Note: Descriptions are shown in the official language in which they were submitted.
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TITLE OF THE INVENTION
NUCLEIC ACID MOLECULES COMPRISING THE PROMOTER FOR THE
PROSTATE CANCER MARKER PCA3 AND USES THEREOF
FIELD OF THE INVENTION
The present invention relates to prostate cancer. More
precisely, the present invention relates to nucleic acid molecules, which have
been isolated and characterized as the promoter for PCA3dd3, a new prostate
cancer antigen. This invention related also to different methods based on the
use
of recombinant DNA technology for prostate tumor diagnosis, prevention,
therapy
and the like.
BACKGROUND OF THE INVENTION
In the United States, according to statistical data published in
1999 by Landis et al. (CA Cancer J. Clin. 1999), prostate cancer is the most
commonly diagnosed cancer and the second leading cause of cancer death,
following lung cancer, in men. These results are not specific to the United
States
as they characterize all western countries. In addition incidence for this
cancer is
rising rapidly in most countries including low-risk populations, namely
populations
from Asian countries (Hsing et al., Int. J. Cancer 2000).
There is at present no effective treatment available for patients with
advanced
and/or hormone-insensitive prostate cancer. Therapeutic modalities for
advanced
prostate cancer are limited to drugs with considerable toxicity. As the
proliferation
rate is low in these cancers, many cytotoxic agents are ineffective (Millikan,
Semin. Oncol. 1999). Thus there is an urgent need to develop new approaches to
treat patients with progressive prostate cancer. For example, gene therapy can
be
done using prostate-specific gene promoters linked to genes that suppress
tumor
cell growth, induce apoptosis and/or kill tumor cells (Tanejaet a/., Cancer
Surv.
1995; and Boulikas, Anticancer Res. 1997). The promoter sequences responsible
for the prostate specific expression of several genes have been cloned and the
unraveling of their transcriptional
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regulation is ongoing. The PSA gene promoter has been most extensively studied
and revealed the existence of a proximal prostate-specific promoter with an
upstream prostate-specific enhancer. Both sequences are required for high,
androgen-regulated activation of PSA expression (Schuur et al., J. Biol. Chem.
1996; Cleutjens et al., Mol. Endocrinol. 1997a; Cleutjens et al., Mol.
Endocrinol.
1997b; Pang et al., Cancer Res. 1997; and Wei et al., Proc. Natl. Acad. Sci.
USA
1997). Using the PSA enhancer-promoter linked up to the HSV-tk gene, encoding
a prodrug-converting enzyme and delivered by the human adenovirus into
prostatic tumor cells growing subcutaneously in nude mice, it has been shown
that prostate tumor cell growth was significantly suppressed and that the life
span
of the animals was prolonged (Gotoh et a/., J. Urol. 1998; and Martiniello-
Wilks
et al., Hum. Gene Ther. 1998). This proof of principle opens the way for
application of promoter-based gene therapy for prostate cancer patients.
A new candidate marker for prostate cancer was discovered a
few years ago by differential display analysis intended to highlight genes
associated with prostate cancer development. This new gene was named PCA3
and also DD3 (Bussemakers, PCT/CA98/00346 1998, Schalken, Eur. Urol. 1998,
Bussemakers et al., Cancer Res. 1999a and Bussemakers, Eur. Urol. 1999b).
PCA3dd3 is located on chromosome 9 and more precisely to region 9q21-22. It
consists of four exons, which give rise, by both alternative splicing and
alternative
poly-adenylation, to differently sized transcripts. By RT-PCR, PCA3dd3
expression
was found to be limited to the prostate tissue and absent in all other tissues
tested, including testis, seminal vesicle, ovary, placenta and bladder. In
addition
northern blot analysis showed that PCA3ad3 is highly expressed in the vast
majority of prostate cancer examined (47 out of 50) whereas no or very low
expression is detected in BPH or normal prostate cells from the same patients.
Of note, there is at least a 20-fold over-expression of PCA3 dd3 in prostate
carcinoma in comparison to normal or BPH tissues. PCA3dd3 expression seems
to increase with tumor grade and is detected in metastatic lesions.
There thus remains a need to identify the promoter responsible
for regulation and expression of PCA3da3 There also remains a need to provide
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regulatory sequences which are prostate cancer tissue specific and to use such
sequences to diagnose, prevent or treat prostate cancer.
The present invention seeks to meet these and other needs.
DEPOSIT OF NUCLEOTIDE SEQUENCE
The nucleotide sequence forthe PCA3aa3 promoter region was
deposited into the GenBankT"" data base under the accession numberAF279290.
SUMMARY OF THE INVENTION
The invention concerns the identification of the promoter
sequence for PCA3dd3 gene. This gene is a new prostate cancer marker, which
overcomes many if not all of the drawbacks of the prostate marker of the prior
art.
The characterization of this promoter is a very useful tool for the
development of
new strategies in order to treat, for example, patients having defects,
diseases or
damages in prostatic cells and especially of patients with advanced prostate
cancer, and particularly androgen-insensitive prostate cancer. Having
identified
PCA3dd3 as a marker for prostate cancer, and having now identified the
promoter
thereof, the present invention provides the means to prevent the progression
of
the cancer into higher grades as well as to revert the cancerous state of the
prostate tumor.
The present invention further relates to the discovery and
characterization of the novel prostate specific promoter sequence of the
PCA3da3
gene, which enables a prostate tissue specific expression of a heterologous
sequence.
The invention in addition provides a recombinant nucleic acid
molecule comprising, 5' to 3', a prostate specific promoter effective to
initiate
transcription in a prostate host cell and a heterologous DNA sequence of
interest.
In one embodiment of the present invention, the prostate cancer specific
promoter is the PCA3dd3 promoter as defined in Figure 1, between position -460
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to +62. However, sequence polymorphisms may exist. In addition, sub-regions
and variants of this sequence may be used in accordance with the present
invention, provided that such sub-regions or variants retain their biological
activity
in enabling a prostate cancer-specific tissue expression of the sequences
operably linked thereto. Indeed, as exemplified hereinbelow, sub-regions of
this
-460 to +62 nucleic acid region still retain prostatic tissue promoter
activity.
The invention also provides a recombinant vector comprising
one of the above-described PCA3dd3 promoter sequences and a heterologous
sequence operably linked thereto.
The invention also provides a cell that contains an
above-described recombinant vector.
The invention further includes antisense nucleic acid
molecules or ribozymes specific to the PCAdd3 promoter sequences of the
present
invention, so as to enable a modulation of the expression of the PCA3dd3
promoter
and thus of the sequence operably linked thereto (e.g. the PCA3 coding
sequences). In one particular embodiment, this antisense comprises a sequence
which is complementary to the sequence spanning nucleotides -70 to -30 as
shown in Figure 1. Antisense sequences complementary to the other promoter
sequences of the present invention are also within the scope of the present
invention.
The invention further relates to a non-human organism that
contains an above-described recombinant vector. In particular, the invention
relates to a non-human organism containing a recombinant vector comprising a
PCA3dd3 promoter sequence operably linked to a heterologous sequence. In a
particular embodiment, the promoter sequence comprises a sequence as set forth
in Figure 1, between position -460 to +62, variants of parts thereof.
The invention provides, in general, isolated nucleic acid
molecules enabling a prostatic tissue specific expression of a heterologous
sequence and to variants or portions thereof, retaining their ability to
enable a
prostate tissue specific expression thereof.
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The invention also provides methods for therapeutic uses
involving all or part of a nucleic acid sequence encoding the PCA3da3 promoter
according to the present invention, variants or parts thereof, operably linked
to a
heterologous sequence.
Having identified and characterized the PCA3ad3 promoter and
having determined and characterized regions thereof which retain their tissue-
specific promoter expression enables the design of diagnostic methods to
determine the promoter activity of PCA3dd3 and hence the cancer-predisposing
or
cancer status of the sample from which the promoter activity is assessed. A
number of methods for determining a promoter activity are available and known
to a person of ordinary skill. A non-limiting example of such a diagnostic
method
to determine the promoter activity of the PCA3da3 promoter in a sample from a
patient can be performed by PCR analysis of the PCA3dd3 promoter using primers
which can distinguish between different methylation states of a sequence (i.e.
CpG; Jarrard et al. 1998, Cancer Res. 58:5310-5314). Such methods could
enable a diagnosis/prognosis of prostate cancer in a patient. Since sodium
bisulfite transforms non-methylated cytosine into thymidine (methylated
cytosine
are not changed into thymidine), a design of PCR primers which can distinguish
between a hypo- versus a hyper-methylated state of the promoter can be carried-
out by a person of ordinary skill. The hypo-methylated state of the promoter
(which correlate with an active state of the promoter) would indicate a
cancerous
state or predisposition thereto of the prostatic sample (e.g. the primer would
have
bound to a sequence in which the Cs would have been changed into Ts). Of
course, other methods of determining the activity of a promoter can be carried-
out.
The invention in addition relates to screening assays to identify
agents which modulate the PCA3dd3 promoter of the present invention comprising
a comparison of the level of expression thereof in the presence as opposed to
in
the absence of a candidate agent. Such assays, which can be adapted by a
person of ordinary skill, can be used to further dissect the structure
function of the
promoter sequence. One type of such assays is exemplified hereinbelow in order
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to more precisely define the sequences and/or boundaries of the PCA3ad3
promoter.
As taught herein, a number of sequences from the PCA3ad3
promoter are shown to retain the prostate-specific promoter activity (see
figure 7).
Thus, the present invention present a number of PCA3dd3 promoter sequences
which can be used to direct the expression of a chosen heterologous sequence.
