Note: Descriptions are shown in the official language in which they were submitted.
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PROSTAPIN GENE AND PROTEIN AND USES THEREOF
FIELD OF THE INVENTION
The invention described herein relates to a novel gene, PROSTAPIN, and its
expression
product; to the expression of PROSTAPIN in normal and prostate cancer cells;
and to
diagnostic, prognostic, and therapeutic compositions and methods useful in the
management of prostate cancer.
BACKGROUND OF THE INVENTION
Prostate cancer is the most frequently diagnosed cancer and second leading
cause of
cancer death in men. Some 45,000 men die annually of this disease and only
lung cancer
has a higher mortality rate. In the United States, the chance of a man
developing invasive
prostate cancer during his lifetime is approximately 1 in 6 or greater.
Numerous
fundamental limitations in the presently available methods used for the
treatment,
diagnosis and prognosis of prostate cancer have rendered it impossible to
effectively
manage.
Surgical prostatectomy, radiation therapy, hormone ablation therapy, and
chemotherapy
are the main components in the current arsenal for treating prostate cancer.
However,
these treatments are ineffective for the 45,000 prostate cancer patients who
die of this
disease every year. While some advances in the treatment of locally confined
tumors
have been achieved, prostate cancer is presently incurable once it has
metastasized.
The major cause of morbidity and mortality from prostate cancer is the result
of
androgen-independent metastatic tumor growth. Patients with metastatic
prostate
cancer are treated by hormonal ablation therapy, but only with short term
success.
Eventually, these patients develop an androgen-refractory state leading to
aggressive
disease progression, ultimately resulting in the development of debilitating
bone and
other metastases causing death. The mechanism by which androgen-independent
growth of prostate tumors occurs is not known. Moreover, there is no method
available
for predicting the emergence of metastatic disease. Metastatic prostate cancer
is
typically diagnosed by open or laparoscopic pelvic lymphadenectomy, whole body
radionuclide scans, skeletal radiography, andlor bone lesion biopsy. As a
result, there is
great interest in defining the molecular basis for advanced staged disease
with the hope
that these insights may improve the therapeutic options for these patients.
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The primary sites of prostate cancer metastasis are the regional lymph nodes
and bone.
Bone metastases occur in sites of hematopoietically active red bone marrow,
including
lumbar vertebral column, ribs, pelvis, proximal long bones, sternum and skull.
Bony
metastases of prostate cancer differ from those of other tumors commonly
colonizing
bone in that they are characterized by a net gain in bone formation
(osteoblastic) rather
than resorption predominant in bone metastases of breast cancer and melanoma.
Until recently, bone metastasis was thought to be a late stage in disease
progression.
However, the recent development of highly sensitive techniques (such as RT-PCR
for
prostate specific genes) to detect prostate cancer cells has revised this
notion. Prostate
cancer cells have been detected in the peripheral blood and bone marrow of
patients
with advanced stage disease using RT-PCR assays for PSA mRNA (Ghossein et al.,
1995;
Seiden et al.,1994; Wood et al.,194; Katz et al., 1994) or immunomagnetic bead
selection
for PSA protein (Brandt et al., 1996). When positive, these tests show that
prostate
cancer cells represent about 0.1-1.0% of the circulating blood cells.
Moreover, it is now
clear that small numbers of prostate cancer cells circulate in the peripheral
blood and
lodge in the bone marrow even in patients with early stage, low risk disease
(Olsson et
al., 1997; Deguchi et al., 1997; Katz et al., 1996). Interestingly, these
cells tend to
disappear in most patients following radical prostatectomy (Melchior et al.,
1997). These
results suggest that the primary tumor site is a constant source for seeding
the marrow,
and that only a small subset of these cells have the capacity to grow into a
metastatic
lesion. This concept is consistent with estimates from animal models for other
tumor
types that only about 1 in 10,000 circulating cancer cells are able to lodge
in and
productively colonize other organs (Fidler et al., 1990). The biological
factors involved in
advanced prostate cancer progression to bone metastasis are unknown. (See
also,
Lalani et al.,1997, Cancer Metastasis Rev.16: 29-66).
A another factor complicating the management of prostate cancer is that
reliable
diagnostic and prognostic markers capable of accurately detecting early-stage
tumors
andlor predicting which patients will progress to advanced stages do not
exist. Early
detection and diagnosis of prostate cancer currently relies on digital rectal
examinations
(DRE), prostate specific antigen (PSA) measurements, transrectal
ultrasonography, and
transrectal needle biopsy. Serum PSA measurements in combination with DRE
represent
the leading diagnostic approach at present. Although a number of prostate
cancer
markers have been identified, and at least one, PSA, is in widespread clinical
use, the
ideal prostate tumor marker has been extremely elusive. Moreover, no marker
capable
of reliably predicting disease progression has been identified.
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There is currently a tremendous worldwide effort aimed at the development of
novel
molecular approaches to prostate cancer diagnosis and treatment. For example,
there is
great interest in identifying truly prostate-specific genes and proteins that
could be used
as diagnostic, prognostic andlor therapeutic targets or reagents. Progress has
been
slow, and despite this intensive worldwide research effort, no effective
biological
approaches for treating prostate cancer have emerged in clinical practice.
Similarly, the
inability to reliably detect early-stage prostate cancer or predict which
patients will
progress to advanced disease persists.
Tumor suppressors are proteins which regulate cell growth. Absence of tumor
suppressors by mutation, deletion, or loss of expression results in the
malignant
phenotype. Numerous tumor suppressor genes have been identified. Two of the
more
well known and studied tumor suppressor genes are the retinoblastoma (Rb) gene
and
the p53 gene, both of which are directly involved in influencing the cell
cycle machinery.
The expression of Rb inhibits cell cycle progression from G, into S phase. The
p53 gene is
the most frequently mutated gene in human cancers, with approximately half of
all tumors
containing abnormal p53 genes. p53 participates in a cell cycle checkpoint
signal
transduction pathway that causes either G, arrest or apoptosis following ONA
damage.
Loss of p53 function during tumorigenesis can result in progression through
the cell
cycle in the face of ONA damage and survival of a cell otherwise destined for
death.
There is no known prostate-specific tumor suppressor. A class of serine
protease
inhibitors known as the serpins includes a protein, maspin, which may function
as a
tumor suppressor in both breast and prostate cancer. Maspin has been shown to
be
down-regulated in breast and prostate carcinoma (Zou et al., 1994, Science
283:52ti),
and overexpression of maspin andlor exogenous addition of maspin has been
shown to
dramatically reduce the tumorigenic properties and metastatic potential of
breast cancer
cells (Zou et al., 1994, Science 283:526). Other members of the serpin family
include
leupin (SCCA2) (Suminami et al., 1991; BBRC 181:51), bomapin (Riewald and
Schleef,
1995, JBC 270:2fi754), and leukocyte elastase inhibitor (Dubin et al., 1993,
Biochem. J.
293:187). The latter, Leukocyte elastase inhibitor (LEI), appears to have a
functional role
in apoptosis. Specifically, LEI is converted to L-DNase II by digestion with
elastase,
whereupon it functions as an endonuclease in DNA degradation during apoptosis
(Torriglia et al., 1998, MCB 18:3612). Several other serpins appear to have a
role in
apoptosis.
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SUMMARY OF THE INVENTION
The present invention relates to a novel member of the serpin family, termed
PROSTAPIN, which is expressed almost exclusively in the prostate. PROSTAPIN
expression is greatly attenuated or completely lost in cells of advanced
prostate tumors
and metastases, while its expression is maintained at or near normal levels in
locally
confined prostate cancers. In addition to transcriptional loss of PROSTAPIN,
there is
initial evidence that the PROSTAPIN gene is substantially mutated in some
advanced
stage prostate tumors. Accordingly, PROSTAPIN may function as a prostate-
specific
tumor suppressor, apoptosis-inducer or apoptosis-modulator. The PROSTAPIN gene
and
protein, as well as factors capable of activating PROSTAPIN expression, may be
useful as
therapeutic agents capable of restoring critical tumor suppressor activity
lost in
advanced prostate cancer. In addition, PROSTAPIN may represent an ideal marker
for
predicting and identifying progression to advanced stage and metastatic
prostate
cancer, and may also be useful for determining susceptibility to advanced
disease and
for gauging prostate tumor aggressiveness.
The invention provides polynucleotides corresponding or complementary to all
or part of
the PROSTAPIN gene, mRNA, andlor coding sequence, preferably in isolated form,
including polynucleotides encoding PROSTAPIN proteins and fragments thereof,
DNA,
RNA, DNAIRNA hybrid, and related molecules, polynucleotides or
oligonucleotides
complementary to the PROSTAPIN gene or mRNA sequence or a part thereof, and
polynucleotides or oligonucleotides which hybridize to the PROSTAPIN gene,
mRNA, or
to PROSTAPIN-encoding polynucleotides. Also provided are means for isolating
cDNAs
and the gene encoding PROSTAPIN, as well as those encoding mutated and other
forms
of PROSTAPIN. Additionally, functionally mutant PROSTAPIN polynucleotides are
provided. Recombinant DNA molecules containing PROSTAPIN pofynucleotides,
cells
transformed or transduced with such molecules, and host-vector systems for the
expression of PROSTAPIN gene products are also provided. The invention further
provides PROSTAPIN proteins and polypeptide fragments thereof. The invention
further
provides antibodies that bind to PROSTAPIN proteins and polypeptide fragments
thereof,
including polyclonal and monoclonal antibodies, murine and other mammalian
antibodies,
chimeric antibodies, humanized and fully human antibodies, and antibodies
labeled with
a detectable marker. The invention further provides methods for detecting the
presence
of PROSTAPIN polynucleotides and proteins in various biological samples, as
well as
methods for identifying cells that express PROSTAPIN. The invention further
provides
methods and assays for determining PROSTAPIN expression status, diagnosing
advanced
prostate cancer, gauging tumor aggressiveness, and predicting susceptibility
to advanced
prostate cancer. The invention further provides various therapeutic
compositions and
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strategies for treating prostate cancer by restoring functional PROSTAPIN to
prostate
tumor cells.
BRIEF DESCRIPTION OF THE FIGURES
FIG. 1. Nucleotide and amino acid sequences of PROSTAPIN (SEQ lD NOS. XX and
XX,
respectively), derived from the overlapping nucleotide sequences of SSH
fragment cDNA
clone 11 P2A6 (5' 471 bp) (SEQ ID NO. XX) and cDNA clone 103 (SEQ ID NO. XX).
Clones
11 P2A6 and 103 overlap across 304 by beginning at position 168. The 5'
untranslated
region indicates two translational STOP signals (indicated by asterisks)
upstream of the
START ATG. The sequence surrounding the start ATG (AAA ATG G) exhibits a Kozak
sequence (A at position -3, and G at position +1 ). The underlined sequence in
the
carboxyl region indicates the putative highly exposed reactive site loop that
is
characteristic of the serpin family.
