCGGTAAGACACGACTTATCGCCACTGGCAGCAGCCACTGGTAACAGGATTAGCAGAGCGAG
G
TATGTAGGCGGTGCTACAGAGTTCTTGAAGTGGTGGCCTAACTACGGCTACACTAGAAGGACAGTATTTGGTATCTGCG
C V
TCTGCTGAAGCCAGTTACCTTCGGAAAAAGAGTTGGTAGCTCTTGATCCGGCAAACAAACCACCGCTGGTAGCGGTGGT
T
TTTTTGTTTGCAAGCAGCAGATTACGCGCAGAAAAAAAGGATCTCAAGAAGATCCTTTGATCTTTTCTACGGGGTCTGA
C
GCTCAGTGGAACGAAAACTCACGTTAAGGGATTTTGGTCATGAGATTATCAAAAAGGATCTTCACCTAGATCCTTTTAA
A
TTAAAAATGAAGTTTTAAATCAATCTAAAGTATATATGAGTAAACTTGGTCTGACAGTTACCAATGCTTAATCAGTGAG
G
CACCTATCTCAGCGATCTGTCTATTTCGTTCATCCATAGTTGCCTGACTCCCCGTCGTGTAGATAACTACGATACGGGA
G -4
GGCTTACCATCTGGCCCCAGTGCTGCAATGATACCGCGAGACCCACGCTCACCGGCTCCAGATTTATCAGCAATAAACC
A
GCCAGCCGGAAGGGCCGAGCGCAGAAGTGGTCCTGCAACTTTATCCGCCTCCATCCAGTCTATTAATTGTTGCCGGGAA
G
CA 02826738 2013-09-05
WO 2007/147265
PCT/CA2007/001134
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0000H4H040H440E-.UUE. ......-...HH40400...,-.00C.)0...C)C.)
,
151
CTTTAAAAAACCTCCCACACCTCCCCCTGAACCTGAAACATAAAATGAATGCAATTGTTGTTGTTAACTTGTTTATTGC
A 0
GCTTATAATGGTTACAAATAAAGCAATAGCATCACAAATTTCACAAATAAAGCATTTTTTTCACTGCAATCTAAGAAAC
C
ATTATTATCATGACATTAACCTATAAAAATAGGCGTATCACGAGGCCCTTTCGTC
SEQ.ID NO.102
a
Sequence information not disclosed by Ambion
SEQ.ID NO.103
GTCAAGAAACCACACTTTA
SEQ.ID NO.104
GTGACATGGAACCCAGCGA
0
SEQ.ID NO.105
ACCGTGGCTGCTCGATAAA
SEQ.ID NO.106
GCCAGAGAGCACAGAAATA
SEQ.ID NO.107
O
GAGGAATGCCTCTAAGAAA
O
SEQ.ID NO.108
GGGAACGAGAAGGGCTTCT
SEQ.ID NO.109
AGCTGGAGGAATGAGAATT
SEQ.ID NO.110
AGGGCCAAAGCTTTCCATA
SEQ.ID NO.111
GGCAGGCTGTCCGCTTAAA
SEQ.ID NO.112
0
GGTCCTTAGGCACCCAGAT
=
=
SEQ.ID NO.113
4-
GCGGAGCCCAGGGAGAATA
SEQ.ID NO.114
GCCCGGATTGATGACATAT
SEQ.ID NO.115
GTGGAGGCTGAGTTTCCAT
0
SEQ.ID NO.116
o
GGATGTTAACCTGCGAAAT
SEQ.ID NO.117
GGTCAGCAGGGTTCATTTA
N.)