Since, as well known in the art, a promoter is defined as a DNA region
involved
in the binding of RNA polymerase to initiate transcription, the PCA3dd3
promoter
sequences characterized herein should not be limited to the DNA sequence which
comprises the +2 to +62 region of Figure 1. Indeed, while most of the
constructs
used to characterize the promoter sequences included the +2 to +62 region, and
hence the mRNA start site and beyond (all part of exon 1), it should be clear
to
the person of ordinary skill that the promoter sequences may end at the -1
position (or +1 position if the transcription start site is included) as shown
in Figure
1. Thus, non-limiting examples of the PCA3ad3 promoter sequences of the
present
invention include a PCA3dd3 sequence comprising, with reference to Figure 1,
nucleotides -89 to -1, -89 to +1, -89 to +62; -152 to -1, -152 to +1, -152 to
+62;
-254 to -1, -254 to +1, -254 to +62; -366 to -1, -366 to +1, -366 to +62; or -
433
to -1, -433 to +1, -433 to +62. Of course, SEQ ID NO: 1 is also within the
scope
of the present invention as well as the PCA3dd3 sequence spanning -460 to -1,
-460 to +1 or -460 to +62.
In order to provide a clear and consistent understanding of
terms used in the present description, a number of definitions are provided
hereinbelow.
As used herein, the terminology "non-malignant prostate or
status" is meant to cover a non-cancerous prostatic state. Thus, these
terminologies are meant to include a normal status as well as a benign
prostatic
status (such as BPH, for example).
The terminology "therapeutic sequence" refers to a DNA
sequence which indirectly or directly has an effect on the host cell in which
it is
expressed. Preferably, this therapeutic sequence encodes an amino acid
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sequence encoding a functional protein which is capable of displaying a
therapeutic effect in the host cell in which the functional promoter activity
of the
PCA3dd3 promoter enables expression of the therapeutic protein and the
therapeutic effect of this protein is effected directly, or indirectly.
The terminology "PCA3dd3 promoter" refers to a sequence from
the PCA3da3 promoter as shown in Figure 1 or Figure 5, or fragments or
derivatives thereof, which retain their prostate tissue specific promoter
activity.
The terminology "enhancer element" is meant to define a
sequence which enhances the activity of a promoter (i.e. increases the rate of
transcription of a sequence downstream of the promoter) which, as opposed to
a promoter, does not possess promoter activity, and which can usually function
irrespective of its location with respect to the promoter (i.e. upstream, or
downstream of the promoter). In one embodiment, the isolated nucleic acid
having promoter activity, as taught herein, may be combined with an enhancer
element, so as to increase the transcriptional activity of the PCA3da3
promoter.
Enhancer elements are well-known in the art. Non-limiting examples of enhancer
elements (or parts thereof) which could be used to increase the promoter
activity
of the PCA3da3 promoter include enhancer elements found in retroviral LTRs
(e.g.
MMTV LTR), or SV40 enhancer sequences and the like.
As well-known in the art, having identified sequences having
prostate tissue specific promoter activity, as well as important regions of
the
PCA3dd3 promoter (e.g. NF-1 box and E-box), a person skilled in the art could
modify the promoter in order to further increase its transcriptional activity.
For
example, (1) multiplying the number of sequences binding prostate cell- and
especially prostate cancer cells-specific or regulatory proteins or factors
which
bind the different elements boxed in Figure 1; (2) adding one or more
enhancers,
could significantly enhance the strength (i.e. the transcriptional activity)
of the
PCA3aa3 promoter. Of course, such modifications should retain the prostate
tissue-specific promoter activity of the PCA3ad3 promoter sequences.
Numerous therapeutic genes are well-known in the art. A non-
limiting list thereof can be found, for example, in US. Patent 5,919,652.
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Therapeutically genes include, without being limited thereto, suicide genes,
which
when produced, will kill the host cell in which it is expressed. Suicide genes
include, for example, enzymes, toxins, tumor suppressor genes and oncogenes.
While the primary purpose of the suicide gene is to kill the host cell in
which it is
expressed, suicide gene could also inhibit the growth of the host cell. It
will be
understood that the inhibitory or killing effect of the suicide gene can be
direct or
indirect.
Non-limiting examples of therapeutic sequences which can be
used in accordance of the present invention include TK, suitable oncogenes and
tumor suppressor genes such as Ras, p53, Rb, Wilm's tumor gene, toxins such
as diphteria toxin, bacterial toxins, tumor necrosis factors, interferons and
the like.
Numerous vectors can be used in accordance with the present
invention in order to express, through the influence of the PCA3dd3 promoter,
a
chosen heterologous sequence, and more particularly, a therapeutic sequence.
Such vectors are very well-known in the art. Non-limiting examples thereof
include: adenovirus-based vectors, retroviral vectors, adenoassociated viral
vectors (AAV), other human and animal viruses, SV40 and HSV-1 (see U.S.P.
5,919,652 for a more extensive list of non-limiting vectors which could be
used in
accordance with the present invention).
Nucleotide sequences are presented herein by single strand, in
the 5' to 3' direction, from left to right, using the one letter nucleotide
symbols as
commonly used in the art and in accordance with the recommendations of the
IUPAC-IUB Biochemical Nomenclature Commission.
Unless defined otherwise, the scientific and technological terms
and nomenclature used herein have the same meaning as commonly understood
by a person of ordinary skill to which this invention pertains. Generally, the
procedures for cell cultures, infection, molecular biology methods and the
like are
common methods used in the art. Such standard techniques can be found in
reference manuals such as for example Sambrook et al. (1989, Molecular Cloning
-A Laboratory Manual, Cold Spring Harbor Laboratories) and Ausubel et al.
(1994,
Current Protocols in Molecular Biology, Wiley, New York).
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The present description refers to a number of routinely used
recombinant DNA (rDNA) technology terms. Nevertheless, definitions of selected
examples of such rDNA terms are provided for clarity and consistency.
As used herein, "nucleic acid molecule", refers to a polymer of
nucleotides. Non-limiting examples thereof include DNA (i.e. genomic DNA,
cDNA) and RNA molecules (i.e. mRNA) as well as chimeras thereof. The nucleic
acid molecule can be obtained by cloning techniques or synthesized. DNA can be
double-stranded or single-stranded (coding strand or non-coding strand
[antisense]).
The term "recombinant DNA" as known in the art refers to a
DNA molecule resulting from the joining of DNA segments. This is often
referred
to as genetic engineering.
The term "DNA segment", is used herein, to refer to a DNA
molecule comprising a linear stretch or sequence of nucleotides. This sequence
when read in accordance with the genetic code, can encode a linear stretch or
sequence of amino acids which can be referred to as a polypeptide, protein,
protein fragment and the like.
The terminology "amplification pair" refers herein to a pair of
oligonucleotides (oligos) of the present invention, which are selected to be
used
together in amplifying a selected nucleic acid sequence by one of a number of
types of amplification processes, preferably a polymerase chain reaction.
Other
types of amplification processes include ligase chain reaction, strand
displacement amplification, or nucleic acid sequence-based amplification, as
explained in greater detail below. As commonly known in the art, the oligos
are
designed to bind to a complementary sequence under selected conditions.
The nucleic acid (i.e. DNA or RNA) for practicing the present
invention may be obtained according to well known methods.
Oligonucleotide probes or primers of the present invention may
be of any suitable length, depending on the particular assay format and the
particular needs and targeted genomes employed. In general, the
oligonucleotide
probes or primers are at least 12 nucleotides in length, preferably between 15
and
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24 molecules, and they may be adapted to be especially suited to a chosen
nucleic acid amplification system. As commonly known in the art, the
oligonucleotide probes and primers can be designed by taking into
consideration
the melting point of hydrizidation thereof with its targeted sequence (see
below
and in Sambrook et al., 1989, Molecular Cloning -A Laboratory Manual, 2nd
Edition, CSH Laboratories; Ausubel et al., 1989, in Current Protocols in
Molecular
Biology, John Wiley & Sons Inc., N.Y.).
The term "oligonucleotide" or "DNA" molecule or sequence
refers to a molecule comprised in general of the deoxyribonucleotides adenine
(A), guanine (G), thymine (T) and/or cytosine (C) (modified or rare
nucleotides can
also be used). When in a double-stranded form, it can comprise or include a
"regulatory element" according to the present invention, as the term is
defined
herein. The term "oligonucleotide" or "DNA" can be found in linear DNA
molecules or fragments, viruses, plasmids, vectors, chromosomes or
synthetically
derived DNA. As used herein, particular double-stranded DNA sequences may
be described according to the normal convention of giving only the sequence in
the 5' to 3' direction. It will also be recognized that "oligonucleotide" can
be (and
usually are) in a single-stranded form.
Probes of the invention can be utilized with naturally occurring
sugar-phosphate backbones as well as modified backbones including
phosphorothioates, dithionates, alkyl phosphonates and a-nucleotides and the
like. Modified sugar-phosphate backbones are generally taught by Miller, 1988,
Ann. Reports Med. Chem. 23:295 and Moran et al., 1987, Nucleic acid molecule.
Acids Res., 14:5019. Probes of the invention can be constructed of either
ribonucleic acid (RNA) or deoxyribonucleic acid (DNA), and preferably of DNA.
The types of detection methods in which probes can be used
include Southern blots (DNA detection), dot or slot blots (DNA, RNA), and
Northern blots (RNA detection). Although less preferred, labeled proteins
could
also be used to detect a particular nucleic acid sequence to which it binds.