FIG. 2. Amino acid sequence alignment of PROSTAP1N with several other Serpin
family
members. The alignment was performed using the PIMA1.4 alignment program of
the
Baylor College of Medicine Search Launcher Web site. The RSL sites are
indicated in
bold. The protease cleavage site, indicated by the P1 and P1' residues, is
boxed.
FIG. 3. Semi-quantitative RT-PCR analysis of PROSTAPIN expression in normal
human
tissue, prostate cancer xenograft tissue, and cell lines using primers derived
from clone
11 P2A6 cONA (SEQ ID NO. XX). First strand cONAs were prepared from 16 normal
tissues, the LAPC xenografts (4AD, 4AI and 9AD) and HeLa cells. Normalization
was
performed by PCR using primers to actin and GAPDH. Expression of PROSTAPIN is
detected only in normal prostate and in the LAPC-9 AD xenograft.
FIG. 4. Northern blot analyses of PROSTAPIN expression in various normal human
tissues and prostate cancer xenografts. A and B: Multiple tissue northern
blots probed
with full length PROSTAPIN cONA clone 103 (SEQ ID NO. XX). Size standards in
kilobases
(kb) are indicated. C: Multiple tissue RNA dot blot (Clontech, Human Master
Blot cat#
7770-1) probed with PROSTAPIN CLONE 103 cDNA probe (SEQ ID NO. XX). D: Normal
prostate and various prostate cancer xenograft Northern blot, showing lack of
expression in the LAPC-4 prostate cancer xenografts and down-regulated
expression in
the LAPC-9 xenograft relative to normal prostate expression levels.
FIG. 5. Loss of PROSTAPIN expression in metastatic prostate cancer. Semi-
quantitative
RT-PCR analysis on normalized first strand cDNAs derived from prostate cancer
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xenografts, cell lines and human tissue specimens. The prostate pool (lane 3)
comprises
several normal prostate cDNAs and was obtained from Clontech (Palo Alto,
California).
Prostate 25 and Prostate 32 were derived from 25 and 32 year old individuals,
respectively (BioChain). Gleason Grade and TMN Stage of the human prostate
tumor
specimens analyzed are indicated. For details, see Example 4.
FIG. 6. Mutant PROSTAPIN gene generated from prostate cancer xenograft LAPC-9
AD:
LAPC-9 AD PROSTAPIN cDNA clone 2 nucleotide (SEQ ID NO. XX) and deduced amino
acid (SEQ ID NO. XX) sequences. Point mutations in the nucleotide sequence and
any
resulting amino acid changes relative to the wild-type PROSTAPIN sequence of
FIG. 1
(SEQ ID NO. XX) are indicated in boldface type and are underlined. A large
insertion
sequence (relative to wild-type PROSTAPIN) is indicated in bold and is
underlined.
FIG. 7. Detection of PROSTAPIN protein in cell membrane fraction. The results
show that
PROSTAPIN is predominantly expressed in the light membrane fraction. See
Example 6
for experimental details.
FIG. 8. Chromosomal mapping of human PROSTAPIN, showing position within serpin
gene cluster on chromosome 18q21.3.
FIG. 9. Southern blot analysis for PROSTAPIN gene. Ten micrograms of each DNA
sample was digested with EcoRl, blotted onto nitrocellulose and probed with
PROSTAPIN
CLONE 103 cDNA probe (SEQ ID NO. XX). (A) Zooblot: Genomic ONAs prepared from
several different organisms including human, monkey, dog, mouse, chicken and
Drosophila. (B) Human BACs 2002H14, 2074J2, 2100H19, and PAC 152122,
containing
the PROSTAPIN gene, a non-specific BAC (2116L1), and a non-specific PAC
(40P22)
(lanes 4 and 5, respectively). (C) Mouse BACs 74e14 (lanes 3 and 5), 21313
(lanes 4 and
6) containing the mouse PROSTAPIN gene probed together with human positive
(BAC
2074J2) and negative (BAC 40P22) controls.
FIG. 10. Intronlexon boundaries of the human wild-type PROSTAPIN gene.
Sequence in
capital letters designate exonic sequences (with the translation below) and
sequence in
lower case letters designate intronic sequences. A total of 6 introns and 7
exons were
identified within the PROSTAPIN coding region.
FIG. 11. Amplification of PROSTAPIN exons from human genomic DNA: An example
of
the PCR products obtained from human genomic DNA using the primers described
in
-Example 8. Human BAC DNA containing the PROSTAPIN gene was used as a positive
control for PCR amplification.
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FIG. 12. Western blot analysis of PROSTAPIN expression in lysates of cells
transfected or
transduced with PROSTAPIN using purified poiyclonal antibody generated against
a
PROSTAPIN-GST fusion (see Example 9).
FIG. 13. Western blot analysis of PROSTAPIN expression in lysates derived from
LAPC
xenografts (LAPC-4 AD, 9AD, and 9AI), prostate cancer cell lines (TsuPrl,
LNCaP, PC-3)
and a prostate tumor-normal matched patient sample cells (see Example 10).
DETAILED DESCRIPTION OF THE INVENTION
Unless otherwise defined, all terms of art, notations and other scientific
terminology used
herein are intended to have the meanings commonly understood by those of skill
in the
art to which this invention pertains. In some cases, terms with commonly
understood
meanings are defined herein for clarity andlor for ready reference, and the
inclusion of
such definitions herein should not necessarily be construed to represent a
substantial
difference over what is generally understood in the art. The techniques and
procedures
described or referenced herein are generally well understood and commonly
employed
using conventionat methodology by those skilled in the art, such as, for
example, the
widely utilized molecular cloning methodologies described in Sambrook et al.,
Molecular
Cloning: A Laboratory Manual 2nd. edition (1989) Cold Spring Harbor Laboratory
Press,
Cold Spring Harbor, N.Y. As appropriate, procedures involving the use of
commercially
available kits and reagents are generally carried out in accordance with
manufacturer
defined protocols andlor parameters unless otherwise noted.
As used herein, the terms "advanced prostate cancer", "locally advanced
prostate
cancer", "advanced disease" and "locally advanced disease" mean prostate
cancers
which have extended through the prostate capsule, and are meant to include
stage C
disease under the American Urological Association (AUA) system, stage C1 - C2
disease
under the Whitmore-Jewett system, and stage T3 - T4 and N+ disease under the
TNM
(tumor, node, metastasis) system. In general, surgery is nat recommended for
patients
with locally advanced disease, and these patients have substantially less
favorable
outcomes compared to patients having clinically localized (organ-confined)
prostate
cancer. Locally advanced disease is clinically identified by palpable evidence
of
induration beyond the lateral border of the prostate, or asymmetry or
induration above
the prostate base. Locally advanced prostate cancer is presently diagnosed
pathologically following radical prostatectomy if the tumor invades or
penetrates the
prostatic capsule, extends into the surgical margin, or invades the seminal
vesicles.
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As used herein, the terms "metastatic prostate cancer" and "metastatic
disease" mean
prostate cancers which have spread to regional lymph nodes or to distant
sites, and are
meant to include stage D disease under the AUA system and stage TxNxM+ under
the
TNM system. As is the case with locally advanced prostate cancer, surgery is
generally
not indicated for patients with metastatic disease, and hormonal (androgen
ablation)
therapy is the preferred treatment modality. Patients with metastatic prostate
cancer
eventually develop an androgen-refractory state within 12 to 18 months of
treatment
initiation, and approximately half of these patients die within 6 months
thereafter. The
most common site for prostate cancer metastasis is bone. Prostate cancer bone
metastases are, on balance, characteristically osteoblastic rather than
osteolytic (i.e.,
resulting in net bone formation). Bone metastases are found most frequently in
the spine,
followed by the femur, pelvis, rib cage, skull and humerus. Other common sites
for
metastasis include lymph nodes, lung, liver and brain. Metastatic prostate
cancer is
typically diagnosed by open or laparoscopic pelvic lymphadenectomy, whole body
radionuclide scans, skeletal radiography, andlor bone lesion biopsy.
As used herein, the term "polynucleotide" means a polymeric form of
nucleotides of at
least 10 bases or base pairs in length, either ribonucleotides or
deoxynucleotides or a
modified form of either type of nucleotide, and is meant to include single and
double
stranded forms of DNA.
As used herein, the term "polypeptide" means a polymer of at least 10 amino
acids.
Throughout the specification, standard three letter or single letter
designations for amino
acids are used.
As used herein, the terms "hybridize", "hybridizing", "hybridizes" and the
like, used in the
context of polynucleotides, are meant to refer to conventional hybridization
conditions,
preferably such as hybridization in 50% formamidel6XSSC10.1% SDSI100 ~glml
ssDNA, in
which temperatures for hybridization are above 37 degrees C and temperatures
for
washing in 0.1XSSCI0.1% SDS are above 55 degrees C, and most preferably to
stringent
hybridization conditions.
Additional definitions are provided throughout the subsections which follow.
PROSTAPIN POLYNUCLEOTIDES
One aspect of the invention provides polynucleotides corresponding or
complementary
to all or part of the PROSTAPIN gene, mRNA, andlor coding sequence, preferably
in
isolated form, including polynucleotides encoding PROSTAPIN proteins and
fragments
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thereof, DNA, RNA, DNAlRNA hybrid, and related molecules, polynucleotides or
oligonucleotides complementary to the PROSTAPIN gene or mRNA sequence or a
part
thereof, and polynucleotides or oligonucleotides which hybridize to the
PROSTAPIN
gene, mRNA, or to PROSTAPIN-encoding polynucieotides (collectively, "PROSTAPIN
polynucleotides").
A PROSTAPIN polynucleotide may comprise a polynucleotide having the sequence
shown in FIG. 1 (SEQ t0 NO. XX), a sequence complementary thereto, or a
polynucleotide
fragment thereof. Another embodiment comprises a polynucelotide which encodes
the
PROSTAPIN protein amino acid sequence shown in FIG. 1 (SEQ lD NO. XX) or a
polynucteotide fragment thereof. Another embodiment comprises a potynucleotide
which is capable of hybridizing under stringent hybridization conditions to
the
PROSTAPIN cONA shown in FIG. 1 (SEQ ID NO. XX) or to a polynucleotide fragment
thereof. In addition, the invention includes polypeptides derived from
PROSTAPIN
mutants, such as the LAPC-9 mutant PROSTAPIN described herein. Such mutant
PROSTAPIN polynucieotides may comprise the sequence of the LAPC-9 PROSTAPIN
mutant, as shown in FIG. 6 (SEQ ID NO. X), or a polypeptide fragment thereof.
A related
embodiment comprises a polynucleotide which is capable of hybridizing under
stringent
hybridization conditions to the PROSTAPIN mutant cDNA shown in FIG. 6 (SEQ ID
NO. XX)
or to a polynucleotide fragment thereof.