SEQ.ID NO.118
GCCTCAGGAACAAGATGAA
SEQ.ID NO.119
GCGCGAGATCCTCTCCATT
SEQ.ID NO.120
GCGCCAGAGGAGCGGGAAG
SEQ.ID NO.121
GCCGCCCAGTTCAATACAA
SEQ.ID NO.122
GAGCTTACAACCTGCCTTA
=
SEQ.ID NO.123
=
GGCGCCCACTACCCAAGAA
4-
SEQ.ID NO.124
0
=
GAGTCAGGGATGGGTCCAT
=
SEQ.ID NO.125
GGGCCAGTCTGTACTCATT
a
!It
SEQ.ID NO.126
GGGAATTCCATCTCCATAT
SEQ.ID NO.127
0
GGCGCAGATCACCCAGAAG
SEQ.ID NO.128
GAGCATCCTGGTGAGGAAT
SEQ.ID NO.129
o
GGTGCCACATGACTAGGAT
SEQ.ID NO.130
GCTGCAGACGTGTATGCATo
SEQ.ID NO.131
GCGGAGGCACTGGGCTTAT
SEQ_ID NO.132
GCCCGCTTACTTCCTGGAG
SEQ.ID NO.133
GCTCTGCTCAAGTTGGATA
SEQ.ID NO.134
=
GCTGCTGCCTTGCAGTTTG
=
SEQ.ID NO.135
c7,
0
GCCCTTACCTGATGCTAAA
=
=
SEQ.ID NO.136
GGCACCTACAAATGTTATA
a
SEQ.ID NO.137
tit
GAGGCCTGGAAGCTCCTAA
SEQ.ID NO.138
GCAGCTTCAGGAGGTTAAA
0
SEQ.ID NO.139
o
GCCGGACCTCTTCATCTTA
SEQ.ID NO.140
GCGTCCATCACGGAAACAT
SEQ.ID NO.141
GTCATCAGGACGTCCATTA
SEQ.ID NO.142
O
GACACGATCTACCCTCAAA
SEQ.ID NO.143
GGGCCATAGGGAAGCTTGA
SEQ.ID NO.144
V
GCCCACGTGTTGAGATCAA
SEQ.ID NO.145
GCTCCCACTGATTCCACAT
=
=
SEQ.ID NO.146
=
GCCAGAGAGTAAAAGGGAT
_______________________________________________________________________________
______________ 0
SEQ.ID NO.147
=
=
GGCATATGGAAGGAGCATT
4-
SEQ.ID NO.148
-4
GTGGTTTGGTTCAGCAGTT
SEQ.ID NO.149
GGCCTCCAGCCACGTAATT
0
SEQ.ID NO.150
o
GGCGCTGCTGCCGCTCATC
SEQ.ID NO.151
GGGCTGGAACTGGACTTCA
CP
SEQ.ID NO.152
GCCCATAAGGATGTTTCCT
SEQ.ID NO.153
O
GCGTCCGGGCCTGTCTTCAACCT
SEQ.ID NO.154
GCCCCACCCTCTACCCCACCACTA
SEQ.ID NO.155
GAGATCCTGATCAAGGTGCAGG
SEQ.ID NO.156
TGCACGCTCACAGCAGTCAGG
=
=
SEQ.ID NO.157
o
AACATGACTAAGATGCCCAACC
4-
SEQ.ID NO.158
r.)