More
recently, PNAs have been described (Nielsen et al. 1999, Current Opin.
Biotechnol. 10:71-75). PNAs could also be used to detect the mRNAs of the
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present invention. Other detection methods include kits containing probes on a
dipstick setup and the like.
Although the present invention is not specifically dependent on
the use of a label for the detection of a particular nucleic acid sequence,
such a
label might be beneficial, by increasing the sensitivity of the detection.
Furthermore, it enables automation. Probes can be labeled according to
numerous well known methods (Sambrook et al., 1989, supra). Non-limiting
examples of labels include 3H, 14C, 32P, and 35S. Non-limiting examples of
detectable markers include ligands, fluorophores, chemiluminescent agents,
enzymes, and antibodies. Other detectable markers for use with probes, which
can enable an increase in sensitivity of the method of the invention, include
biotin
and radionucleotides. It will become evident to the person of ordinary skill
that the
choice of a particular label dictates the manner in which it is bound to the
probe.
As commonly known, radioactive nucleotides can be
incorporated into probes of the invention by several methods. Non-limiting
examples thereof include kinasing the 5' ends of the probes using gamma 32P
ATP and polynucleotide kinase, using the Klenow fragment of Pol I of E. coli
in
the presence of radioactive dNTP (i.e. uniformly labeled DNA probe using
random
oligonucleotide primers in low-melt gels), using the SP6/T7 system to
transcribe
a DNA segment in the presence of one or more radioactive NTP, and the like.
As used herein, "oligonucleotides" or "oligos" define a molecule
having two or more nucleotides (ribo or deoxyribonucleotides). The size of the
oligo will be dictated by the particular situation and ultimately on the
particular use
thereof and adapted accordingly by the person of ordinary skill. An
oligonucleotide
can be synthetised chemically or derived by cloning according to well known
methods.
As used herein, a "primer" defines an oligonucleotide which is
capable of annealing to a target sequence, thereby creating a double stranded
region which can serve as an initiation point for DNA synthesis under suitable
conditions.
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Amplification of a selected, ortarget, nucleic acid sequence may
be carried out by a number of suitable methods. See generally Kwoh et al.,
1990,
Am. Biotechnol. Lab. 8:14-25. Numerous amplification techniques have been
described and can be readily adapted to suit particular needs of a person of
ordinary skill. Non-limiting examples of amplification techniques include
polymerase chain reaction (PCR), ligase chain reaction (LCR), strand
displacement amplification (SDA), transcription-based amplification, the Qp
replicase system and NASBA (Kwoh et al., 1989, Proc. Natl. Acad. Sci. USA 86,
1173-1177; Lizardi et al., 1988, BioTechnology 6:1197-1202; Malek et al.,
1994,
Methods Mol. Biol., 28:253-260; and Sambrook et al., 1989, supra). Preferably,
amplification will be carried out using PCR.
Polymerase chain reaction (PCR) is carried out in accordance
with known techniques. See, e.g., U.S. Pat. Nos. 4,683,195; 4,683,202;
4,800,159; and 4,965,188. In general, PCR involves, a treatment of a nucleic
acid
sample (e.g., in the presence of a heat stable DNA polymerase) under
hybridizing
conditions, with one oligonucleotide primer for each strand of the specific
sequence to be detected. An extension product of each primer which is
synthesized is complementary to each of the two nucleic acid strands, with the
primers sufficiently complementary to each strand of the specific sequence to
hybridize therewith. The extension product synthesized from each primer can
also
serve as a template for further synthesis of extension products using the same
primers. Following a sufficient number of rounds of synthesis of extension
products, the sample is analysed to assess whether the sequence or sequences
to be detected are present. Detection of the amplified sequence may be carried
out by visualization following EtBr staining of the DNA following gel
electrophores,
or using a detectable label in accordance with known techniques, and the like.
For
a review on PCR techniques (see PCR Protocols, A Guide to Methods and
Amplifications, Michael et al. Eds, Acad. Press, 1990).
As used herein, the term "gene" is well known in the art and
relates to a nucleic acid sequence defining a single protein or polypeptide. A
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"structural gene" defines a DNA sequence which is transcribed into RNA and
translated into a protein having a specific amino acid sequence thereby giving
rise
the a specific polypeptide or protein. It will be readily recognized by the
person of
ordinary skill, that the nucleic acid sequence of the present invention can be
incorporated into anyone of numerous established kit formats which are well
known in the art.
A "heterologous" (i.e. a heterologous gene) region of a DNA
molecule is a subsegment segment of DNA within a larger segment that is not
found in association therewith in nature. The term "heterologous" can be
similarly
used to define two polypeptidic segments not joined together in nature.
Non-limiting examples of heterologous genes include reporter genes such as
luciferase, chloramphenicol acetyl transferase, beta-galactosidase, growth
hormone, green fluorescence protein (U.S.P. 5,968,8750) and the like which can
be juxtaposed or joined to heterologous control regions or to heterologous
polypeptides. In accordance with one embodiment and in particular for the
screening assays, such a reporter gene can enable a quantification of the
PCA3aa3 promoter activity. Preferably the heterologous sequence encodes a
therapeutic sequence.
The term "vector" is commonly known in the art and defines a
plasmid DNA, phage DNA, viral DNA and the like, which can serve as a DNA
vehicle into which DNA of the present invention can be cloned. Numerous types
of vectors exist and are well known in the art.
The term "expression" defines the process by which a gene is
transcribed into mRNA (transcription), the mRNA is then being translated
(translation) into one polypeptide (or protein) or more.
The terminology "expression vector" defines a vector or vehicle
as described above but designed to enable the expression of an inserted
sequence following transformation into a host. The cloned gene (inserted
sequence) is usually placed under the control of control element sequences
such
as promoter sequences. The placing of a cloned gene under such control
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sequences is often refered to as being operably linked to control elements or
sequences.
Operably linked sequences may also include two segments that
are transcribed onto the same RNA transcript. Thus, two sequences, such as a
promoter and a "reporter sequence" or "therapeutic sequence" are operably
linked
if transcription commencing in the promoter will produce an RNA transcript of
the
reporter sequence or therapeutic sequence. In order to be "operably linked" it
is
not necessary that two sequences be immediately adjacent to one another.
Expression control sequences will vary depending on whether
the vector is designed to express the operably linked gene in a prokaryotic or
eukaryotic host or both (shuttle vectors) and can additionally contain
transcriptional elements such as enhancer elements, termination sequences,
tissue-specificity elements, and/or translational initiation and termination
sites. In
accordance with a preferred embodiment of the present invention, the promoter
sequences of the present invention find utility in the expression of a
heterologous
sequence in a prostate cell or more preferably a prostate cancer cell. Of
course,
the present invention also finds utility in a prostate cancer tissue-derived
cell (e.g.
metastatis).
The DNA construct can be a vector comprising a promoter that
is operably linked to an oligonucleotide sequence of the present invention,
which
is in turn, operably linked to a heterologous gene, such as the gene for the
luciferase reporter molecule. "Promoter" refers to a DNA regulatory region
capable of binding directly or indirectly to RNA polymerase in a cell and
initiating
transcription of a downstream (3' direction) coding sequence. For purposes of
the
present invention, the promoter is bound at its 3' terminus by the
transcription
initiation site and extends upstream (5direction) to include the minimum
number
of bases or elements necessary to initiate transcription at levels detectable
above
background. Within the promoter will be found a transcription initiation site
(conveniently defined by mapping with S1 nuclease), as well as protein binding
domains (consensus sequences) responsible for the binding of RNA polymerase.
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Eukaryotic promoters will often, but not always, contain "TATA" boxes and
"CCAT" boxes.
As used herein, the designation "functional derivative" denotes,
in the context of a functional derivative of a sequence whether an nucleic
acid or
amino acid sequence, a molecule that retains a biological activity (either
function
or structural) that is substantially similar to that of the original sequence.
This
functional derivative or equivalent may be a natural derivative or may be
prepared
synthetically. Such derivatives include more precisely PCA3dd3 sequences
having
substitutions, deletions, or additions of one or more nucleotides
(duplications),
provided that the promoter activity and the prostate-specific activity thereof
are
conserved. The term "functional derivatives" is intended to include
"fragments",
"segments", "variants", "analogs" or "chemical derivatives" of the subject
matter
of the present invention.
Thus, the term "variant" refers herein to a nucleic acid molecule
which is substantially similar in structure and biological activity to the
nucleic acid
of the present invention.
The functional derivatives of the present invention can be
synthesized chemically or produced through recombinant DNA technology. AII
these methods are well known in the art.
One skilled in the art will realize that genomes often contain
slight allelic variations between individuals. Therefore, the isolated nucleic
acid
molecule is also intended to include allelic variations, so long as the
sequence is
a functional derivative of the PCA3aa3 promoter sequence. When a PCA3 allele
does not encode the identical sequence to that found in SEQ ID No:1, it can be
isolated and identified as a PCA3dd3 promoter using the same techniques used
herein, and especially PCR techniques to amplify the appropriate region with
primers based on the sequences disclosed herein.
As used herein, "chemical derivatives" is meant to cover
additional chemical moieties not normally part of the subject matter of the
invention. Such moieties could affect the physico-chemical characteristic of
the
derivative (i.e. solubility, absorption, half life and the like, decrease of
toxicity).
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Such moieties are examplified in Remington's Pharmaceutical Sciences (1980).
Methods of coupling these chemical-physical moieties to a polypeptide are well
known in the art.
The term "allele" defines an alternative form of a gene which
occupies a given locus on a chromosome.