Specifically contemplated are genomic DNA, cDNAs, ribozymes, and antisense
molecules,
as well as nucleic acid molecules based on an alternative backbone or
including alternative
bases, whether derived from natural sources or synthesized. For example,
antisense
molecules can be RNAs or other molecules, including peptide nucleic acids
(PNAs) or
non-nucleic acid molecules such as phosphorothioate derivatives, that
specifically bind
DNA or RNA in a base pair-dependent manner. A skilled artisan can readily
obtain these
classes of nucleic acid molecules using the PROSTAPIN polynucleotides and
polynucieotide sequences disclosed herein.
Further specific embodiments of this aspect of the invention include primers
and primer
pairs, which allow the specific amplification of the polynucleotides of the
invention or of
any specific parts thereof, and probes that selectively or specifically
hybridize to nucleic
acid molecules of the invention or to any part thereof. Probes may be labeled
with a
detectable market, such as, for example, a radioisotope, fluorescent compound,
bioluminescent compound, a chemiluminescent compound, metal chelator or
enzyme.
Such probes and primers can be used to detect the presence of a PROSTAPIN
polynucleotide in a sample and as a means for detecting a cell expressing a
PROSTAPIN
protein. Examples of such probes include polypeptides comprising all or part
of the cDNA
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sequence shown in FIG. 1 (SEQ ID NO. XX). Examples of primer pairs capable of
specifically amplifying PROSTAPIN mRNA are described in the examples which
follow. As
will be understood by the skilled artisan, a great many different primers and
probes may be
prepared based on the sequences provided in FIG. 1 (SEQ ID NO. XX) and used
effectively
to amplify andlor detect PROSTAPIN.
As used herein, a polynucleotide is said to be "isolated" when it is
substantially separated
from contaminant polynucleotides which correspond or are complementary to
genes other
than the PROSTAPIN gene or which encode polypeptides other than PROSTAPIN gene
product or fragments thereof. A skilled artisan can readily employ nucleic
acid isolation
procedures to obtain an isolated PROSTAPIN polynucleotide.
The PROSTAPIN polynucleotides of the invention are useful for a variety of
purposes,
including but not limited to their use as probes and primers for the
amplification andlor
detection of the PROSTAPIN gene(s), mRNA(s), or fragments thereof; as reagents
for the
diagnosis andlor prognosis of prostate cancer; as coding sequences capable of
directing
the expression of PROSTAPIN polypeptides; as tools for modulating or
inhibiting the
expression of the PROSTAPIN genes) andlor translation of the PROSTAPIN
transcript(s); and as therapeutic agents.
METHODS FOR ISOLATING PROSTAPIN-ENCODING NUCLEIC ACID MOLECULES
The PROSTAPIN cDNA sequences described herein enables the isolation of other
polynucieotides encoding the PROSTAPIN gene product(s), as well as the
isolation of
polynucleotides encoding PROSTAPIN gene product homologues, alternatively
sliced
isoforms, allelic variants, and mutant forms of the PROSTAPIN gene product.
Various
molecular cloning methods that can be employed to isolate full length cDNAs
encoding the
PROSTAPIN gene are well known (See, for example, Sambrook, J. et al.,
Molecular Cloning:
A Laboratory Manual, 2d edition., Cold Spring Harbor Press, New York, 1989;
Current
Protocols in Molecular Biology. Ausubel et al., Eds., Wiley and Sons, 1995).
For example,
lambda phage cloning methodologies may be conveniently employed, using
commercially
available cloning systems (e.g., Lambda ZAP Express, Stratagene). Preferably,
cDNA
libraries may be generated from normal testis tissue, placental tissue,
prostate cancer cell
lines, prostate cancer xenografts or another PROSTAPIN-expressing source.
Phage
clones containing PROSTAPIN gene cDNAs may be identified by probing with
labeled
PROSTAPIN cDNA or a fragment thereof. For example, in one embodiment, the
PROSTAPIN cDNA of FIG.1 or a portion thereof can be synthesized and used as a
probe to
retrieve overlapping and full length cDNAs corresponding to the PROSTAPIN-1
gene. The
PROSTAPIN gene itself may be isolated by screening genomic DNA libraries,
bacterial
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art~cial chromosome libraries (BACs), yeast artificial chromosome libraries
(YACs), and
the like, with PROSTAPIN DNA probes or primers.
RECOMBINANT DNA MOLECULES AND HOST-VECTOR SYSTEMS
The invention also provides recombinant DNA or RNA molecules containing a
PROSTAPIN
polynucleotide, including but not limited to phages, plasmids, phagemids,
cosmids, YACs,
BACs, as well as various viral and non-viral vectors well known in the art,
and cells
transformed or transfected with such recombinant ONA or RNA molecules. As used
herein,
a recombinant DNA or RNA molecule is a DNA or RNA molecule that has been
subjected to
molecular manipulation in vitro. Methods for generating such molecules are
well known
(see, for exampte, Sambrook et a1,1989, supra).
The invention further provides a host-vector system comprising a recombinant
DNA
molecule containing a PROSTAPtN polynucleotide within a suitable prokaryotic
or
eukaryotic host cell. Examples of suitable eukaryotic host cells include a
yeast cell, a
plant cell, or an animal cell, such as a mammalian cell. Examples of suitable
mammalian
cells include various prostate cancer cell lines such LnCaP, PC-3, DU145, LAPC-
4,
TsuPrl, other transfectable or transducibie prostate cancer cell lines, as
well as a
number of mammalian cells routinely used for the expression of recombinant
proteins
(e.g., COS, CHO, 293, 293T cells). More particularly, a polynucleotide
comprising the
coding sequence of PROSTAPIN may be used to generate PROSTAPIN proteins or
fragments thereof using any number of host-vector systems routinely used and
widely
known in the art.
A wide range of host vector systems suitable for the expression of PROSTAPIN
proteins or
fragments thereof are available, see for example, Sambrook et al., 1989,
supra; Current
Protocols in Molecular Biology,1995, supra). Preferred vectors for mammalian
expression
include but are not limited to pcDNA 3.1 myc-His-tag (Invitrogen) and the
retroviral vector
pSRatkneo (Muller et al., 1991, MCB 11:1785). Using these expression vectors,
PROSTAPIN may be preferably expressed in several prostate cancer cell lines,
including
for example PC-3, LNCaP and TsuPrl. The host-vector systems of the invention
are
useful for the production of a PROSTAPIN protein or fragment thereof. Such
host-vector
systems may be employed to study the functional properties of PROSTAPIN and
PROSTAPIN mutations.
Proteins encoded by the PROSTAPIN gene, or by fragments thereof, will have a
variety of
uses, including but not limited to generating antibodies, as therapeutic
agents, and in
methods for identifying ligands and other agents and cellular constituents
that bind to a
PROSTAPIN gene product. Antibodies raised against PROSTAPIN proteins or
fragments
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thereof may be useful in diagnostic and prognostic assays, imaging
methodologies, and
therapeutic methods in the management of prostate cancer. Various
immunological
assays useful for the detection of PROSTAPIN proteins are contemplated,
including but not
limited to various types of radioimmunoassays, enryme-linked immunosorbent
assays
(ELISA), enzyme-linked immunofluorescent assays (ELIFA), immunocytochemical
methods,
and the like. Such antibodies may be labeled and used as immunological imaging
reagents
capable of detecting prostate cells (e.g., in radioscintigraphic imaging
methods).
PROSTAPIN PROTEINS
Another aspect of the present invention provides various PROSTAPIN proteins
and
polypeptide fragments thereof. As used herein, PROSTAPIN refers to a protein
that has or
includes the amino acid sequence of human PROSTAPIN as provided in FIG. 1 (SE4
ID NO.
XX), the amino acid sequence of other mammalian PROSTAPIN homologues, as well
as
allelic variants and conservative substitution mutants of these proteins that
have
PROSTAPIN activity. The PROSTAPIN proteins of the invention include those
specifically
identified herein, as well as allelic variants, conservative substitution
variants and
homologs that can be isolatedlgenerated and characterized without undue
experimentation
following the methods outlined below. Such PROSTAPIN proteins will be
collectively
referred to as the PROSTAPIN proteins, the proteins of the invention, or
PROSTAPIN. As
used herein, the term "PROSTAPIN polypeptide" refers to a polypeptide fragment
or a
PROSTAPIN protein of at least 10 amino acids, preferably at least 15 amino
acids, and more
preferably at least 20 amino acids.
A specific embodiment of a PROSTAPIN protein comprises a polypeptide having
the amino
acid sequence shown in FIG. 1 (SEQ ID NO. XX). As used herein, the term "wild-
type
PROSTAPIN" is meant to refer to a protein having the amino acid sequence
depicted in FIG.
1 (SEQ ID NO. XX). In general, for example, naturally occurring allelic
variants of human
PROSTAPIN will share significant homology (e.g., 70 - 90%) to the PROSTAPIN
amino acid
sequence provided in FIG. 1. Typically, allelic variants of the PROSTAPIN
protein will
contain conservative amino acid substitutions from the PROSTAPIN sequence
herein
described or will contain a substitution of an amino acid from a corresponding
position in a
PROSTAPIN homologue.
One class of PROSTAPIN allelic variants will be proteins that share a high
degree of
homology with at least a small region of the PROSTAPIN amino acid sequence,
but will
further contain a radical departure form the sequence, such as a non-
conservative
substitution, truncation, insertion or frame shift. Such alleles are termed
mutant alleles of
PROSTAPIN and represent proteins that typically do not perform the same
biological
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functions. Mutant PROSTAPIN proteins having altered biological function are
also included
within the scope of the invention. As used herein, the term "functional
mutant", when used
to modify the term PROSTAPIN, is meant to refer to a PROSTAPIN polypeptide
which
contains one or more mutations that alter or eliminate PROSTAPIN biological
activity,
including the LAPC-9 mutant described herein (FIG. 6; SEQ ID NO. XX). Thus,
the invention
also provides mutant PROSTAPIN proteins and mutant PROSTAPIN polypeptides,
such as
those corresponding to the amino acid sequences encoded by the IAPC-9
PROSTAPIN
mutant as shown in FIG. 6 (SEQ ID NO. XX).
Conservative amino acid substitutions can frequently be made in a protein
without
altering either the conformation or the function of the protein. Such changes
include
substituting any of isoleucine (I), valine (V), and leucine (L) for any other
of these
hydrophobic amino acids; aspartic acid (D) for glutamic acid (E) and vice
versa;
glutamine (Q) for asparagine (N) and vice versa; and serine (S) for threonine
(T) and vice
versa. Other substitutions can also be considered conservative, depending on
the
environment of the particular amino acid and its role in the three-dimensional
structure of
the protein. For example, glycine (G) and alanine (A) can frequently be
interchangeable,
as can alanine (A) and valine (V). Methionine (M), which is relatively
hydrophobic, can
frequently be interchanged with leucine and isoleucine, and sometimes with
valine.
Lysine (K) and arginine (R) are frequently interchangeable in locations in
which the
significant feature of the amino acid residue is its charge and the differing
pK's of these
two amino acid residues are not significant. Still other changes can be
considered
"conservative" in particular environments.