=
AATCTCCTTCACCTCCACTACTG
=
SEQ.ID NO.159
=
k,4
AAGCATAGCCATAGGTGATTGG
a
fai
SEQ.ID NO.160
ACAGGTATCAGACAAGGGAGCAG
SEQ.ID NO.161
0
TTACGACCTATTTCTCCGTGG
SEQ.ID NO.162
AATGCAATAATTGGCCACTGC
SEQ.ID NO.163
ACACATCAAACTGCTTATCCAGG
SEQ.ID NO.164
O
ACTGATGTGAAAATGCACATCC
SEQ.ID NO.165
0
ATGGCTCATACAGCACTCAGG
SEQ.ID NO.166
GAACTGTCACTCCGGAAAGCCT
SEQ.ID NO.167
V
.TGAAGGTCGGAGTCAACGGATTTGGT
SEQ.ID NO.168
,CATGTGGGCCATGAGGTCCACCAC
=
=
SEQ.ID NO.169
SEQ.ID NO.170
ccetaatgectccaacaataactgttgactttttattttcagtcagagaagcctggcaaccaagaactgtttttttggt
g MKILILGIFLFLCSTPAWAKEKHYYIGIIET
4-
gtttacgagaact taactgaat tggaaaatatttgctttaatgaaacaatt
tactcttgtgcaacactaaattgtgtcaa TWDYASDHGEKKL I SVDTEHSNI YLQNGPDR
t caagcaaataaggaagaaagtct tatttataaaattgcctgctcctgatt ttacttcatt tct
tctcaggctccaagaa IGRLYKICALYLQYTDETERTTIEKPVWLGEL
ggggaaaaaaatgaagattttgatacttggtatttttctgtttttatgtagtaccccagcctgggcgaaagaaaagcat
t GPI IKAETGDKVYVHLKNLASRPYTFHSHGI
at tacattggaat tat tgaaacgact tgggattatgcc
tctgaccatggggaaaagaaacttatttctgttgacacggaa TYYKEHEGAIYPDNTTDFQRADDKVYPGEQY
catt ccaatatc tat ct t caaaatggcccagatagaat tgggagac tatataagaaggccc t
ttatcttcagtacacaga TYMLLATEEQSPGEGDGNCVTR I YESHIDAP
tgaaacctttaggacaactatagaaaaaccggtctggettgggtttt
taggccctattatcaaagctgaaactggagata KDIASGLI GPLI I CKKD S LDKEKEKHIDREF
aagtttatgtacact taaaaaacct tgcctctaggcce
tacacctttcattcacatggaataacttactataaggaacat VVMFSVVDENFSWYLEDNIKTYCSEPEKVDK
gagggggccatctaccctgataacaccacagat tt t caaagagcagatgacaaagtatat
ccaggagagcagtatacata DNEDFQESNRMYSVNGYTEGSLPGLSMCAED
catgttgcttgccactgaagaacaaagtcctggggaaggagatggcaattgtgtgactaggatttaccat
tcccacattg RVKWYLFGMGNEVDVHAAFFHGQALTNKNYR
atgctccaaaagatattgcctcaggactcatcggacctttaataatctgtaaaaaagattctctagataaagaaaaaga
a IDTINLFPATLFDAYMVAQNPGEWMLSCQNL
aaacatattgaccgagaatt tgtggtgatgttttctgtggtggatgaaaatt teagct
ggtacctagaagacaacattaa NHLKAGLQAFFQVQECNKSSSKDNIRGKHVR
aacctactgct cagaaccagagaaagttgacaaagacaacgaagact
tccaggagagtaacagaatgtattctgtgaatg HYTIAAEEIIWNYAFSGIDIFTKENLTAPGS
gatacact tttggaagtc
tcccaggactctccatgtgtgctgaagacagagtaaaatggtacctttttggtatgggtaat
DSAVFFEQGTTR/GGSYKKLVYREYTDASFT
ga.agttgatgtgcacgcagctt tctt tcacgggcaagcactgactaacaagaactaccgt at
tgacacaatcaacctctt NRKERGPEEEHLGILGPVIWAEVGDTIRVTF n.)
co
tcctgctaccctgtttgatgcttatatggtggcccagaaccctggagaatggatgctcagctgteagaatctaaaccat
c HNKGAYPLSIEPIGVRFNKNNEGTYYS PNYN n.)