As commonly known, a "mutation" is a detectable change in the
genetic material which can be transmitted to a daughter cell. As well known, a
mutation can be, for example, a detectable change in one or more
deoxyribonucleotide. For example, nucleotides can be added, deleted,
substituted
for, inverted, or transposed to a new position. Spontaneous mutations and
experimentally induced mutations exist. The result of a mutations of nucleic
acid
molecule is a mutant nucleic acid molecule. A mutant polypeptide can be
encoded
from this mutant nucleic acid molecule.
As used herein, the term "purified" refers to a molecule having
been separated from a cellular component. Thus, for example, a "purified
protein"
has been purified to a level not found in nature. A "substantially pure"
molecule
is a molecule that is lacking in all other cellular components.
As used herein, the terms "molecule", "compound", or "agent"
are used interchangeably and broadly to refer to natural, synthetic or
semi-synthetic molecules or compounds. The term "molecule" therefore denotes
for example chemicals, macromolecules, cell or tissue extracts (from plants or
animals) and the like. Non limiting examples of molecules include nucleic acid
molecules, peptides, ligands, antibodies, carbohydrates and pharmaceutical
agents. The agents can be selected and screened by a variety of means
including random screening, rational selection and by rational design using
for
example protein or ligand modelling methods such as computer modelling. The
terms "rationally selected" or "rationally designed" are meant to define
compounds
which have been chosen based on the configuration of the interaction domains
of the present invention. As will be understood by the person of ordinary
skill,
macromolecules having non-naturally occurring modifications are also within
the
scope of the term "molecule". For example, peptidomimetics, well known in the
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pharmaceutical industry and generally referred to as peptide analogs can be
generated by modelling as mentioned above. The molecules identified in
accordance with the teachings of the present invention have a therapeutic
value
in diseases or conditions in which the physiology or homeastasis of the
prostate
cell and/or tissue or cell derived therefrom is compromised. In a preferred
embodiment, the defect of the cell or tissue is associated with a defect in
the
expression of PCA3 mRNAs. Alternatively, the molecules identified in
accordance
with the teachings of the present invention find utility in the development of
compounds which can modulate the activity of the PCA3aa3 promoter sequences.
The present invention also provides antisense nucleic acid
molecules which can be used for example to decrease or abrogate the activity
of
the PCA3dd3 promoter. An antisense nucleic acid molecule according to the
present invention refers to a molecule capable of forming a stable duplex or
triplex
with a portion of its targeted nucleic acid sequence (DNA or RNA). The use of
antisense nucleic acid molecules and the design and modification of such
molecules is well known in the art as described for example in WO 96/32966, WO
96/11266, WO 94/15646, WO 93/08845 and USP 5,593,974. Antisense nucleic
acid molecules according to the present invention can be derived from the
nucleic
acid sequences and modified in accordance to well known methods. For example,
some antisense molecules can be designed to be more resistant to degradation
to increase their affinity to their targeted sequence, to affect their
transport to
chosen cell types or cell compartments, and/or to enhance their lipid
solubility by
using nucleotide analogs and/or substituting chosen chemical fragments
thereof,
as commonly known in the art.
An indicator cell in accordance with the present invention can
be used to identify antagonists. For example, the test molecule or molecules
are
incubated with the host cell in conjunction with one or more agonists held at
a
fixed concentration. An indication and relative strength of the antagonistic
properties of the molecule(s) can be provided by comparing the level of gene
expression in the indicator cell in the presence of the agonist, in the
absence of
test molecules vs in the presence thereof. Of course, the antagonistic effect
of
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a molecule can also be determined in the absence of agonist, simply by
comparing the level of expression of the reporter gene product in the presence
and absence of the test molecule(s).
It shall be understood that the 'fn vivo "experimental model can
also be used to carry out an 'in vitro "assay. For example, cellular extracts
from
the indicator cells can be prepared and used in one of the aforementioned 'in
vitro "tests.
As used herein the recitation "indicator cells" refers to cells that
contain a PCA3dd3 promoter sequence operably linked to a detectable
heterologous sequence. In some embodiment, the protein encoded by the
heterologous sequence can be coupled to an identifiable or selectable
phenotype
or characteristic. Such indicator cells can be used in the screening assays of
the
present invention. In certain embodiments, the indicator cells have been
engineered so as to express a chosen derivative, fragment, homolog, or mutant
of the PCA3dd3 promoter of the present invention. The cells must be chosen so
as
to enable a detectable promoter activity of the PCA3dd3 promoter. Preferably,
the
cell is a prostate cell, more preferably the prostate cell is a prostate
cancer cell.
For certainty, the sequences and polypeptides useful to
practice the invention include without being limited thereto mutants,
homologs,
subtypes, alleles and the like. It shall be understood that generally, the
sequences of the present invention should encode a functional (albeit
defective)
PCA3dd3 promoter. It will be clear to the person of ordinary skill that
whether the
PCA3dd3 promoter sequence of the present invention, variant, derivative, or
fragment thereof retains its promoter activity can be readily determined by
using
the teachings and assays of the present invention and the general teachings of
the art.
As exemplified herein below, the PCA3aa3 promoter of the
present invention can be modified, for example by in vitro mutagenesis (e.g.
point
mutations, Example 6) or by deletion analysis (Example 4), to dissect the
structure-function relationship thereof and permit a better design and
identification
of modulating compounds.
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A host cell or indicator cell has been "transfected" by
exogenous or heterologous DNA (e.g. a DNA construct) when such DNA has
been introduced inside the cell. The transfecting DNA may or may not be
integrated (covalently linked) into chromosomal DNA making up the genome of
the cell. A stably transfected cell is one in which the transfecting DNA has
become integrated into a chromosome so that it is inherited by daughter cells
through chromosome replication. This stability is demonstrated by the ability
of
the eukaryotic cell to establish cell lines or clones comprised of a
population of
daughter cells containing the transfecting DNA. Transfection methods are well
known in the art (Sambrook et al., 1989, supra; Ausubel et al., 1994 supra).
From the specification and appended claims, the term
therapeutic agent should be taken in a broad sense so as to also include a
combination of at least two such therapeutic agents. Further, the DNA segments
or proteins according to the present invention can be introduced into
individuals
in a number of ways. For example, prostatic cells can be isolated from the
afflicted individual, transformed with a DNA construct according to the
invention
and reintroduced to the afflicted individual in a number of ways.
Alternatively, the
DNA construct can be administered directly to the afflicted individual. The
DNA
construct can also be delivered through a vehicle such as a liposome, which
can
be designed to be targeted to a specific cell type, and engineered to be
administered through different routes.
For administration to humans, the prescribing medical
professional will ultimately determine the appropriate form and dosage for a
given
patient, and this can be expected to vary according to the chosen therapeutic
regimen (i.e. DNA construct, protein, cells), the response and condition of
the
patient as well as the severity of the disease.
Composition within the scope of the present invention should
contain the active agent (i.e. recombinant vector, nucleic acid, and molecule)
in
an amount effective to achieve the desired therapeutic effect while avoiding
adverse side effects. Typically, the nucleic acids in accordance with the
present
invention can be administered to mammals (i.e. humans) in doses ranging from
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0.005 to 1 mg per kg of body weight per day of the mammal which is
treated. Pharmaceutically acceptable preparations and salts of the active
agent are within the scope of the present invention and are well known in
the art (Remington's Pharmaceutical Science, 16th Ed., Mack Ed.). For
the administration of polypeptides, antagonists, agonists and the like, the
amount administered should be chosen so as to avoid adverse side
effects. The dosage will be adapted by the clinician in accordance with
conventional factors such as the extent of the disease and different
parameters from the patient. Typically, 0.001 to 50 mg/kg/day will be
administered to the mammal.
The present invention also relates to a kit comprising the
oligonucleotide primer of the present invention, which are specific to either
one of the PCA3 mRNA lacking the additional sequence of the present
invention or the PCA3 mRNA containing the additional sequence of the
present invention.
The present invention thus provides the means to
specifically express a heterologous sequence of interest into prostate cells
or prostate-derived cell and more particularly in prostate cancer cells.
Thus, the present invention provides the means to directly or indirectly
correct a disease state in a prostate or prostate-derived cell. The highly
prostate specific expression of the PCA3dd3 promoter is herein
demonstrated.
Thus, in accordance with a preferred embodiment of the
present invention, there is provided a method of expressing specifically in
prostate cells or prostate-derived cells, a chosen heterologous sequence,
and preferably a therapeutic sequence, capable of directly or indirectly
improving the diseased state in the prostate cell or prostate-derived cells.
In a particularly preferred embodiment, the present invention provides the
means to specifically kill prostate cancer cells or prostate cancer-derived
cells, through an introduction thereinto of a recombinant vector comprising
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a PCA31d3 promoter sequence of the present invention, operably linked to
a suicide gene.
In accordance with a first broad aspect, the present
invention relates to isolated nucleic acid sequence for use as a promoter
sequence, comprising a sequence as set forth between nucleotide positions
372 to 460 of SEQ ID NO:1.
In accordance with a second broad aspect, the present
invention also relates to a method of modulating the transcription of a
heterologous sequence in a prostate cell in vitro. This method comprises the
joining of a heterologous sequence downstream of a sequence as set forth
between nucleotide positions 372 to 460 of SEQ ID NO:1 so as to subject the
heterologous sequence to the control of the promoter sequence. The
introduction of the joined heterologous sequence in a cell enabling the
promoter activity of the nucleic acid and an assessment of the level of
transcription of the heterologous sequence.