PROSTAPIN proteins may be embodied in many forms, preferably in isolated form.
As
used herein, a protein is said to be "isolated" when physical, mechanical or
chemical
methods are employed to remove the PROSTAP1N protein from cellular
constituents that
are normally associated with the protein. A skilled artisan can readily employ
standard
pur~cation methods to obtain an isolated PROSTAPIN protein. A purified
PROSTAPIN
protein molecule will be substantially free of other proteins or molecules
which impair the
binding of PROSTAPIN to antibody or other ligand. The nature and degree of
isolation and
purification will depend on the intended use. Embodiments of the PROSTAPIN
protein
include a purified PROSTAPIN protein and a functional, soluble PROSTAPIN
protein. In
one form, such functional, soluble PROSTAPIN proteins or fragments thereof
retain the
ability to bind antibody or other ligand.
The invention also provides PROSTAP1N polypeptides comprising biologically
active
fragments of the PROSTAPIN amino acid sequence, such as a poiypeptide
corresponding
to part of the amino acid sequence shown in FIG. 1 (SEQ !D' NO. XX). Such
polypeptides
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WO 99158560 PCT/US99/07123
of the invention exhibit properties of PROSTAPIN, such as the ability to
elicit the
generation of antibodies which specifically bind an epitope associated with
PROSTAPIN.
PROSTAPIN poiypeptides can be generated using standard peptide synthesis
technology
S and the amino acid sequences of the human PROSTAPIN protein disclosed
herein.
Alternatively, recombinant methods can be used to generate nucleic acid
molecules that
encode a poiypeptide fragment of the PROSTAPIN protein. In this regard, the
PROSTAPIN-
encoding nucleic acid molecules described herein provide means for generating
defined
fragments of PROSTAPIN. PROSTAPIN polypeptides are particularly useful in
generating
domain speck antibodies, identifying agents or cellular factors that bind to
PROSTAPIN or
a PROSTAPIN domain, and in prostate cancer therapeutic strategies which
comprise the
restoration of PROSTAPIN functionality. PROSTAPIN polypeptides containing
particularly
interesting structures can be predicted andlor identified using various
analytical
techniques well known in the art, including, for example, the methods of Chou-
Fasman,
Gamier-Robson, Kyte-Ooolittle, Eisenberg, Karplus-Schultz or Jameson-Wolf
analysis, or
on the basis of immunogenicity. Fragments containing such structures are
particularly
useful in generating subunit specific anti-PROSTAPIN antibodies or in
identifying cellular
factors that bind to PROSTAPIN.
PIZOSTAPIN ANTIBODIES
Another aspect of the invention provides antibodies that bind to PROSTAPIN
proteins and
polypeptides. The most preferred antibodies will selectively bind to PROSTAPIN
and will
not bind (or will bind weakly) to non-PROSTAPIN proteins and polypeptides.
Anti-
PROSTAPIN antibodies that are particularly contemplated include monoclonal and
polyclonal antibodies as well as fragments containing the antigen binding
domain andlor
one or more complement determining regions of these antibodies. As used
herein, an
antibody fragment is defined as at least a portion of the variable region of
the
immunoglobulin molecule which binds to its target, i.e., the antigen binding
region.
As used herein, a PROSTAPIN antibody is an antibody which (1 ) was raised
against a
preparation comprising a PROSTAPIN protein, a PROSTAPIN polypeptide, a mutant
PROSTAPIN protein or polypeptide, a fusion protein comprising any of the
foregoing, a cell
preparation containing PROSTAPIN protein or polypeptide, a cell engineered to
express a
PROSTAPIN protein or polypeptide, or a similar PROSTAPIN immunogen, andlor (2)
binds
to a PROSTAPIN andlor mutant PROSTAPIN protein or polypeptide.
PROSTAPIN antibodies of the invention may be particularly useful in prostate
cancer
diagnostic and prognostic assays, imaging methodologies, and therapeutic
strategies.
The invention provides various immunological assays useful for the detection
and
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WO 99/58560 PCT/US99/07123
quantification of PROSTAPIN and mutant PROSTAPIN proteins and polypeptides.
Such
assays generally comprise one or more PROSTAPIN antibodies capable of
recognizing and
binding a PROSTAPIN or mutant PROSTAPIN protein, as appropriate, and may be
performed within various immunological assay formats well known in the art,
including but
not limited to various types of radioimmunoassays, enzyme-linked immunosorbent
assays
(ELISA), enzyme-linked immunofluorescent assays (ELIFA), and the like. In
addition,
immunological imaging methods capable of detecting prostate cancer are also
provided by
the invention, including but limited to radioscintigraphic imaging methods
using labeled
PROSTAPIN antibodies. Such assays may be clinically useful in the detection,
monitoring,
and prognosis of prostate cancer, particularly advanced prostate cancer.
PROSTAPIN antibodies may also be used in methods for purifying PROSTAPIN and
mutant
PROSTAPIN proteins and polypeptides and for isolating PROSTAPIN homologues and
related molecules. For example, in one embodiment, the method of purifying a
PROSTAPIN
protein comprises incubating a PROSTAPIN antibody, which has been coupted to a
solid
matrix, with a lysate or other solution containing PROSTAPIN under conditions
which
permit the PROSTAPIN antibody to bind to PROSTAPIN; washing the solid matrix
to
eliminate impurities; and eluting the PROSTAPIN from the coupled antibody.
Other uses of
the PROSTAPIN antibodies of the invention include generating anti-idiotypic
antibodies
that mimic the PROSTAPIN protein.
Various methods for the preparation of antibodies are well known in the art.
For example,
antibodies may be prepared by immunizing a suitable mammalian host using a
PROSTAPIN
protein, peptide, or fragment, in isolated or immunoconjugated form
(Antibodies: A
Laboratory Manual, CSH Press, Eds., Harlow, and Lane (1988); Harlow,
Antibodies, Cold
Spring Harbor Press, NY (1989)). In addition, fusion proteins of PROSTAPIN may
also be
used, such as a PROSTAPIN GST~fusion protein. In a particular embodiment, a
GST fusion
protein comprising all or most of the open reading frame amino acid sequence
of FIG. 1
may be produced and used as an immunogen to generate appropriate antibodies.
Cells
expressing or overexpressing PROSTAPIN may also be used for immunizations.
Similarly,
any cell engineered to express PROSTAPIN may be used. This strategy may result
in the
production of monoclonal antibodies with enhanced capacities for recognizing
endogenous
PROSTAPIN.
The amino acid sequence of PROSTAPIN as shown in FIG. 1 (SEQ ID NO. XX) may be
used
to select specific regions of the PROSTAPIN protein for generating antibodies.
For
example, hydrophobicity and hydrophilicity analyses of the PROSTAPIN amino
acid
sequence may be used to identify hydrophilic regions in the PROSTAPIN
structure.
Regions of the PROSTAPIN protein that show immunogenic structure, as well as
other
CA 02324206 2000-09-29
WO 99/58560 PCT/US99/07123
regions and domains, can readily be identified using various other methods
known in the
art, such as Chou-Fasman, Garnier-Robson, Kyte-Doolittle, Eisenberg, Karplus-
Schultz or
Jameson-Wolf analysis. For the generation of antibodies which specifically
recognize a
mutant PROSTAPIN protein, amino acid sequences unique to the mutant (relative
to wild
type PROSTAPIN) are preferable. For example, for generating antibodies to the
LAPC-9
mutant PROSTAPIN protein, the inserted or unique amino acid sequences shown in
FIG. 6
(SEQ 10 NO. XX) may be used to select specific regions.
Methods for preparing a protein or polypeptide for use as an immunogen and for
preparing
immunogenic conjugates of a protein with a carrier such as BSA, KLH, or other
carrier
proteins are well known in the art. In some circumstances, direct conjugation
using, for
example, carbodiimide reagents may be used; in other instances finking
reagents, such as
those supplied by Pierce Chemical Co., Rockford, IL, may be effective.
Administration of a
PROSTAPIN immunogen is conducted generally by injection over a suitable time
period and
with use of a suitable adjuvant, as is generally understood in the art. During
the
immunization schedule, titers of antibodies can be taken to determine adequacy
of antibody
formation.
PROSTAPIN monoclonal antibodies are preferred and may be produced by various
means
well known in the art. For example, immortalized cell lines which secrete a
desired
monoclonal antibody may be prepared using the standard method of Kohler and
Milstein or
modifications which effect immortalization of lymphocytes or spleen cells, as
is generally
known. The immortalized cell lines secreting the desired antibodies are
screened by
immunoassay in which the antigen is the PROSTAPIN protein or PROSTAPIN
fragment.
When the appropriate immortalized cell culture secreting the desired antibody
is identified,
the cells can be cultured either in vitro or by production in ascites fluid.
The desired monoclonal antibodies are then recovered from the culture
supernatant or
from the ascites supernatant. Fragments of the monoclonals or the polyclonal
antiserum
which contain the immunologically significant portion can be used as
antagonists, as well
as the intact antibodies. Use of immunologically reactive fragments, such as
the Fab, Scfy,
or F(ab')Z fragments is often preferable, especially in a therapeutic context,
as these
fragments are generally less immunogenic than the whole immunoglobulin.
The antibodies or fragments may also be produced, using current technology, by
recombinant means. Regions that bind spec~cally to the desired regions of the
PROSTAPIN protein can also be produced in the context of chimeric or CDR
grafted
antibodies of multiple species origin. Humanized or human PROSTAPIN antibodies
may
also be produced and are preferred for use in therapeutic contexts. Various
approaches
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WO 99/58560 PCT/US99/07123
for producing such humanized antibodies are known, and include chimeric and
CDR
grafting methods; methods for producing fully human monoclonal antibodies
include
phage display and transgenic methods (for review, see Vaughan et al., 1998,
Nature
Biotechnology 16: 535-539).
Fully human PROSTAPIN monoclonal antibodies may be generated using cloning
technologies employing large human Ig gene combinatorial libraries (i.e.,
phage
display)(Griffiths and Hoogenboom, Building an in vitro immune system: human
antibodies
from phage display libraries. In: Protein Engineering of Antibody Molecules
for
Prophylactic and Therapeutic Applications in Man. Clark, M. (Ed.), Nottingham
Academic,
pp 45-64 (1993); Burton and Barbas, Human Antibodies from combinatorial
libraries. Id., pp
65-82). Fully human PROSTAP1N monoclonal antibodies may also be produced using
transgenic mice engineered to contain human immunoglobulin gene loci as
described in
PCT Patent Application W098124893, Jakobovits et al., published December 3,
1997 (see
also, Jakobovits, 1998, Exp. Opin. Invest. Drugs 7(4): 607-614). This method
avoids the in
vitro manipulation required with phage display technology and efficiently
produces high
affinity authentic human antibodies.