tgaaagccggtttgcaagcctttttccaggtccaggagtgtaacaagtcttcatcaaaggataatatccgtgggaagca
t PQSRSVPPSASHVAPTETFTYEWTVPKEVGP
gttagacactactacat
tgccgctgaggaaatcatctggaactatgctccctctggtatagacatcttcactaaagaaaa
TNADPVCLAKMYYSAVDPTKD I FTGLI GPM1C
cc
co
cttaacagcacctggaagtgactcagcggtgtttt ttgaacaaggtaccacaagaattggaggctct
tataaaaagctgg ICKKGSLHANGRQKDVD KEEYLEPTVED ENE n.)
t t tat cgtgagtacacagat gcct ccttcacaaatcgaaaggagagaggccctgaagaagagcat
ettggcatcctgggt SLLLEDNIRMFTTAPDQVDKEDEDFQE SNKM
cctgtcatttgggcagaggtgggagacaccatcagagtaaccttccataacaaaggagcatatccectcagtattgagc
c HSMINIGEMYGNQPGLTMCKGDSVVW YLFSAGN
gattggggtgagat
tcaataagaacaacgagggcacatactattccccaaattacaacccccagagcagaagtgtgcctc EADVHG
YFSGNTYL WRGERRDTANLFPQTS
cttcagcctcccatg tag cacccacagaaacattcacctat gaatggactgtccccaaagaagtaggacccac
taatgca LTLHMWPDTEGTFNVECLTTDHYTGGMKQKY
gatcctgtgtgtctagctaagatgtattattctgctgtggatcccactaaagatatattcactgggcttattgggccaa
t TVNQCRRQSEDSTFYLGERTYY I AAVEVEWD
gaaaatatgcaagaaaggaagt t tacatgcaaatgggagacagaaagatgtagacaaggaat t c tat ttgt
t tcctacag YSPQREWEKELHHLQEQNVSNAFLDKGEFYI
tatttgatgagaatgagagtttactcctggaagataatat tagaatgtttacaactgcacctgat
caggtggataaggaa GSKYKKVVYRQYTDSTFRVPVERKAEEEHLG
gatgaagactttcaggaatctaataaaatgcactccatgaatggattcatgtatgggaatcagccgggtctcactatgt
g ILGPQLHADVGDKVKIIFKNMATRPYSIHAH
caaaggagat tcggtcgtgtggtacttat
tcagcgccggaaatgaggccgatgtacatggaatatacttttcaggaaaca GVQTESSTVTPTLPGETLTYVWKI
PERSGAG
catatctgtggagaggagaacggagagacacagcaaacetcttccctcaaacaagtc t t a cgc t
ccacatgtggcctgac TEDSACI PWAYYSTVDQVKDL YSGL I GPLIV
acagaggggacttttaatgttgaatgccttacaactgatcattacacaggcggcatgaagcaaaaatatactgtgaacc
a CRRPYLKVENPRRKLEFALLFLVEDENESWY
atgcaggcggcagtctgaggattecaccttctacctgggagagaggacatactatatcgcagcageggaggtggaatgg
g LDDNIKTYSDHPEKVNKDDEEFIESNKMHAI
attattccccacaaagggagtgggaaaaggagctgcatcat t
tacaagagcagaatgtttcaaatgcatttttagataag NGRMFGNLQGLTMHVGDEVNWYLMGMGNEID
ggagagttttacataggctcaaagtacaagaaagttgtgtatcggcagtatactgatagcacattccgtgttccagtgg
a LHTVHFHGHSFQYKHRGVYSSDVFDIFPGTY
gagaaaagctgaagaagaa ca ctgggaat t ctaggtccacaactt catgcagatgt
tggagacaaagtcaaaat tat ct QTLEMFPR TPGIWLLHCHVTDH I HAGMETTY
ttaaaaacatggccacaaggccc tactcaatacatgcccatggggtacaaacagagagtt
ctacagttactccaacatta TVLQNEDTKSG
ccaggtgaaactctcact tacgta tggaaaatcccagaaagatctggagc tggaacagaggattc
tgcttgtattccatg
ggettattattcaactgtggatcaagttaaggacctctacagtggattaattggccecctgattgtttgtcgaagacct
t
(4.