In accordance with a third broad aspect, the present invention
further relates to a method of modulating the transcription of a heterologous
sequence in a prostate cell in vitro. This method comprises a joining of the
heterologous sequence downstream of a sequence selected from the group
consisting of: 372 to 461 of SEQ ID NO:1; 372 to 522 of SEQ ID NO:1; 309 to
460 of SEQ ID NO:1; 309 to 461 of SEQ ID NO:1; 309 to 522 of SEQ ID NO:1;
207 to 460 of SEQ ID NO:1; 207 to 461 of SEQ ID NO:1; 207 to 522 of SEQ
ID NO:1; 95 to 460 of SEQ ID NO:1; 95 to 461 of SEQ ID NO:1; 95 to 522 of
SEQ ID NO:1; 28 to 460 of SEQ ID NO:1; 28 to 461 of SEQ ID NO:1; 28 to
522 of SEQ ID NO:1; 1 to 460 of SEQ ID NO:1; 1 to 461 of SEQ ID NO:1; and
1 to 522 of SEQ ID NO:1. This is done so as to subject the heterologous
sequence to the control of the promoter sequence. The introduction of the
thereby joined heterologous sequence in a cell enabling the promoter activity
of the nucleic acid and an assessment of the level of transcription of the
heterologous sequence.
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In accordance with a fourth broad aspect, the present invention
also relates to a method of modulating an expression of a transcript in a
prostate cancer cell in vitro. This method comprises an administration in the
cell of an antisense molecule capable of modulating the transcriptional
activity
of a nucleic acid sequence as set forth between nucleotide positions 372 to
460 of SEQ ID NO:1.
In accordance with another broad aspect, the present invention
relates to a method of identifying an agent which modulates an expression of a
transcript in a prostate cell. This method comprises an assessment of a
transcriptional activity of a nucleic acid sequence as set forth between
nucleotide positions 372 to 460 of SEQ ID NO:1 in vitro, in the presence,
versus the absence of a candidate compound, wherein a compound which
modulates the transcriptional activity of the promoter sequence is selected
when the transcriptional activity is significantly different in the presence
of the
compound, as compared to in the absence thereof.
In accordance with a further broad aspect, the present
invention also relates to a method of modulating expression of a transcript
in a prostate cancer cell in vitro. The method comprises the administration
in the cell of an antisense molecule capable of modulating the
transcriptional activity of a nucleic acid sequence selected from the group
consisting of: 372 to 461 of SEQ ID NO:1; 372 to 522 of SEQ ID NO:1; 309
to 460 of SEQ ID NO:1; 309 to 461 of SEQ ID NO:1; 309 to 522 of SEQ ID
NO:1; 207 to 460 of SEQ ID NO:1; 207 to 461 of SEQ ID NO:1; 207 to 522
of SEQ ID NO:1; 95 to 460 of SEQ ID NO:1; 95 to 461 of SEQ ID NO:1; 95
to 522 of SEQ ID NO:1; 28 to 460 of SEQ ID NO:1; 28 to 461 of SEQ ID
NO:1; 28 to 522 of SEQ ID NO:1; 1 to 460 of SEQ ID NO:1; 1 to 461 of
SEQ ID NO:1; and 1 to 522 of SEQ ID NO:1.
In accordance with yet another broad aspect, the present
invention relates to a method of identifying an agent which modulates
expression of a transcript in a prostate cell. This method comprises an
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assessment of a transcriptional activity of a promoter sequence in the
presence versus the absence of a candidate compound, wherein the
promoter sequence comprises a sequence selected from the group
consisting of: 372 to 461 of SEQ ID NO:1; 372 to 522 of SEQ ID NO:1; 309
to 460 of SEQ ID NO:1; 309 to 461 of SEQ ID NO:1; 309 to 522 of SEQ ID
NO:1; 207 to 460 of SEQ ID NO:1; 207 to 461 of SEQ ID NO:1; 207 to 522
of SEQ ID NO:1; 95 to 460 of SEQ ID NO:1; 95 to 461 of SEQ ID NO:1; 95
to 522 of SEQ ID NO:1; 28 to 460 of SEQ ID NO:1; 28 to 461 of SEQ ID
NO:1; 28 to 522 of SEQ ID NO:1; 1 to 460 of SEQ ID NO:1; 1 to 461 of
SEQ ID NO:1; and 1 to 522 of SEQ ID NO:1. A compound which
modulates the transcriptional activity of the promoter sequence is selected
when the transcriptional activity of the promoter sequence is significantly
different in the presence of the compound, as compared to in the absence
thereof.
In accordance with yet another broad aspect, the present
invention also relates to a method for diagnosing prostate cancer or a
predisposition thereto in a nucleic acid prostatic sample of a patient. The
method comprises assessing in the sample, the promoter activity of a
promoter sequence comprising a sequence selected from the group
consisting of: 372 to 461 of SEQ ID NO:1; 372 to 522 of SEQ ID NO:1; 309
to 460 of SEQ ID NO:1; 309 to 461 of SEQ ID NO:1; 309 to 522 of SEQ ID
NO:1; 207 to 460 of SEQ ID NO:1; 207 to 461 of SEQ ID NO:1; 207 to 522
of SEQ ID NO:1; 95 to 460 of SEQ ID NO:1; 95 to 461 of SEQ ID NO:1; 95
to 522 of SEQ ID NO:1; 28 to 460 of SEQ ID NO:1; 28 to 461 of SEQ ID
NO:1; 28 to 522 of SEQ ID NO:1; 1 to 460 of SEQ ID NO:1; 1 to 461 of
SEQ ID NO:1; and 1 to 522 of SEQ ID NO:1. The assessment of an active
state of the promoter sequence, as compared to an inactive state thereof,
indicates a cancerous state of the prostatic sample or a predisposition of
the sample to develop into a cancerous state.
Further objects and advantages of the present invention
will be clear from the description that follows.
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BRIEF DESCRIPTION OF THE DRAWINGS
Having thus generally described the invention, reference will
now be made to the accompanying drawings, showing by way of illustration a
preferred embodiment thereof, and in which:
Figure 1 shows the nucleotide sequence of the PCA3dd3
promoter and exon 1 regions (SEQ ID NO:1). (A) Restriction map of PCA3ad3
genomic clones pFS28, pGV61, and pME4.6. The location of exon 1 is indicated
by a shaded box. (B) Nucleotide sequence of the genomic region surrounding the
transcription start site (position 1,'') of the PCA3d13 gene. The exon 1
sequence is
underlined. Potential transcription factor binding sites are identified by the
MatlnspectorTM program using the TRANSFAC 3.5 matrices (Quandt et a/.,
Nucleic Acids Res. 1995), with a core similarity of 1.00 and a matrix
similarity of
over 0.90, and are indicated by boxes. Abbreviations: CCAAT: CAAT-box;
C/EBPbeta: CCAAT/enhancer binding protein beta; E-box: bHLH binding site;
FKHL: forkhead homologue; FREAC: forkhead related activator; NF-1: nuclear
factor 1; Thl/E47: Thingl/E47 (bHLH) heterodimer;
Figure 2 shows the nucleotide sequences of the specific
oligonucleotides (SEQ ID NO:2 to SEQ ID NO:4) used in the determination of the
PCA3ad3 transcription start site. BUS2 and BUS7 sequences are PCA3dd3 specific
antisense oligonucleotides used for primer extension analysis. The BUS21
sequence is a PCA3dd3 specific oligonucleotide used for RNase protection
assay;
Figure 3 shows the results of two complementary experiments
(Primer extension analysis and RNase protection assay) for the determination
of
the PCA3aa3 transcription start site. (A) Primer extension analysis of the
PCA3ad3
5'-flanking region. Twenty micrograms of total RNA from prostatic
adenocarcinoma (lanes 4, 5, 7, 8, and 9), liver (lane 3 and 10), or lung
(lanes 2
and 11) tissue or tRNA (lanes 1 and 6) were used for primer extension using
oligonucleotides BUS7 (lanes 1-5) and BUS2 (lanes 6-11) as primers as
indicated. Primer extension products are indicated by arrows (F-, BUS2, and
F-//+, BUS7 products). A DNA sequence ladder of the PCA3dd3 genomic clone
pME1.5S3 primed with BUS7 was used as a size marker. (B) For RNase (S1
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nuclease) protection analysis, a 252-bp radiolabeled single-stranded DNA probe
was synthesized from plasmid pME4.6, containing exon 1 of PCA3dd3 and it's 5'-
flanking region, using BUS21 as a primer. The Hincll-digested probe was
hybridized to 40 micrograms total RNA from prostatic adenocarcinoma (lanes 1-
4), liver (lane 5), or lung (lane 6) tissue or tRNA (lane 7) and treated with
S1
nuclease at 37 C (lanes 1-7) or at 30 C (data not shown). Arrows mark
protected
fragments. A DNA sequence ladder of the PCA3dd3 genomic clone pME4.6 primed
with BUS21 was used as a size marker;
Figure 4 shows the nucleotide sequence surrounding the
PCA3aa3 transcription start site (SEQ ID NO:5). Primer extension products are
indicated by triangles (closed triangle, major start site; open triangle,
minor site),
and S1 nuclease protected fragments by diamonds (closed diamond, major start
site; open diamond, minor site);
Figure 5 shows the PCA3ad3 promoter activity in the two
human prostate cancer cell lines TSU-prl and LN-CaP after transient
transfection.
PCA3dd3 promoter constructs used for transient transfections are shown at the
left.