Reactivity of PROSTAPIN antibodies with PROSTAPIN protein or mutant PROSTAPIN
protein, as appropriate, may be established by a number of well known means,
including
Western blot, immunoprecipitation, ELISA, and FACS analyses using, as
appropriate,
PROSTAPIN proteins, peptides, PROSTAPIN-expressing cells or extracts thereof.
A PROSTAPIN antibody or a fragment thereof may be labeled with a detectable
marker
and used for targeting the detectable marker to a PROSTAPIN positive cell
(Vitetta, E.S.
et al., 1993, Immunotoxin therapy, in DeVita, Jr., V.T. et al., eds, Cancer:
Principles and
Practice of Oncology, 4th ed., J.B. Lippincott Co., Philadelphia, 2624-2636).
Suitable
detectable markers include, but are not limited to, a radioisotope, a
fluorescent
compound, a bioluminescent compound, chemiluminescent compound, a metal
chelator
or an enzyme.
METHODS FOR THE DETECTION OF PROSTAPIN
Another aspect of the present invention relates to methods for detecting
PROSTAPIN
polynucleotides and PROSTAPIN proteins, as well as methods for identifying a
cell which
expresses PROSTAPIN.
More particularly, the invention provides assays for the detection of
PROSTAPIN
polynucleotides in a biological sample, such as serum, bone, prostate, and
other tissues,
urine, cell preparations, and the like. Detectable PROSTAPIN polynucleotides
include, for
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CA 02324206 2000-09-29
WO 99/58560 PCT/US99/07123
example, a PROSTAPIN gene or fragments thereof, PROSTAPIN mRNA, alternative
splice
variant PROSTAPIN mRNAs, and recombinant ONA or RNA molecules containing a
PROSTAPIN polynucleotide. A number of methods for amplifying andlor detecting
the
presence of PROSTAPIN polynucleotides are well known in the art and may be
employed in
the practice of this aspect of the invention.
In one embodiment, a method for detecting PROSTAPIN mRNA in a biological
sample
comprises producing cDNA from the sample by reverse transcription using at
least one
primer; amplifying the cDNA so produced using PROSTAPIN polynucleotides as
sense
and antisense primers to amplify PROSTAPIN cDNAs therein; and detecting the
presence
of the amplified PROSTAPIN cDNA. In another embodiment, a method of detecting
the
PROSTAPIN gene in a biological sample comprises first isolating genomic DNA
from the
sample; amplifying the isolated genomic DNA using PROSTAPIN polynucleotides as
sense and antisense primers to amplify the PROSTAPIN gene therein; and
detecting the
presence of the amplified PROSTAPIN gene. Any number of appropriate sense and
antisense probe combinations may be designed from the nucleotide sequence
provided
in FIG. 1 (SEQ ID NO. XX) and used for this purpose, as will be understood by
those skilled
in the art.
The invention also provides assays for detecting the presence of a PROSTAPIN
protein in a
tissue of other biological sample such as serum, bone, prostate, and other
tissues, urine,
cell preparations, and the like. Methods for detecting a PROSTAPIN protein are
also well
known and include, for example, immunoprecipitation, immunohistochemical
analysis,
Western Blot analysis, molecular binding assays, ELISA, E~1FA and the like.
For example, in one embodiment, a method of detecting the presence of a
PROSTAPIN
protein in a biological sample comprises first contacting the sample with a
PROSTAPIN
antibody, a PROSTAPIN-reactive fragment thereof, or a recombinant protein
containing
an antigen binding region of a PROSTAPIN antibody; and then detecting the
binding of
PROSTAPIN protein in the sample thereto.
Methods for identifying a cell which expresses PROSTAPIN are also provided. in
one
embodiment, an assay for identifying a cell which expresses a PROSTAPIN gene
comprises
detecting the presence of PROSTAPIN mRNA in the cell. Methods for the
detection of
particular mRNAs in cells are well known and include, for example,
hybridization assays
using complementary DNA probes (such as in situ hybridization using labeled
PROSTAPIN
rtboprobes, Northern blot and related techniques) and various nucleic acid
amplification
assays (such as RT-PCR using complementary primers specific for PROSTAPIN, and
other
amplification type detection methods, such as, for example, branched DNA,
SISBA, TMA
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WO 99/58560 PC'f/US99/07123
and the like). Alternatively, an assay for identifying a cell which expresses
a PROSTAPIN
gene comprises detecting the presence of PROSTAPIN protein in the cell or
secreted by
the cell. Various methods for the detection of proteins are well known in the
art and may be
employed for the detection of PROSTAPIN proteins and PROSTAPIN expressing
cells.
PROSTAPIN expression analysis may also be useful as a tool for identifying and
evaluating
agents which modulate PROSTAPIN gene expression. PROSTAPIN expression is
significantly reduced in prostate cancer samples, prostate cancer xenografts
and cell
lines. The mechanism of inactivation is unclear, since southern blotting of
DNA derived
from the xenografts (LAPC-4 AD, 4AI, 9AD), prostate cancer cell lines (PC-3,
DU145,
LNCaP) and normal human DNA show no remarkable differences in intensity or
banding
pattern. Similar observations were made for the tumor suppressor gene
PTENIMMAC1,
which encodes a dual-specificity phosphatase (Whang et al., 1998, PNAS 95:
5246).
PTENIMMAC1 mRNA expression was restored in nonexpressing prostate cancer cells
by
in vitro treatment with the demethylating agent 5-azadeoxycytidine (Whang et
al., 1998,
PNAS 95: 5246). This suggests that methylation was responsible for silencing
of the
PTENIMMAC1 gene. A similar mechanism of transcriptional inactivation may
explain loss
of PROSTAPIN expression in some of the prostate cancer specimens.
Identification of a
molecule or biological agent that could reactivate PROSTAPIN expression may be
of
therapeutic value in the treatment of prostate cancer. Such an agent may be
identified
by using a screen that allows for recognizing the acquisition of PROSTAPIN
expression
by RT-PCR, nucleic acid hybridization or antibody binding.
As will be appreciated, the foregoing methods may be applied to the detection
of mutant
PROSTAPIN poiynucleotides and proteins using, as appropriate, probes, primers,
antibodies and other binding agents capable of detecting such mutant forms.
ASSAYS FOR DETERMINING PROSTAPIN EXPRESSION STATUS
PROSTAPIN gene expression appears to be lost or greatly attenuated in advanced
prostate
cancers. Thus, determining the status of PROSTAP1N expression in an individual
may be
used to diagnose advanced stage prostate cancer as well as provide prognostic
information useful in defining appropriate therapeutic options. Similarly, the
expression
status of PROSTAPIN may provide information useful for predicting
susceptibility to
advanced stage disease, rate of progression, andlor tumor aggressiveness. The
invention
provides methods and assays for determining PROSTAPIN expression status,
diagnosing
advanced prostate cancer, and predicting susceptibility to advanced prostate
cancer.
PROSTAPIN expression status is meant to include quantitative andlor
qualitative aspects,
i.e., the level of wild type PROSTAPIN expression as well as the presence of
functional
PROSTAPIN mutations.
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WO 99/58560 PCT/US99/07123
In one aspect, the invention provides assays useful in determining the
presence of
advanced stage prostate cancer in an individual. Presently, advanced stage
prostate
cancer is commonly diagnosed by pathological examination of prostate and
surrounding
tissues surgically removed during radical prostatectomy. Unfortunately, in
most cases of
advanced stage prostate cancer, surgery is not desirable, but is generally
performed
because there is no method of reliably distinguishing between advanced and
localized
prostate cancer other than pathological examination of surgically removed
tissues. In
other words, for most patients who learn that they have advanced prostate
cancer, the
undesirable surgical option has already been performed. The invention provides
a
means of distinguishing between advanced prostate cancer and locally confined
prostate
cancer by assaying for PROSTAPIN expression. The means comprises detecting a
marked loss or absence of wild type PROSTAPIN expression in prostate tumor
tissues
and cells relative to expression levels in normal prostate tissue and cells.
As
demonstrated in the Examples which follow, PROSTAPIN mRNA is expressed at
easily
detectable levels in all normal prostate tissues and all locally confined
prostate cancer
tissues tested. In contrast, wild type PROSTAPIN expression is either
completely
undetectable or greatly attenuated in all advanced stage prostate tumor
specimens, cell
lines derived from prostate cancer metastases, and SCID mouse xenografts
derived from
human prostate cancer metastases.
In one embodiment, a method or assay for identifying the presence of advanced
prostate
cancer comprises determining the level of PROSTAPIN mRNA expressed by cells in
a test
sample, preferably a prostate, prostate tumor, lymph, bone or peripheral blood
sample; and
2$ comparing the level so determined to the level of PROSTAPIN expressed in
normal
prostate, preferably a comparable known normal prostate tissue sample. The
absence or
substantial attenuation of PROSTAPIN mRNA expression in the test sample
relative to
normal prostate indicates the presence of advanced prostate cancer.
Attenuation of
PROSTAPIN mRNA expression is "substantial" when expression is reduced by at
least
about 10%, and preferably by about 30-50% or more, relative to PROSTAPIN mRNA
expression levels detectable in normal prostate.
in a related embodiment, PROSTAPIN expression status may be determined at the
protein
level rather than at the nucleic acid level. For example, such a method or
assay would
comprise determining the level of PROSTAPIN protein expressed by cells in a
test sample,
preferably a prostate, prostate tumor, lymph, bone or peripheral blood sample;
and
comparing the level so determined to the level of PROSTAPIN expressed in
normal
prostate, preferably a comparable known normal prostate tissue sample. The
absence or
substantial attenuation of PROSTAPIN protein expression in the test sample
relative to
CA 02324206 2000-09-29
WO 99/58560 PCT/US99/07123
normal prostate indicates the presence of advanced prostate cancer. PROSTAPIN
antibodies or binding partners capable of detecting PROSTAPIN protein
expression may be
used in a variety of assay formats well known in the art for this purpose.
A specific, preferred embodiment comprises determining the expression status
of a
patient's PROSTAPIN mRNA or PROTEIN in the cells of a known prostate tumor
sample and
comparing the level of PROSTAPIN expression so determined to the level
expressed by
normal prostate cells, the presence of comparable expression levels being
indicative of a
locally confined or less advanced stage. In this regard, prostate tumor cells
may be
"known" by virtue of their origin, e.g., biopsied from a tumor mass, or by the
presence of
one or more molecular markers of prostate cancer cells. A number of such
molecular
markers are known, including for example PSCA and PSMA.
Assaying the expression status of a prostate cancer marker and PROSTAPIN in
the same
tissue sample, preferably simultaneously, may be particularly useful where
tumor origin of
the sample cannot be assured. In such cases, the presence of a known prostate
cancer
molecular marker in the sample can be used to identify the sample as prostate
cancer,
while the level of PROSTAPIN expressed in the same sample may be used as a
tool for
determining the presence of advanced prostate cancer (as well as
susceptibility to
advanced prostate cancer and tumor aggressiveness). In a speck embodiment,
expression of PSCA and PROSTAPIN in a sample tissue are assayed together. PSCA
is
widely over-expressed across all stages and grades of prostate cancer. Thus
the presence
of PSCA over-expression relative to expression levels in normal prostate may
be used to
reliably identify samples which comprise prostate cancer cells.