acttgaaagtattcaatcccagaaggaaactggaatttgcccttctgtttctagtttttgatgagaatgaatcttggta
c
ttagatgacaacatcaaaacatactctgatcaccccgagaaagtaaacaaagatgatgaggaattcatagaaagcaata
a 0
aatgcatgctattaatggaagaatgtttggaaacctacaaggcctcacaatgcacgtgggagatgaagtcaactggtat
c
tgatgggaatgggcaatgaaatagacttacacactgtacattttcacggccatagcttccaatacaagcacaggggagt
t =
tatagttctgatgtctttgacattttccctggaacataccaaaccctagaaatgtttccaagaacacctggaatttggt
t
actccactgccatgtgaccgaccacattcatgctggaatggaaaccacttacaccgttctacaaaatgaagacaccaaa
t
ctggctgaatgaaataaattggtgataagtggaaaaaagagaaaaaccaatgattcataacaatgtatgtgaaagtgta
a
aatagaatgttactttggaatgactataaacattaaaagaagactggaagcatacaactttgtacatttgtgggggaaa
a
ctattaattttttgcaaatggaaagatcaacagactatataatgatacatgactgacacttgtacactaggtaataaaa
c
tgattcatacagtctaatgatatcaccgctgttagggttttataaaactgcatttaaaaaaagatctatgaccagatat
t
ctcctgggtgctcctcaaaggaacactattaaggttcattgaaatgttttcaatcattgccttcccattgatecttcta
a
catgctgttgacatcacacctaatattcagagggaatgggcaaggtatgagggaaggaaataaaaaataaaataaataa
a
atagaatgacacaaatttgagttttgtgaacccctgaacagatggtcttaaggacgttatctggaactggagaaaagca
g 0
agttgagagacaattctatagattaaatectggtaaggacaaacattgccattagaagaaaagcttcaaaatagacctg
t
ggcagatgtcacatgagtagaatttctgcccagccttaactgcattcagaggataatatcaatgaactaaacttgaact
a
aaaattttttaaacaaaaagttataaatgaagacacatggttgtgaatacaatgatgtatttctttattttcacataca
c
tctagctaaaagagcaagagtacacatcaacaaaaatggaaacaaggctttggctgaaaaaaacatgcatttgacaaat
c
atgttaatagctagacaagaagaaagttagctttgtaaacttctacttcatttgattcagagaaacagagcatgagttt
t
cttaaaagtaacaagaaaa
o
SEQ.ID NO.171
o
GCTTAAAAGAGTCCTCCTGTGGC
SEQ.ID NO.172
TGGACATTGTTCTTAAAGTGTGG
SEQ.ID NO.173
AGGTTTTATGGCCACCGTCAG
SEQ.ID NO.174
ATCCTATACCGCTCGGTTATGC
SEQ.ID NO.175
>
GGGCGGCGGCTCTTTCCTCCTC
SEQ.ID N0.176
a
CA 02826738 2013-09-05
WO 2007/147265
PCT/CA2007/001134
4 o u U H
U 4 E-, E-t U E.
0 H H 4 0 0 H 0
4
< E-, 0 4F1 H 4 E.
0 CD U gt E. 0 U E. U 0
U H E. 0 E. 4 CD C./ H 0 U 0
E.