Thin lines indicate vector sequences, boxes indicate the human growth hormone
(hGH) gene, and the hatched boxes the PCA3 dd3 promoter fragments. Positions
relative to the transcription initiation site are indicated. Human growth
hormone
production in LNCaP cells (PCA3dd3-positive, black bars) and in TSU-pr1 cells
(PCA3dd3-negative, light bars) were calculated using pTKGH (hGH gene
expression driven by the HSV-tk promoter) as a reference. Error bars indicate
the
standard error of the mean of three independent experiments done in
duplicates;
Figure 6 illustrates the specificity of the PCA3dd3 promoter
activity in cell lines from different tissue origin. The pDDGH-1.9 and pDDGH-
1.12
PCA3dd3-promoter constructs were transiently transfected into LNCaP (L), TSU-
pr1 (T), A431 (A), SKRC-7 (SK), HT-29 (H), and SW800 (SW) cells. PCA3dd3
promoter activity, i.e. hGH production, was determined as described in Figure
5;
and
Figure 7 shows the effects of site directed mutagenesis on the
PCA3dd3 promoter activity. (A) PCA3dd3 minimal promoter constructs (pDDGH-1.9
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and -1.12) are shown as lines with numbers corresponding to their positions
relative to the transcription start site. Mutant constructs are shown below.
The
position of the mutations with respect to the transcription start site and the
substituted bases are indicated to the left of each construct. (B) Promoter
mutants
were transiently transfected into LNCaP cells and hGH production and promoter
activities were determined as described in Figure 5.
Other objects, advantages and features of the present
invention will become more apparent upon reading of the following non-
restrictive
description of preferred embodiments with reference to the accompanying
drawing which is exemplary and should not be interpreted as limiting the scope
of
the present invention.
DESCRIPTION OF THE PREFERRED EMBODIMENT
PCA31a3 gene has been shown to be sensitive and specific
marker for diagnosis and prostate cancer in a patient. The characterization of
its
promoter, sequence deposited into the GenBankT"" data base, opens the way to
the specific expression of heterologous sequence in a prostate or prostate-
derived cell and more particularly prostate cancer cells and to development of
therapies against prostate cancer.
The present invention is illustrated in further detail by the
following non-limiting examples.
EXAMPLE 1
Isolation and sequence analysis of PCA3aa3 promoter clones
Genomic clones IambdaFlX-ME3, -ME4 and -IH1, containing
the 5' end of the human PCA3aa3 cDNA were obtained previously (Bussemakers,
PCT/CA98/00346 1998; and Bussemakers et al., Cancer Res. 1999a). Lambda
phage DNA was endonuclease digested and subcloned in plasmid vectors
pGEM-3Zf(+) or pT2. Double stranded plasmid DNA was isolated by standard
procedures, and sequenced using the Thermo SequenaseTM cycle sequencing kit
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(Amersham) and Texas RedTM labeled universal primers. Sequencing products
were separated and analyzed using the VistraTM DNA Sequencer 725.
In figure 1A, a restriction map is shown of the resulting clones
pFS28, pGV61 and pME4.6, containing PCA3dd3 exon 1 and its 5'-flanking
sequences. The nucleotide sequence of the 5'-flanking region was determined
(Fig. 1 B) and is deposited in the GenBank database under GenBank Accession
Number AF279290. Comparison of the PCA3dd3 5'-flanking nucleotide sequence
with sequences in the non-redundant nucleotide databases and the eukaryotic
promoter database, using BLAST (Altschul et al., Nucleic Acids Res. 1997),
revealed no homology to any gene, c.q. promoter sequences described.
Identification of potential transcription factor (TF) binding sites by the
MatlnspectorTM program, using the TRANSFAC 3.5 matrices (Quandt et al.,
Nucleic Acids Res. 1995), revealed no canonical binding sites at consensus
positions, i.e. no initiator nor TATA boxes could be identified. Only a single
CAAT
element at a non-consensus position (-374 to -378) was found. Other potential
TF
binding sites are shown in figure 1 B.
The structure function relationship of the PCA3dd3 promoter had
thus to be dissected using molecular biology methods as shown below.
EXAMPLE 2
Sequences of the oligonucleotides used in
the determination of the PCA3aa3 transcription start site
The nucleotide sequences of the specific oligonucleotides used
in the determination of the PCA3da3transcription start site are shown in
Figure 2
(SEQ ID NO:2 to SEQ ID NO:4). BUS2 (SEQ ID NO:2) and BUS7 (SEQ ID NO:3)
sequences are PCA3da3 specific antisense oligonucleotides used for primer
extension analysis. The BUS21 (SEQ ID NO:4) sequence is a PCA3 dd3 specific
oligonucleotide used for RNase protection assay.
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EXAMPLE 3
Determination of the PCA3aa3 start site
The isolated human PCA3dd3 cDNAs were shown to possess
different 5' ends, associated with their different lengths (Bussemakers et
al.,
Cancer Res. 1999a). In order to precisely define the PCA3aa3 transcription
start
site, primer extension analysis and RNase protection assays, using total RNA
from human prostate cancer tissue and normal human liver and lung tissue as a
negative control, were performed.
For the primer extension analysis, PCA3da3 exon 1 (BUS2) and
exon 3 (BUS7)-specific oligonucleotides were used as a primer. The use of the
exon 3-specific primer, BUS7, is validated, since exon 2 is present in only a
minority of transcripts (<5%) due to alternative splicing (Bussemakers et al.,
Cancer Res. 1999a). Briefly, total RNA of human tissue was isolated using
guanidium isothiocyanate by standard procedures. PCA3dd3-specific antisense
oligonucleotides BUS2 (5'-CTCTGTATCATCAGGTCCTTCC-3', position +120 to
+99) and BUS7 (5'-CTGGAAATGTGCAAAAACAT-3', position +420 to +401)
were [gamma 32P]ATP end-labeled (3000 mCi/mmol, Amersham) and annealed
with 20 micrograms of RNA at 30 C in 30 microliters hybridization buffer (40
mM
PIPES pH 6.7, 1 mM EDTA, 0.5 M NaCI, 80% formamide). Primers were
extended with 200 units of SuperScriptTM II-MMLV reverse transcriptase (Gibco
BRL) in 25 microliters RT buffer (Gibco BRL), supplemented with 0.5 mM dNTPs,
10 mM DTT, 0.5 micrograms actinomycin D, and 1 unit/microliter RNase
inhibitor,
for 1.5 hours at 42 C. RNase A-treated primer extension products were resolved
by 6% polyacrylamide-urea gel electrophoresis. PCA3ad3 genomic sequence
ladders generated with the same oligonucleotide primers were used as size
markers. Gels were dried and exposed to HyperfilmTM (Amersham) using two
amplifying screens at -80 C for approximately three days.
For the RNase protection assay, a DNA fragment
encompassing the presumed transcription start site was used as a probe. A 517
bps Sau3AI DNA fragment from clone pME4.6 was ligated into the BamHI site of
M13mp18. Single-stranded M13 DNA was isolated, annealed with the PCA3dd3
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exon 1-specific oligonucleotide BUS21 (5'-
CCTTCCCACCATGCAGATCTTCCTGGTCTCCCTCGG CTGCAGCCA-CACAA-
3'), and extended using [gamma 32P]dATP and Klenow DNA polymerase. The
radiolabeled probe was linearized with Hincll and purified from a denaturing
5%
polyacrylamide-urea gel. All reactions were performed according to standard
protocols. The probe (105 cpm) was annealed overnight with 40 micrograms total
RNA at 30 C in 30 microliters hybridization buffer (40 mM PIPES pH 6.7, 1 mM
EDTA, 0.5 M NaCl, 80% formamide). DNA-RNA hybrids were digested with 300
units S1 nuclease (Amersham) in 300 microliters S1 buffer (0.28 M NaCI, 0.05 M
NaAc pH 4.5, 4.5 mM ZnSO4, supplemented with 20 micrograms/mi single-
stranded herring sperm DNA) for 60 minutes at 30 C or 37 C. Digestions were
stopped by the addition of stop buffer (4 M NH4Ac, 50 mM EDTA, 50
micrograms/mi tRNA). Protected DNA fragments were resolved by 6%
polyacrylamide-urea gel electrophoresis. A PCA3aa3 genomic sequence ladder
generated with the BUS21 primer was used as size marker. Gels were dried and
exposed to HyperfilmTM (Amersham) using two amplifying screens at -80 C for
approximately three days.
The results of a representative experiment for the primer
extension analysis are shown in Figure 3A. Two prostate-specific extension
products could be identified in figure 3A (lanes 4, 5, 7, 8 and 9). The
transcription
start site deduced from the longest extension product was designated +1, and
consequently, the smaller fragment was initiated from an alternative start
site at
position +34. As shown in Figure 3B, RNase protection analysis, using S1
nuclease, revealed two prostate-specific S1-protected fragments (Fig. 3B,
lanes 1
to 4). The most abundant product was initiated from position +3 with respect
to
the transcription start site identified by primer extension analysis (position
+1).
The minor S1 protected fragment, was initiated from an alternative start site
at
position +12. The identified major and minor transcription start sites are
shown
above the PCA3 dd3 nucleotide sequence in figure 4.