Depending on the method of detection used, a single sample may be
heterogeneous for the
expression of the known tumor marker. For example, a sample may be shown to
contain
some regions of cells expressing (or over-expressing, as appropriate) the
marker while
other regions do not express the marker (or expressing normal levels of the
marker). In
such cases, it may be most appropriate to use the level of PROSTAPIN
expression in the
regions showing expression or over-expression of the marker in order to
reliably determine
that patient's prostate cancer stage. In other cases, the tissue sampled is
inherently
heterogeneous for a number of cell types, such as, for example, blood. Here,
the presence
of the known prostate cancer marker may be used to identify andlor isolate the
prostate
cancer cells from other cells present in the sample. The PROSTAPIN expression
status in
the known prostate cancer marker positive cells should be used for staging
purposes. This
type of combined analysis may be used not only for determining locally
confined cancers,
but also for determining advanced stage cancers, aggressiveness and
susceptibility to
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WO 99/58560 PCT/US99/07123
advanced stage cancer, by assaying PROSTAPiN expression as described above
together
with a known prostate cancer marker.
Peripheral blood may be conveniently assayed by the combined analysis
described above
using RT-PCR to detect and quantify the expression of PROSTAPIN and known
prostate
tumor marker mRNAs. RT-PCR amplification of a known tumor marker mRNA combined
with the absence or attenuation of RT-PCR amplifiable PROSTAPIN mRNA (relative
to
normal prostate expression levels) provides an indication of the presence of
advanced
prostate cancer and may provide information concerning the aggressiveness of
the
originating tumor. RT-PCR detection assays for tumor cells in peripheral blood
are
currently being evaluated for use in the diagnosis and management of a number
of human
solid tumors. In the prostate cancer field, these include RT-PCR assays for
the detection of
cells expressing PSA and PSM (Verkaik et al.,1997, Urol. Res. 25: 373-384;
Ghossein et al.,
1995, J. Clin. Oncol. 13: 1195-2000; Heston et al., 1995, Clin. Chem. 41: 1687-
1688). RT-
PCR assays are well known in the art. Semi-quantitative RT-PCR assays for
PROSTAPIN
expression are described in greater detail by way of the examples which
follow. Such
assays may also be employed for the detection (and quantitation) of a known
prostate
tumor marker.
A related aspect of the invention is directed to predicting susceptibility to
developing
advanced prostate cancer in an individual. In one embodiment, a method for
predicting
susceptibility to advanced prostate cancer comprises determining the level of
PROSTAPIN
mRNA or PROSTAPIN protein expressed by cells in a first prostate or prostate
tumor
sample, comparing the level so determined to the level of PROSTAPIN mRNA or
PROSTAPIN protein expressed in a second normal prostate tissue, the absence or
substantial attenuation of PROSTAPIN mRNA or PROSTAPIN protein expression in
the first
sample relative to the second sample indicating susceptibility to advanced
prostate cancer,
wherein the degree of attenuated PROSTAPIN expression relative to normal
prostate is
proportional to the degree of susceptibility to advanced prostate cancer.
Yet another related aspect of the invention is directed to methods for gauging
prostate
tumor aggressiveness. In one embodiment, a method for gauging aggressiveness
of a
prostate tumor comprises determining the level of PROSTAPIN mRNA or PROSTAPIN
protein expressed by cells in a sample of the prostate tumor, comparing the
level so
determined to the level of PROSTAPIN mRNA or PROSTAPIN protein expressed in a
normal
prostate tissue taken from the same individual or a normal prostate tissue
reference
sample, wherein the degree of PROSTAPIN mRNA or PROSTAPIN protein expression
loss
in the prostate tumor sample relative to the normal prostate sample
proportionally
indicating degree of aggressiveness.
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WO 99/58560 PCTNS99/07123
Methods for detecting and quantifying the expression of PROSTAPIN mRNA or
protein are
described herein and use standard nucleic acid and protein detection and
quantification
technologies well known in the art. Standard methods for the detection and
quant~cation
of PROSTAPIN mRNA include in situ hybridization using labeled PROSTAPIN
riboprobes,
Northern blot and related techniques using PROSTAPIN polynucleotide probes, RT-
PCR
analysis using primers specific for PROSTAPIN, and other amplification type
detection
methods, such as, for example, branched DNA, SISBA, TMA and the like. In a
specific
embodiment, semi-quantitative RT-PCR may be used to detect and quantify
PROSTAPIN
mRNA expression as described in the Examples which follow. Any number of
primers
capable of amplifying PROSTAPIN may be used for this purpose, including but
not limited to
the various primer sets specifically described herein. Standard methods for
the detection
and quantification of protein may be used for this purpose. In a specific
embodiment,
polyclonal or monoclonal antibodies spec~cally reactive with the wild-type
PROSTAPIN
protein may be used in an immunohistochemical assay of biopsied tissue.
Since loss of PROSTAPIN expression appears to correlate with advanced disease
stage,
the expression of normal levels of PROSTAPIN by prostate cancer cells may also
be useful
in identifying locally confined prostate cancer. The invention provides
methods and assays
for identifying locally confined prostate cancer comprising determining the
expression
status of a patient's PROSTAPIN mRNA or PROTEIN in the cells of a known
prostate tumor
sample and comparing the level of PROSTAPIN expression so determined to the
level
expressed by normal prostate cells, the presence of comparable expression
levels being
indicative of a locally confined or less advanced stage.
In addition to the methods and assays described above, wherein the expression
levels of
PROSTAPIN are determined and evaluated, the invention also provides methods
and
assays capable of detecting functional mutations of the PROSTAPIN gene.
Similar to the
loss or attenuation of PROSTAPIN expression, the presence of a functional
PROSTAPIN
mutation also correlates with advanced prostate cancer and may be used to
distinguish
advanced from locally confined prostate cancers, predict aggressiveness, and
determine
susceptibility to advanced prostate cancer. The general molecular diagnostic
methods
described above may be used for this purpose, provided that the means used to
detect
expression are capable of specifically identifying a PROSTAPIN mutation
expected to result
in a loss of PROSTAPIN function. In this regard, for detection of PROSTAPIN
mutant
mRNAs, molecular probes or primers specifically designed to hybridize to or
amplify the
mutant PROSTAPIN, but not wild-type PROSTAPIN are used. In a specific
embodiment, a
hybridization probe comprising the nucleotide sequence of the LAPC-9 PROSTAPIN
mutant
as shown in FIG. 6 (SEQ ID NO. XX) may be used. Alternatively, a probe
comprising a
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WO 99/58560 PC1'/US99/07123
fragment of the sequence shown in FIG. 6 (SEQ ID NO. XX) which contains enough
of the
mutant sequence to render it capable of specifically hybridizing to mutant but
not wild type
PROSTAPIN may be used. In another embodiment, primers designed to PCR amplify
polynucleotides containing the sequences specific to the LAPC-9 mutant
PROSTAPIN
sequence shown in FIG. 6 (SEQ ID NO. XX), may be used to detect the expression
of a
functional PROSTAPIN mutant. In another embodiment, primers designed to
amplify
polynucieotides corresponding to either wild type or mutant PROSTAPIN may be
used to
amplify PROSTAPIN sequences which may then be sequenced and analyzed for the
presence of mutations. Functional PROSTAPIN mutants may also be identified at
the
genomic level, by direct sequencing or by SSCP analysis of genomic DNA to
identify
PROSTAPIN mutations or polymorphisms that correlate with prostate cancer.
Mutant or
polymorphic exons can be sequenced and compared to wild type PROSTAPIN using
standard technologies. In one embodiment, the primer pairs described in
Example 8 may
be used to sequence particular PROSTAPIN axons.
THERAPEUTIC APPLICATIONS OF PjtOSTAPIN
Loss of wild type PROSTAPIN expression or the expression of functionally
mutant
PROSTAPIN correlates with advanced and metastatic prostate cancer.
Structurally,
PROSTAPIN is a member of a family of proteins which contain both tumor
suppressors
(e.g., maspin) and proteins involved in apoptosis (e.g., LEI). Accordingly,
the PROSTAPIN
protein may function as a prostate-specific tumor suppressor, apoptosis-
inducer or
apoptosis-modulator, or may have another biological activity involved in
modulating
prostate cancer progression. Therapeutic strategies which restore functional
PROSTAPIN
to prostate tumor cells may result in inhibition of primary prostate tumors
and prostate
cancer metastasis, tumor regression, andlor an inhibition in the rate or
extent of disease
progression.
Various strategies for restoring normal PROSTAPIN function in vivo are
available, including
protein therapy and gene therapy methods. For gene therapy, a vector
comprising a
polynucleotide encoding wild type PROSTAPIN or a peptide mimetic with
PROSTAPIN
biological activity may be administered to the prostate cancer patient such
that the vector
makes contact with the prostate tumor cells. Preferably, the vector will be
capable of
integrating the PROSTAPIN gene into the patient's tumor cells (e.g.,
retroviral vectors)
andJor is capable of highly efficient in vivo transduction (e.g., adenoviral
vectors). The
vector may be delivered via any route which results in the vector making
contact with the
tumor cells. A preferred route of administration is by intraprostatic
injection. Multiple
injections may be required to account for clearance of the initial dose and
achieve more
uniform distribution of the vector to the tumor. Alternatively, compositions
comprising the
wild type PROSTAPIN protein or a peptide mimetic or a small molecule mimetic
may be
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WO 99/58560 PCTlUS99/07123
administered to a patient such that the composition makes contact with the
tumor cells. In
addition, methods capable of inducing transcription of functional PROSTAPIN in
vivo may
be employed.
Preferably, functional PROSTAPIN restoration is accomplished via gene transfer
methods,
such as those further described below. For example, if PROSTAPIN functions as
an
apoptosis-inducing gene, gene therapy transfer of PROSTAPIN into prostate
tumor cells
may be used to trigger apoptosis of the tumor cells. If PROSTAPIN functions as
a prostate-
spec~c tumor suppressor gene, in vivo PROSTAPIN gene restoration therapy may
be
useful to slow or reverse prostate cancer cell growth.
A PROSTAPIN polynucleotide encoding wild type PROSTAPIN may be operably linked
to
a promoter capable of driving the expression of functional PROSTAPIN within
the cells of
the target tumor and utilized for gene therapy. Preferably, expression of the
PROSTAPIN
gene will be regulated by a prostate-specific promoter is utilized. An example
of a
preferred promoter is the PSA promoter.
Various gene therapy vectors may be used to deliver the PROSTAPIN gene into
the cells
of the target tissue (e.g., prostate, prostate tumors, prostate metastasis),
wherein
PROSTAPIN protein is expressed and exerts PROSTAPIN functionality. There are a
great
many viral vectors well known in the gene therapy field which may be utilized,
including
but not limited to adenoviral, retroviral, and vaccinia vectors. See, for
example, Jolly, D.