U H E. 0 0 U U 0 H E--,
u o u u E. P u Ft E-1 P u
4 o EA 4 o EA o u o 0 U 0
E. E. H H 0 4 0 4 4 0 E-. 0
Kt r- (..) co H cr) H o 0 H C.) cv 0 rn C.) ,I. 0 Ln
4 µ.1) H N Hi a)
C) N H N C.) cc, 4 co 0 co C) co C..7 co C.) co 0 co
U co (,) co
U =-f U H H H H H U H U H H H 0 H 0 H 0 H 0 H 0 H
0 = 0 = U = 0 . 0 . U = U = 4 . U = E. = 4 - 0 =
0 0 H 0 U 0 H 0 H 0 U 0 0 0 U 0 4 0 U 0 0 0 CD 0
0 Z 4 Z U Z U Z 0 Z 0 Z 0 Z 0 Z U Z 0 Z H Z 4 Z
0 0 0 H g E. 4 0 0 H u 0
u A 0 A 0 A H A E-. A A 0 A u A 0 A E. A 0 n 0 A
0 H H. H H H (,) H 0 H ect H Hi H 4 1¨i 0 I¨I 0 H
[¨t 1-4 F:4 H
4 ' U = H = 4 = 4 = 0 ' H . H. . 0 . 0 . C) . 0 =
H 0 < 0 H 0 U 0 H 0 U 0 U 0 Cl 0 4 0 U 0 Ci 0 0 0
U rrl U 41 C.) CA U al 4 41 4 53 4 C.4 4 t4 0 W H
41 U 41 U 41
0 rn KC cn 0 cn u co u cn pcc Ln F, co u cn u ul K4
cn C cn E--. cn
160
CA 02826738 2013-09-05
WO 2007/147265
PCT/CA2007/001134
,
(.9 4 u 4
E. 0 0 E. U
U 0 0 KC E. 0 U 0 U
E, U P 0 0 KC 0 0 < 0 KC
0
F 0 CJ 4 P 0 E. 0 E. H
0 U < 0 U E-4 4 E. 0 E. E.
U 0 E. 0 CD 0 0 U E. E. 0 H
0 E--, U U 0 4 4 0 4 0 H 0
4 4 U 4 E. 0 H E-. u H 0 El
0 0 E. 0 0 d P El 0 C.7 U H
0 al 4 o < ,-, U CN 4 SI .1* 0 1.11 4 Lo U r- P co
U N P o
E-, 03 St 0N 0 'M St ON H En 0 a) 4 cm 4 a) E-, cn
4 cc) E-, 01 U 0
E, ,--4 OH 0 ,--I H 0 ,4 H OH C) i-4 CD ,--i CD ,-
4 CD ,--1 0C'>
U= E.= U= U= 0= 4 = 0= U= U= H = H= E.=
E-40 PO 00 40 00 <0 00 <0 PO 4 0 HO 00
OZ 4 Z UZ HZ E.-1Z OZ UZ o=CZ HZ UZ UZ OZ
CJ E. U it H it 0 u 0 E. u 0
on CJ0 on ito 00 00 00 00 00 o 4n E.0
OH u v-i EH H E-41-1 OH 1-1 C.DH 41-I K4H OH
c_.) H
0 = o= H= U= H = U it= 0= O= u= o= o=
E-= 01 4 01 4 0 4 01 H 0 U 0 itCY U 01 4 0 4 0 E-, 01 C)01
E=14 OW OW OW 0L..1 OW OW OW Ur4 KCC4 UC4 UO1
CJ Ci) F, tr) E, VI 0 Cr) 0 ta 0) cil C./ c.c) 0 cc]
E. cil U Crl 4 01 0 Ci)
161
CA 02826738 2013-09-05
WO 2007/147265
PCT/CA2007/001134
_
1
1
O u F.
0 E. 0 u E.
4
E. E. 4
E. u u 0
0 0 0 E. E-, U 4 4
cn
0 0 E-= 0 0 E. 0 4 E. E. çCi 0
E. O 4 E. 4 u F 0 4 u Ci4
E. F 0 u 0 4 E. 0 0 4 0 E-
0 0 E. E. 0 0 0 4
E. 4 0
CJ F E. g4cl E. 0 0 0 u 0
E. E. u 0 0 E. E. 0 0 0
EH ( \I E-rn 0 =ri= On 0o E. s F:C co 0 cr% F 0
=--i 0 rv
00 00 00 Fo Oo 00 40 uo uo 0-1 -1 0-I
0 rs, E. EN 0 N U N CD N E.0 N 4 N E. (NI 0 oi 0 r1
N E-1 N
4 = 0 = 0 = H = a( . U = K.) = 0 = 0 = 04 = U = 0 =
C-) 0'04 0 00 HO 40 FO E0 00 00 00 (JO 00
OZ A=CZ UZ UZ OZ OZ 5Z OZ E.Z E-4Z UZ UZ
4 0 E 0 0 0 E. u u E.