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EXAMPLE 4
Transcriptional activity of the human PCA3aa3 promoter
In order to demonstrate promoter activity, the PCA3ad3 5'-
flanking region was cloned upstream of the human growth hormone (hGH)
reporter gene (construct pDDGH-1.9, position -433 to +62). Briefly, the
promoteriess plasmid pOGH (Nichols Institute) was used for cloning PCA3dd3
promoter fragments into the polylinker upstream of the human growth hormone
gene. PCA3dd3 promoter fragments were produced by PCR, using 5' Hindlil-
tagged and 3' BamHl-tagged primers and normal human genomic DNA as a
template. Subsequently, the fragments were cleaved with Hindlll and BamHl and
cloned into pOGH, using standard procedure. Mutant promoter constructs were
generated using the "GeneEditor" in vitro site-directed mutagenesis system
(Promega). Since the hGH secretion capacity of different cell lines may vary,
for
each cell line the activity of the PCA3ad3-hGH constructs was compared to that
of
plasmid pTKGH containing the human growth hormone gene driven by the
constitutive promoter of the HSV-thymidine kinase gene (Selden et a/., Mol.
Cell.
Biol. 1986).
Then these constructs were used in transient transfection of the
two following cell lines: PC-346C, a derivative of the human prostate
adenocarcinoma cell lines LNCaP (kindly provided by Dr. W. van Weerden; Dept.
of Urology, Erasmus University Rotterdam, The Netherlands) and TSU-prl. Both
cell lines were grown in RPMI 1640 medium, supplemented with 10% foetal calf
serum (Gibco BRL), in an atmosphere of 5% CO2 and 37 C. For transient
transfection, LNCaP cells were seeded at a density of 1 x 106 cells per 10-cm
dish
two days prior to transfection and TSU-prl cells were seeded at a density of 5
x
105 cells per 10-cm dish one day before transfection. For each transfection 3
micrograms of the appropriate PCA3dd3-hGH construct and 2.3 micrograms
pCH110 (internal marker) were complexed with FuGENE-6T"'reagent (Boeringer
Mannheim) in serum-free medium for 15 minutes at room temperature. The
FuGENE-6TM/DNA complexes were added to the cell cultures, and cells were
grown for an additional 72 hours. Transfections were performed at least three
times in duplica. After transfection, medium was collected and stored at -20 C
CA 02357073 2005-06-08
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until use. Human growth hormone secretion in the medium was determined using
the two-site fluoroimmunometric DelfiaTM hGH assay kit (Wallac Oy, Turku,
Finland), according to the manufacturer's instructions. hGH values were
normalized to the beta-galactosidase activities measured in the corresponding
cell
extracts. hGH values and relative induction values are expressed as mean and
standard error of the mean (SEM).
When pDDGH-1.9 was transfected into LNCaP cells, a human
prostate carcinoma cell line expressing PCA3ad3 mRNA, weak promoter activity
was seen (Figure 5), i.e. hGH production was about 20% of the HSV-tk driven
hGH production. Promoters are known to function unidirectionally. Therefore,
the
pDDGH-1.9 promoter sequences were cloned in the reverse orientation upstream
of the hGH gene (pDDGH-2.1, pos. +62 to -433). This pDDGH-2.1 promoter
construct was inactive in LNCaP cells, moreover, the hGH production was below
that found in cells transfected with the promoterless pOGH construct.
Sequences
upstream of the 500-bps PCA3dd3 promoter (-433 to +62) had no effect on the
PCA3dd3 promoter activity (Figure 5, construct pDDGH-1.5, pos. -1,900 to +62).
To investigate whether regions within the 500-bps PCA3 dd3
promoter contributed to PCA3dd3 promoter activity, a series of 5' deletion
constructs
were generated. Transfection of the deletion constructs pDDGH-1.10, -1.11 and -
1.12 into LNCaP cells resulted in an increased promoter activity, compared to
the
activity observed in pDDGH-1.9 (Figure 5). Construct pDDGH-1.12 (pos. -152 to
+62) displayed the highest PCA3dd3 promoter activity of all constructs tested.
Shortening the latter construct led to a decreased promoter activity (pDDGH-
1.16)
and a complete loss of activity in construct pDDGH-1.18 (pos. -41 to +62).
EXAMPLE 5
Cell-type specificity of PCA3dd3 promoter activity
To define the specificity of the observed PCA3dd3 promoter
activity, promoter constructs pDDGH-1.9, displaying basal activity, and pDDGH-
1.12, displaying maximum activity, were transfected into cell lines of
different
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tissue origin. Then, the production of human growth hormone was measured in
each cell line. The protocol for these experiments was described in Example 4.
The different cell lines used for these experiments were as follow: PC-346C, a
derivative of the human prostate adenocarcinoma cell line LNCaP; TSU-prl, a
human prostate cancer cell line; SW800, a human bladder cancer cell line;
HT29, a
human colon carcinoma cell line; SKRC-7, a renal cell carcinoma cell line and
A431, a
vulval epidermoid cancer cell line.
PCA3dd3 promoter activity of pDDGH-1.9 was found in LNCaP
cells (Figure 6), but not in A431 (vulval carcinoma), HT-29 (colon carcinoma),
SKRC-7 (renal cell carcinoma) and SW800 (bladder carcinoma) cells.
Importantly,
this promoter construct is also silent in prostate carcinoma cell lines that
do not
express PCA3dd3 mRNA (TSU-prl, Figure 6, and PC-3, data not shown). The
increased promoter activity of the truncated promoter construct pDDGH-1.12,
however, was also observed in the PCA3dd3-negative cell lines, although the
maximum promoter activity was significantly lower than in LNCaP cells.
EXAMPLE 6
Site directed mutagenesis and effects on PCA3aa3
promoter activity
In order to further dissect the PCA3dd3 promoter element and to
identify the potential significance of the promoter activity, several base
substitution
mutants in the reporter constructs pDDGH-1.9 and pDDGH-1.12 were created.
Base substitutions were introduced in those motifs that were predicted by the
MatlnspectorTM program, using the TRANSFAC 3.5 matrices (Quandt et al.,
Nucleic Acids Res. 1995), with a core similarity of 1.00 and a matrix
similarity of
over 0.90 (see Figure 1 B). The positions and nature of the base substitutions
are
shown in figure 7A. Promoter mutants were transiently transfected into LNCaP
cells. Then hGH production and promoter activities were measured as described
in Example 4. Base substitutions in the proximal region of the promoter
clearly
reduced its activity (Figure 7B). Mutations in the NF-1 sequence motif
(between -
61 to -60) reduced promoter activity of the 1.9/60a and 1.12/60a constructs to
CA 02357073 2001-09-07
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27% and 18% respectively, and mutations in the E-box motif (between -35 to -
33) reduced transcription from reporter constructs 1.9/60c and 1.12/60c to 43%
and 67% respectively. These data show that the proximal region of PCA3dd3
promoter is functionally involved in the initiation of transcription of this
gene,
corroborating the 5' deletion analysis, shown in Figure 5 and the significant
decrease in promoter activity of the -89 to +62 construct (pDDGH-1.16) and the
silencing of the promoter activity in the -41 to +62 construct (pDDGH-1.18).
As
well, these results highlight region -70 to -30 as harboring sequences having
a
significant prostate-specific promoter activity.
The PCA3d13 gene was previously shown to be highly
overexpressed in the majority of prostatic adenocarcinomas (Bussemakers et
al.,
Cancer Res. 1999a). These data suggest that PCA3da3 mRNA expression is
regulated by a unique prostate-cancer-specific transcriptional mechanism.
Nucleotide sequence analysis did not reveal any obvious promoter elements. No
known initiator motif, no TATA-box, no CAAT-box, and no GC-rich regions were
found at consensus positions within the PCA3dd3 promoter. In addition, the
PCA3a13 promoter initiates transcription mainly at a single site, but lacks
characterized initiator elements. A few TATA-less promoters have been
described
that show the properties of the PCA3dd3 promoter, initiator-less and not GC-
rich
(Brakebusch et al., J. Biol. Chem. 1997).
In any event, the data herein presented suggest that the
PCA3da3 gene promoter is tissue and cell-type specific and, therefore, is a
genuine prostate-cancer-specific promoter. The absolute promoter activities of
the
PCA3da3 promoter constructs tested, however, are rather low compared to the
HSV-tk promoter activity. This correlates with the low level of endogenous
PCA3dd3 mRNA expression observed in LNCaP cells, in contrast to the high
PCA3dd3 expression in prostate cancer cells.
CONCLUSION
CA 02357073 2001-09-07
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In conclusion, the present invention shows the cloning and the
initial characterization of the PCA3dd3 gene promoter. This promoter could be
an
interesting tool for therapeutic application.
Although the present invention has been described
hereinabove by way of preferred embodiments thereof, it can be modified
without
departing from the spirit and nature of the subject invention as defined in
the
appended claims.