Cancer Gene Therapy, vol. 1, pages 51-64 (1994).
Preferred viral vectors include adenovirus, more preferably in non-replicating
or
replication defective forms. For example, replication defective adenovirus
vectors in
which the E1A and E1B regions of the adenovirus genome have been deleted may
be
used. Adenovirus type 5 of subgroup C is most preferred for generating
replication-
defective adenovirus vectors for PROSTAPIN gene therapy, although adenoviruses
of
any of the 42 different serotypes or subgroups A-F may be employed.
As is generally known, various cell lines may be used to propagate recombinant
adenoviruses, so long as they complement any replication defect which may be
present.
A preferred cell line is the human 293 cell line, although other replication
permissive cell
lines may be employed as appropriate. Other complementary combinations of
viruses
and host cells may be employed in connection with the present invention; for
example
adenovirus lacking functional E2 in combination with E2-expressing cells,
adenovirus
lacking functional E4 in combination with E4-expressing cells, and the like.
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WO 99/58560 PCT/US99/07123
For additional information concerning construction, propagation, purification,
and use of
adenoviruses, see, for example, Horwitz, M. S. Adenoviridae and their
Replication, In:
Fields, B. N. and Knipe, D. M., eds., Fundamental Virology, 2nd ed. New York,
N.Y., Raven
Press, Ltd., pages 771-813 (1991 ); and Howley, P. M. Papillomavirinae and
their
S Replication, In: Fields, B. N. and Knipe, D. M., eds., Fundamental Virology,
2nd ed. New
York, N.Y., Raven Press, Ltd., pages 743-767 (1991 ).
In one embodiment, the PROSTAPIN clone 103 cDNA (SEQ ID NO. XX) is inserted
into a
replication defective adenovirus in which the E1A and E18 regions have been
deleted.
Recombinant adenovirus containing the PROSTAPIN cDNA is then propagated in 293
cells and purified according to standard methods. Purified recombinant
adenovirus may
then be delivered to the target tissue via an appropriate route which will
result in delivery
of the recombinant adenovirus to the cells of the target tissue. Where the
target tissue is
the prostate or a locally confined primary prostate tumor, recombinant
adenovirus may
be injected intraprostaticaily, preferably in multiple doses. Where the tissue
target is one
or more tumors in an individual with advanced prostate cancer, a more systemic
route of
administration, either alone or in combination with a direct delivery method
(e.g.,
intraprostatic injection), may be used. For example, recombinant adenovirus
may be
injected directly into the lymph and/or vascular system in order to target
tumors within
lymphatic system or bone marrow as appropriate.
In another embodiment, a polynucleotide encoding a PROSTAPIN protein in which
the
RSL site is deleted may be used to construct an adenovirus. The resulting
recombinant
adenovirus may be used to study PROSTAPIN function and, specifically, the
function of
the RSL, by comparing the activities of the RSL-deleted PROSTAPIN and wild
type
PROSTAPIN proteins expressed in prostate cancer and other cell lines or in
appropriate
animal models. As an example, adenoviruses encoding wild type PROSTAPIN and
RSL-
deleted PROSTAPIN may be used to compare the effects of the encoded proteins
on
tumor cell growth by expressing these two forms of PROSTAPIN in prostate
cancer
xenograft models. Examples of prostate cancer cells into which these forms of
PROSTAPIN may be introduced by the recombinant adenoviruses include LAPC-4,
LAPC-
9, LnCap, PC-3. Xenograft tumors may be conveniently generated by
subcutaneous,
orthotopic or intraosseous injection of the vector-transduced cells into SCID
or other
immune deficient mice.
Examples of retroviral vectors in which the PROSTAPIN gene may be inserted
include,
Moloney murine leukemia virus (MoMuLV), Harvey murine sarcoma virus (HaMuSV),
murine mammary tumor virus (MuMTV), and Rous Sarcoma Virus (RSV). Most
preferably,
a non-human primate retroviral vector is employed, such as the gibbon ape
leukemia
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virus (GaLV). In addition, a number of other retroviral vectors capable of
incorporating
multiple genes, including selectable markers and target-specific factors, may
be
employed. Retroviral vectors may be engineered to include a polynucieotide
encoding a
protein which is specifically reactive with prostate cancer cells, such as,
for example,
polynucleotides encoding prostate cancer cell specific antibodies or fragments
thereof.
In addition to viral vectors, PROSTAPIN polynucleotides may be delivered to
target tumor
and surrounding tissue via liposomes. For example, liposomes comprised of
DOTMA,
such as the LipofectinTM products available from Vical, Inc. (San Diego, CA)
may be used.
A variety of transfection techniques are known and may be used. For delivering
liposomes containing PROSTAPIN polynucieotides, injection into the site of the
target
tumor or systemic injection methods may used. Where, for example, the target
tumor is a
primary prostate tumor, direct injection int the prostate is preferred. As
another
example, where the target comprises lymph andlor bone metastases, injection
into the
lymphatic system andlor arterial system, respectively, may be preferred.
Liposomes
may be enhanced to increase their tissue specificity by coupling the liposome
to a
specific ligand such as a monoclonal antibody, sugar, glycolipid, or protein,
or by
changing the composition or size of the liposome in order to achieve targeting
to organs
and cell types other than the naturally occurring sites of localization. In
one embodiment,
the liposome may be couples to a monoclonal antibody which recognizes a cell
surface
prostate tumor antigen, such as PSCA. Methods for covalently attaching
antibodies or
fragments thereof to a liposome bilayer are known.
IDENTIFICATION OF PROSTAPIN TARGET PROTEASE
The target of PROSTAPIN is likely to be a protease that plays a functional
role in prostate
cancer metastasis. The PROSTAPIN gene andlor protein may be used as tools to
identify
this protease. One method involves screening a yeast two-hybrid cDNA library
with the
prostapin gene as a bait, or by screening a cONA expression library using
prostapin
protein as a probe. Alternatively, prostapin protein may be used to study the
biochemical
interaction with a panel of known proteases, such as: Prostate Specific
Antigen, human
Kallikrein 2, urokinase type plasminogen activator, tissue plasminogen
activator,
plasmin, granzyme B, thrombin, cathepsins B, L and D, and human neutrophi!
elastase.
KITS
For use in the diagnostic and therapeutic applications described or suggested
above,
kits are also provided by the invention. Such kits may comprise a carrier
means being
compartmentalized to receive in close confinement one or more container means
such as
vials, tubes, and the like, each of the container means comprising one of the
separate
elements to be used in the method. For example, one of the container means may
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WO 99/58560 PCT/US99/07123
comprise a probe which is or can be detectably labeled. Such probe may be an
antibody
or polynucleotide specific for a PROSTAPIN protein or a PROSTAPIN gene or
message,
respectively. Where the kit utilizes nucleic acid hybridization to detect the
target nucleic
acid, the kit may also have containers containing nucleotides) for
amplification of the
target nucleic acid sequence andlor a container comprising a reporter-means,
such as a
biotin-binding protein, such as avidin or streptavidin, bound to a reporter
molecule, such
as an enzymatic, florescent, or radionucleotide ~abel.
EXAMPLES
Various aspects of the invention are further described and illustrated by way
of the
several examples which follow, none of which are intended to limit the scope
of the
invention.
EXAMPLE 1:
ISOLATION OF PROSTAPIN cDNA FRAGMENT AND EXPRESSION ANALYSIS
MATERIALS AND METHODS
LAPC Xenografts:
LAPC xenografts were obtained from Dr. Charles Sawyers (UCLA) and generated as
described (Klein et al, 1997, Nature Med. 3: 402-408). Androgen dependent and
independent LAPC-4 xenografts LAPC-4 AD and AI, respectively) and LAPC-9 AD
xenografts were grown in male SCID mice and were passaged as small tissue
chunks in
recipient males. LAPC-4 A1 xenografts were derived from LAPC-4 AD tumors. Male
mice
bearing LAPC-4 AD tumors were castrated and maintained for 2-3 months. After
the
LAPC-4 tumors re-grew, the tumors were harvested and passaged in castrated
males or
in female SCID mice.
Cell Lines:
The human cell lines HeLa (cervical carcinoma), 293 (embryonic kidney), A431
(epidermoid carcinoma), Co1o205 (colon carcinoma), KCL22 (lymphoid blast
crisis of
chronic myeiogenous leukemia), LnCaP (prostate cancer), DU145 (prostate
cancer) and
PC-3 (prostate cancer) were obtained from the ATCC. The LAPC-4 cell line,
derived from
the LAPC-4 AD xenograft, was generated as described (Klein et al., 1997,
supra) and
obtained from Dr. Robert Reiter (UCLA). All cell lines were maintained in DMEM
with 5%
fetal calf serum.
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RNA Isolation:
Tumor tissue and cell lines were homogenized in Trizol reagent (Life
Technologies, Gibco
BRL) using 10 ml! g tissue or 10 ml! 10° cells to isolate total RNA.
Poly A RNA was purified
from total RNA using Qiagen's Oligotex mRNA Mini and Midi kits. Total and mRNA
were
quantified by spectrophotometric analysis (O.D. 260!280 nm) and analyzed by
gel
electrophoresis.
Oligonucieotides:
The following HPLC purified oligonucleotides were used.
RSACDN~cDNA synthesis primer):
5'TTTTGTACAAGCTT3o3'
Ada-ptor 1:
5'CTAATACGACTCACTATAGGGCTCGAGCGGCCGCCCGGGCAGGT3'
3'GGCCCGTCCAS'
Adaptor 2:
5'GTAATACGACTCACTATAGGGCAGCGTGGTCGCGGCCGAGGT3'
3'CGGCTCCAS'
PCR arimer 1:
5'CTAATACGACTCACTATAGGGC3'
Nested ~orimer (NP)1:
5'TCGAGCGGCCGCCCGGGCAGGT3'
Nested I rio mer yNP),2:
5'AGCGTGGTCGCGGCCGAGGT3'
Suppression Subtractive Hyrbridizatiq_n:
Suppression Subtractive Hybridization (SSH) was used to identify cDNAs
corresponding
to genes which may be down-regulated in androgen independent prostate cancer
compared to androgen dependent prostate cancer.
Double stranded cDNAs corresponding to the LAPC-4 AD xenograft (tester) and
the
LAPC-4 AI xenograft (driver) were synthesized from 2 ~g of poly(A)' RNA
isolated from
xenograft tissue, as described above, using CLONTECH's PCR-Select cDNA
Subtraction
Kit and 1 ng of oligonucleotide RSACDN as primer. First- and second-strand
synthesis
were carried out as described in the Kit's user manual protocol (CLONTECH
Protocol No.
PT1117-1, Catalog No. K1804-1 ). The resulting cDNA was digested with Rsa I
for 3 hrs. at
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WO 99/58560 PCT/US99/07123
37°C. Digested cDNA was extracted with phenollchloroform (1:1 ) and
ethanol
precipitated.