c..in on UC1 00 on 00 R0 E.0 4 a) alPO
E.1-1 U H 4 H EH KC H EH U H C) H C) H H çCiH
4 ===1
4 = U= O= E.= c.J= (..)= O= O= F = U= = 0.
00 00 E0 ,rC 01 E0 0 0 00 00 00 00 E0 KC Co
Fc4 E. f=1 OW UW Ow C..)w Fril E.ra Prri 4 w 4 w
0 w
KC cf) KC u) Oí) 4 cr) H u) 4 cr) Fri] 4c.n Om CJ
U) OCT) 0C/)
162
CA 02826738 2013-09-05
WO 2007/147265
PCT/CA2007/001134
_
}
r
E-.
E.
0
4
U
U
0
* U 4
E. H
0 0 0 U E. 0
U 4 4 0 E. U U 4 4 U
< U 0 4
0 E.
H 0 0
U
U
H
4
0 E. U H E.
0 E. 1 U E. E-, E. 0 0 E. U
4 4 0 4 E-I c.., u E--i E. E. 4
E. 0 4 U 4 4 4 0 E. u U E.
O 0 0 4 U u U E-, 0 U 0 4
U E. 0 U U C.7 0 U E. U E. U
H cr. E. cf. 4 in E. ko U r-- 0 co 0 crl 4 o U ,-
1 0N U M
H.-4 0 r-i E. r-1 4 ,--t 4 ,-I 4 H H E. N UN
UN UN ON
UN UN ON pCN UN ON ON ON E-4N KCN 4N HN
O= g= E-.= U= U= 4. 0. H= 0= U= O= U=
40 00 0 HO 00 00 00 UO UO E-,0 E-.0 00
oz 0 Z sz 4 Z OZ UZ OZ UZ E-,Z UZ OZ OZ
O E-, U U 4 0 E-1 F U 0 U
00 00 c.90 00 UM 00 E.0 00 00 E.0 E-.0 00
PH 4H E.H. 0H 4H 0H 0H 4H 0H HH UH UH
0 = 0 = E. = 0 = U = 0 = E. = 0 = U = 0 = 0 = 0 =
4 0 4 0 U0 0 0 00 00 E. Cd 4 0 004 4 0 00 00
O[13 OW Q[1) 4 CA c..)C4 0 47 0 Ell OW 4 C4 4
41 HU) 4 ra
E. ri) F. o) Ht/) 0t/) 0(1) 0(/) Q(/) 4 CO E. Er]
0 ri) u ts) E-. En
163
CA 02826738 2013-09-05
WO 2007/147265 PCT/CA2007/001134
,
U
0
E-
E.
E.
U
4
U
1
0
E-,
H C.) U
U 4 0U
H 0 'C'5 U U U U
KC KC U 0 4 H 1-4 U
CJ C) 0 0 U 0 4 0 U E. E.
4 0 E. E. U U U E, E-. E--, 0 0
U 4 0 0 E. H U CD 4 4 H
U 0 4 U u 4 4 0 0 4 C) u
O u u 0 E. U u 0 0
U 4 u 4 E. 4 0 4 CD CD E.