CA 02357073 2001-09-07
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SEQUENCE LISTING
SEQ ID NO 1: PCA3"j promoter and exon 1
1 CACTAGAGGA GCACCTTAGG AATTGACCTG TGGATCTCAA CTTCGTTAGG
51 GTTAAAAGAT TATTTGTTGG GCAAGGGTAG GACCAATAAC CTCATTCACA
101 ATGCATTCAT TGATTCGTTG ATTCACAGAG CAAATACTTC TGAACAACTC
151 CTGTGTTTCT GGCACTGTTC TAGGCACCAG TGATATAGGA GCCAACAAGA
201 CAGACATGTC ACTGCTCTCA TGGAGCTGCA TTTCAGTGCA TGGAGGCAGA
251 AAACAAACAA ACAAATAAAT AAATAAATAA ATAAATAAGA TAATTTTTAA
301 TAGCAACGTG TCAACATAGT GTGACGGGAA GGAGCATGAT GAGACAGAAG
351 GAAGGTTTAA ACTGGGAAAT CTGAGAAATG GTATGGTTGT ATGTGGGTTG
401 GCATTCTTGC ATGATGGGAG TGGCCACCTG CTTTCATATT CTGAAGTCAG
451 AGTGTTCCAG ACAGAAGAAA TAGCAAGTGC CGAGAAGCTG GCATCAGAAA
exon 1
501 AACAGAGGGG AGATTTGTGT GGCTGCAGCC GAGGGAGACC AGGAAGATCT
exon 1
551 GCATGGTGGG AAGGACCTGA TGATACAGAG
exon 1
SEQ ID NO 2: PCA3113 specific antisense oligonucleotide BUS2
5'-CTCTGTATCATCAGGTCCTTCC-3'
SEQ ID NO 3: PCA3aa3 specific antisense oligonucleotide BUS7
5'-CTGGAAATGTGCAAAAACAT-3'
SEQ ID NO 4: PCA3"3 specific oligonucleotide BUS21
5'-CCTTCCCACCATGCAGATCTTCCTGGTCTCCCTCGGCTGCAGCCACACAA-3'
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SEQ ID NO 5: Nucleotide sequence surrounding the PCA3aa3
transcription start site
-10AGTGTTCCAG ACAGAAGAAA TAGCAAGTGC CGAGAAGCTG GCATCAGAAA +40
I
+1
CA 02357073 2001-09-07
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REFERENCES
Altschul et al. 1997, Nucleic Acids Res. 25 (17):3389-3402.
Boulikas T. 1997, Anticancer Res. 17 (3A):1471-1505.
Brakebusch et al. 1997, J. Biol. Chem. 272 (6):3674-3682.
Bussemakers M.J.G. 1998, WO 98/45420, PCT/CA98/00346.
Bussemakers et al. 1999a, Cancer Res. 59 (23):5975-5979.
Bussemakers M.J.G. 1999b, Eur. Urol. 35 (5-6):408-412.
Cleutjens et al. 1997a, Mol. Endocrinol. 11 (2):148-161.
Cleutjens et al. 1997b, Mol. Endocrinol. 11 (9):1256-1265.
Gotoh et al., 1998, J. Urol. 160 (1):220-229.
Hsing et al. 2000, Int. J. Cancer 85 (1):60-67.
Landis et al. 1999, CA Cancer J. Clin. 49: 8-31.
Martiniello-Wilks et al. 1998, Hum. Gene Ther. 9(11):1617-1626.
Millikan R.E. 1999, Semin Oncol. 26 (2):185-191
Pang et al. 1997, Cancer Res. 57 (3):495-499.
Quandt et al. 1995, Nucleic Acids Res. 23 (23):4878-4884.
Schalken J. 1998, Eur. Urol. 34 (suppl 3):3-6.
Schuur et al. 1996, J. Biol. Chem. 271 (12):7043-7051.
Taneja et al. 1995, Cancer Surv. 23:247-266.
Wei et al. 1997, Proc. Natl. Acad.Sci. USA 94 (12):6369-6374.
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SEQUENCE LISTING
(1) GENERAL INFORMATION:
(i) APPLICANT: BUSSEMAKERS, Marion J.
VERHAEGH, Gerald
SCHALKEN, Jack A.
(ii) TITLE OF INVENTION: Nucleic Acid Molecules Comprising the
Promoter for PCA3dd3, a New Prostate Antigen, and Uses
Thereof
(iii) NUMBER OF SEQUENCES: 7
(iv) CORRESPONDENCE ADDRESS:
(A) ADDRESSEE: GOUDREAU GAGE DUBUC
(B) STREET: 3400-Stock Exchange Tower, P.O. Box 242,800
Place Victoria
(C) CITY: Montreal
(D) STATE: Quebec
(E) COUNTRY: Canada
(F) ZIP: H4Z lE9
(v) COMPUTER READABLE FORM:
(A) MEDIUM TYPE: Floppy disk
(B) COMPUTER: IBM PC compatible
(C) OPERATING SYSTEM: PC-DOS/MS-DOS
(D) SOFTWARE: PatentIn Release #1.0, Version #1.30
(vi) CURRENT APPLICATION DATA:
(A) APPLICATION NUMBER: CA 2,357,073
(B) FILING DATE: 07-SEP-2001
(C) CLASSIFICATION:
(vii) PRIOR APPLICATION DATA:
(A) APPLICATION NUMBER: JP 2001-164963
(B) FILING DATE: 31-MAY-2001
(ix) TELECOMMUNICATION INFORMATION:
(A) TELEPHONE: (514) 397-7449
(B) TELEFAX: (514) 397-4382
(2) INFORMATION FOR SEQ ID NO:1:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 580 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Homo sapiens
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:1:
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CACTAGAGGA GCACCTTAGG AATTGACCTG TGGATCTCAA CTTCGTTAGG GTTAAAAGAT 60
TATTTGTTGG GCAAGGGTAG GACCAATAAC CTCATTCACA ATGCATTCAT TGATTCGTTG 120
ATTCACAGAG CAAATACTTC TGAACAACTC CTGTGTTTCT GGCACTGTTC TAGGCACCAG 180
TGATATAGGA GCCAACAAGA CAGACATGTC ACTGCTCTCA TGGAGCTGCA TTTCAGTGCA 240
TGGAGGCAGA AAACAAACAA ACAAATAAAT AAATAAATAA ATAAATAAGA TAATTTTTAA 300
TAGCAACGTG TCAACATAGT GTGACGGGAA GGAGCATGAT GAGACAGAAG GAAGGTTTAA 360
ACTGGGAAAT CTGAGAAATG GTATGGTTGT ATGTGGGTTG GCATTCTTGC ATGATGGGAG 420
TGGCCACCTG CTTTCATATT CTGAAGTCAG AGTGTTCCAG ACAGAAGAAA TAGCAAGTGC 480
CGAGAAGCTG GCATCAGAAA AACAGAGGGG AGATTTGTGT GGCTGCAGCC GAGGGAGACC 540
AGGAAGATCT GCATGGTGGG AAGGACCTGA TGATACAGAG 580
(2) INFORMATION FOR SEQ ID NO:2:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 22 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: other nucleic acid
(A) DESCRIPTION: /desc = "Oligonucleotide"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:
CTCTGTATCA TCAGGTCCTT CC 22
(2) INFORMATION FOR SEQ ID NO:3:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: other nucleic acid
(A) DESCRIPTION: /desc = "Oligonucleotide"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:
CTGGAAATGT GCAAAAACAT 20
(2) INFORMATION FOR SEQ ID NO:4:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 50 base pairs
(B) TYPE: nucleic acid
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(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: other nucleic acid
(A) DESCRIPTION: /desc = "Oligonucleotide"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:
CCTTCCCACC ATGCAGATCT TCCTGGTCTC CCTCGGCTGC AGCCACACAA 50
(2) INFORMATION FOR SEQ ID NO:5:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 50 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Homo sapiens
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:
AGTGTTCCAG ACAGAAGAAA TAGCAAGTGC CGAGAAGCTG GCATCAGAAA 50
(2) INFORMATION FOR SEQ ID NO:6:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 522 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Homo sapiens
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:
CACTAGAGGA GCACCTTAGG AATTGACCTG TGGATCTCAA CTTCGTTAGG GTTAAAAGAT 60
TATTTGTTGG GCAAGGGTAG GACCAATAAC CTCATTCACA ATGCATTCAT TGATTCGTTG 120
ATTCACAGAG CAAATACTTC TGAACAACTC CTGTGTTTCT GGCACTGTTC TAGGCACCAG 180
TGATATAGGA GCCAACAAGA CAGACATGTC ACTGCTCTCA TGGAGCTGCA TTTCAGTGCA 240
TGGAGGCAGA AAACAAACAA ACAAATAAAT AAATAAATAA ATAAATAAGA TAATTTTTAA 300
TAGCAACGTG TCAACATAGT GTGACGGGAA GGAGCATGAT GAGACAGAAG GAAGGTTTAA 360
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ACTGGGAAAT CTGAGAAATG GTATGGTTGT ATGTGGGTTC ACATTCTTGC ATGATGGGAG 420
TGGCCACCTG CTTTCATATT CTGAAGTCAG AGTGTTCCAG ACAGAAGAAA TAGCAAGTGC 480
CGAGAAGCTG GCATCAGAAA AACAGAGGGG AGATTTGTGT GG 522
(2) INFORMATION FOR SEQ ID NO:7:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 522 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Homo sapiens
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:
CACTAGAGGA GCACCTTAGG AATTGACCTG TGGATCTCAA CTTCGTTAGG GTTAAAAGAT 60
TATTTGTTGG GCAAGGGTAG GACCAATAAC CTCATTCACA ATGCATTCAT TGATTCGTTG 120
ATTCACAGAG CAAATACTTC TGAACAACTC CTGTGTTTCT GGCACTGTTC TAGGCACCAG 180
TGATATAGGA GCCAACAAGA CAGACATGTC ACTGCTCTCA TGGAGCTGCA TTTCAGTGCA 240
TGGAGGCAGA AAACAAACAA ACAAATAAAT AAATAAATAA ATAAATAAGA TAATTTTTAA 300
TAGCAACGTG TCAACATAGT GTGACGGGAA GGAGCATGAT GAGACAGAAG GAAGGTTTAA 360
ACTGGGAAAT CTGAGAAATG GTATGGTTGT ATGTGGGTTG GCATTCTTGC ATGATGGGAG 420
TGGCCGTATG CTTTCATATT CTGAAGTCAG AGTGTTCCAG ACAGAAGAAA TAGCAAGTGC 480
CGAGAAGCTG GCATCAGAAA AACAGAGGGG AGATTTGTGT GG 522