CD 0 0 0 CD CD E. 0 0 U 4 U
U1-1) E-4 k43 ON 4 co Pa\ (Jo, 0,-1 E^. N U rn F4
.4µ 0 Ln u lO
UN E.N ON ON E.CV OM C.) ri 4 cr) E. (--. 0
fft g 0) E. m
O cv H cv 0 cv C7 cv (9(N 4 N ON ON ON E. cv
U (NI UN
0 = 0= 4 = U= H. PC= U= CD= 4 = F.= -.4= U=
Ci 0 4 0 00 E-0 E-.0 E.0 CD 0 CD 0 0 0 00 PC 0-04 0
Z 0Z Z E. Z U Z CJ Z 4 Z E. Z 0 Z E. Z CJ Z 4 Z
E-. 0 u 4 0 0 CJ E. E. 0 4 U
0 E4 n 4 A 40 E--. n 0 A H A F. A u n F-.0
4 H OH (.7 1-4 U H U H 0 I-4 E-1 H Kt H H E-4 1-4
g I-, 4 H
0 = 0 = 0 = 4 = 0 = 0 . Ci = Ei.
.= = U = (.) =
-40 00 00 40 40 40 00 E-I0 00 /0 00 40
gq rrl OW E.P1 C1c4 OW OW OW <al 4 ta r4 0r4
E--.41
E. cc) E. cil 0(/) (9 (/) U(/) 0(11 P cn E4 rn 0(11
0(1) 14 (ri 0(I)
164
CA 02826738 2013-09-05
WO 2007/147265
PCT/CA2007/001134
,
U 0 0
o o o o g
E, 0 4 0 E-1 0 U
4 E. 0 a E. 4 4 0 0 0 4
O c) 0 u u 0 E4 O 0 E-
O E-.
4 0 E, E-, U U 0 4 g
,4 E. 0 4 C) U E-, E. 0 U 0
u u E. 0 U 0 0 g g g E.
O u H E. E. E. U E. U E. u E.
U 0 0 4 4 4 E-4 4 E-, u iJ E.
Hr- H cc, E. o) 00 E. ,--. U N 0( 0 sr 4 Ln 4 vr)
r-- 4 op
Om (7 rn Um 0 .:t. 0'. E4'' 0 ,t, 4 .1. U .1.
E. .1. 0 ,t. ,..0 Tr.
4 N E--, cv Aq r9 E.N ON <N1 UN <N UN ON 0(N 0N
< = U = < = 0 ' F4 = U = E-. = E. = E. = E-, = E. = C' = ,
O 0 4 0 0 0 U 0 00 E.0 00 HO 00 U0 40 HO
UZ 0Z *Z 4Z UZ <4,Z OZ uZ E-.Z c)Z CJZ UZ
E. E. E. 4 U E. 0 E. u 4 u
CJa 00 HQ 40 HQ 40 Ua 00 E.0 OC) 00 Ha
F. 1--1 E.H OH E-tH OH OH r:41-1 OH FCH OH OH
H H
KC= U= U= U= 0 = U= U= 0= 0 . < = E. = E. =
O0' F-40 E. 0 HO 4 0 HO E. 0 E. 0 U 01 00 HO E-. 0
414 OW OW 443 E-141 OW OW ,441 F,[13 4[4 E.[=1 Cul
O(1) 4 cn .J c1 U co 4 u) E. c E. rn CJ cr) 0c1
-4 cn u cn u ci)
165
GTAGAGAGTTTATTTGGGCCAAG
SEQ.ID NO.249
0
=
CATCTATGGTAACTACAATCG
o
SEQ.ID NO.250
GTAGAAGTCACTGATCAGACAC
SEQ.ID NO.251
CTGCCTGCCAACCTTTCCATTTCT
SEQ.ID NO.252
,TGAGCAGCCACAGCAGCATTAGG
SEQ.ID NO.253
0
CACCTGATCAGGTGGATAAGG
SEQ.ID NO.254
TCCCAGGTAGAAGGTGGAATCC
1-3
r)
o
o
Cr>
o
CA 02826738 2013-09-05
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PCT/CA2007/001134
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