Driver cDNA (LAPC-4 AI) was generated by combining in a 1:1 ratio Rsa I
digested LAPC-
4 AI cDNA with a mix of digested cDNAs derived from human benign prostatic
hyperplasia (BPH), the human cell lines HeLA, 293, A431, Co1o205, and mouse
liver.
Tester cONA (LAPC-4 AO) was generated by diluting 1 ul of Rsa i digested LAPC-
4 AD
cDNA (400 ng) in 5 ~I of water. The diluted cDNA (2 ~I, 160 ng) was then
ligated to 2 ~I of
adaptor 1 and adaptor 2 {10 ~M), in separate ligation reactions, in a total
volume of 10 ~I
at 16°C overnight, using 400 a of T4 DNA ligase (CLONTECH). Ligation
was terminated
with 1 ~I of 0.2 M EDTA and heating at 72°C for 5 min.
The first hybridization was performed by adding 1.5 ~I (600 ng) of driver cDNA
to each of
two tubes containing 1.5 ~i {20 ng) adaptor 1- and adaptor 2- ligated tester
cDNA. In a
final volume of 4 ~I, the samples were overlayed with mineral oil, denatured
in an MJ
Research thermal cycler at 98°C for 1.5 minutes, and then were allowed
to hybridize for 8
hrs at 68°C. The two hybridizations were then mixed together with an
additional 1 ~I of
fresh denatured driver cDNA and were allowed to hybridize overnight at
68°C. The
second hybridization was then diluted in 200 ~I of 20 mM Hepes, pH 8.3, 50 mM
NaCi, 0.2
mM EDTA, heated at 70°C for 7 min. and stored at-20°C.
PCR Amplification. Cloning and Seauencing of Gene Fraaments Generated from
SSH:
To amplify gene fragments resulting from SSH reactions, two PCR amplifications
were
performed. In the primary PCR reaction 1 ~I of the diluted final hybridization
mix was
added to 1 Irl of PCR primer 1 (10 ~M), 0.5 wl dNTP mix (10 ~M), 2.5 ~I 10 x
reaction buffer
(CLONTECH) and 0.5 ~I 50 x Advantage cDNA polymerase Mix (CLONTECH) in a final
volume of 25 ~I. PCR 1 was conducted using the following conditions:
75°C for 5 min.,
94°C for 25 sec., then 27 cycles of 94°C for 10 sec, 66°C
for 30 sec, 72°C for 1.5 min. Five
separate primary PCR reactions were performed for each experiment. The
products
were pooled and diluted 1:10 with water. For the secondary PCR reaction, 1 ~1
from the
pooled and diluted primary PCR reaction was added to the same reaction mix as
used for
PCR 1, except that primers NP1 and NP2 (10 ~M) were used instead of PCR primer
1.
PCR 2 was performed using 10-12 cycles of 94°C for 10 sec, 68°C
for 30 sec, 72°C for 1.5
minutes. The PCR products were analyzed using 2% agarose gel electrophoresis.
The PCR products were inserted into pCR2.1 using the TIA vector cloning kit
(Invitrogen).
Transformed E. coli were subjected to bluelwhite and ampicillin selection.
White colonies
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were picked and arrayed into 96 well plates and were grown in liquid culture
overnight.
To identify inserts, PCR amplification was performed on 1 ml of bacterial
culture using
the conditions of PCR1 and NP1 and NP2 as primers. PCR products were analyzed
using
2% agarose gel electrophoresis.
Bacterial clones were stored in 20% glycerol in a 96 well format. Plasmid DNA
was
prepared, sequenced, and subjected to nucleic acid homology searches of the
GenBank,
dBest, and NCI-CGAP databases.
RT-PCR Expression Analyrsis:
First strand cDNAs were generated from 1 ~g of mRNA with oligo (dT)12-18
priming using
the Gibco-BRL Superscript Preamplification system. The manufacturers protocol
was
used and included an incubation for 50 min at 42°C with reverse
transcriptase followed
by RNAse H treatment at 3?°C for 20 min. After completing the reaction,
the volume was
increased to 200 ~I with water prior to normalization. First strand cONAs from
16
different normal human tissues were obtained from Clontech.
Normalization of the first strand cDNAs from multiple tissues was performed by
using the
primers 5'atatcgccgcgctcgtcgtcgacaa3' and 5'agccacacgcagctcattgtagaagg 3' to
amplify p-actin. First strand cONA (5 ~I) was amplified in a total volume of
50 ~I
containing 0.4 ~M primers, 0.2 ~M each dNTPs, 1XPCR buffer (Clontech, 10 mM
Tris-HCL,
1.5 mM MgCIZ, 50 mM KCI, pH8.3) and 1X Klentaq DNA polymerase (Clontech). Five
~I of
the PCR reaction was removed at 18, 20, and 22 cycles and used for agarose gel
electrophoresis. PCR was performed using an MJ Research thermal cycles under
the
following conditions: initial denaturation was at 94°C for 15 sec,
followed by a 18, 20, and
22 cycles of 94°C for 15, 65°C for 2 min, 72°C for 5 sec.
A final extension at 72°C was
carried out for 2 min. After agarose gel electrophoresis, the band intensities
of the 283
by ~i-actin bands from multiple tissues were compared by visual inspection.
Dilution
factors for the first strand cONAs were calculated to result in equal ~3-actin
band
intensities in all tissues after 22 cycles of PCR. Three rounds of
normalization were
required to achieve equal band intensities in all tissues after 22 cycles of
PCR.
To determine expression levels of the 11 P2A6 gene, 5 ~I of normalized first
strand cDNA
was analyzed by PCR using 25, 30, and 35 cycles of amplification using the
following
primer pairs, which were designed with the assistance of (MIT; for details,
see,
www.genome.wi.mit.edu):
5'- GAG TCT GGC TGG TTG ATT TGA GAG -3' (SEQ ID NO. XX)
5'- CCA GTC TAA CTT GCC ACT CTG TGA -3'
(SEQ 10 NO. XX)
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Semi quantitative expression analysis was achieved by comparing the PCR
products at
cycle numbers that give light band intensities.
S
RESULTS:
Several SSH experiments were conducted as described in the Materials and
Methods,
supra, and led to the isolation of numerous candidate gene fragment clones.
All
candidate clones were sequenced and subjected to homology analysis against all
sequences in the major public gene and EST databases in order to provide
information on
the identity of the corresponding gene and to help guide the decision to
analyze a
particular gene for differential expression. In general, gene fragments which
had no
homology to any known sequence in any of the searched databases, and thus
considered
to represent novel genes, as well as gene fragments showing homology to
previously
1 S sequenced expressed sequence tags (ESTs), were subjected to differential
expression
analysis by RT-PCR andlor Northern analysis.
One of the gene fragment cDNA clones showing no homology to any known gene or
EST
sequence was designated 11 P2A6. The isolated 11 P2A6 cDNA (SEQ ID NO. XX) was
471
by in length and has the nucleotide sequence of nucleotide residues 1 through
471 in the
PROSTAPIN cDNA sequence shown in FIG.1 (SEQ ID NO. XX).
Differential expression analysis by RT-PCR showed that the 11P2A6 (PROSTAPIN)
gene
is expressed at approximately equal levels in the LAPC-9 AD xenograft and in
normal
2S prostate tissue, but at greatly reduced levels in the LAPC-4 AI xenograft
and at
undetectable levels in the LAPC-4 AD xenograft (FIG. 3, panel A). RT-PCR
expression
analysis of first strand cDNAs from 16 normal tissues detected expression of
the 11 P2A6
(PROSTAPIN) gene only in prostate tissue after 30 cycles of PCR amplification,
while
lower level expression was detected in lung and placenta after 35 cycles (FIG.
3, panels B
and C).
EXAMPLE 2:
ISOLATION ANO STRUCTURAL ANALYSIS OF
FULL LENGTH cDNA ENCODING HUMAN PROSTAPIN
3S
The full length cDNA encoding the gene corresponding to the 11P2A6 clone
(Example 1,
above) was isolated as follows. A normal human prostate cDNA library
(Clontech) was
screened with a probe comprising the 11 P2A6 cDNA (SEQ ID NO. XX). Several
positive
clones were identified, and the largest of these, clone 103, was sequenced.
Clone 103
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WO 99/58560 PCT/US99/07123
(SEQ ID NO XX) contains an open reading frame encoding a 379 amino acid
protein (see
FIG. 1 ). Amino acid homology analysis of the clone 103 sequence revealed 30-
40%
homology to a class of serine protease inhibitors known as serpins.
Accordingly, the
gene corresponding to clones 11 P2A6 and clone 103 (and the encoded protein)
were
named "PROSTAPIN" (PROSTAte serine Protease INhibitor). PROSTAPIN is most
closely
associated wikh the serpin family member human LEI, which may have a role in
apoptosis
(Torriglia et al.,1998, MCB 18:3612).
The nucleotide sequences of PROSTAPIN clones 103 (SEQ ID NO. XX) and 11 P2A6
(SEQ
10 NO. XX), which overlap in the 5' non-coding region of the PROSTAPIN gene,
were
combined to form the contiguous PROSTAPIN nucleotide sequence depicted in FIG.
1
(SEQ ID NO. XX). The 5' untranslated region contains two translational stop
signals
(indicated by asterisks in FIG. 1 A) upstream of the start ATG, which falls
within the Kozak
sequence 5'-AAA ATG G-3'. A highly conserved reactive site loop characteristic
of the
serpin family is located in the carboxy-terminal region of PROSTAPIN
(underlined
sequence in FIG.1 ).
The serpin protease inhibitory domain is known as the reactive~site loop (RSL)
and is
located 30-50 residues from the carboxyl-terminus. The RSL is about 15-20
amino acids
in length and contains a hinge region and a variable region. The hinge region
confers
stability to the serpin-protease complex. As is evident from the amino acid
alignment of
various serpin family members shown in FIG. 2, the RSL hinge region is highly
conserved
among the serpins. The RSL variable region contains the reactive site amino
acid P1,
which determines specificity of inhibition. During inhibition, the RSL binds
to the
protease active site, undergoes nucleophillic attack by the catalytic serine
residue,
resulting in cleavage of the serpin at P1-P1'. The PROSTAPIN RSL hinge region
amino
acid sequence (GTEAAAATG) is highly homologous to all other serpins analyzed,
with the
exception of maspin (FIG. 2). The PROSTAPIN RSL variable region amino acid
sequence
is distinct, with a lysine at P1 and a serine at P1'. This indicates that
PROSTAPIN most
likely targets a different protease than do the other serpins.
PROSTAPIN clone 103 (SEQ tD NO. XX) has been deposited with the American Type
Culture Collection ("ATCC") (Mannassas, VA) as plasmid pProstapin on May 15,
1998 as
ATCC Accession Number 98757.
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EXAMPLE 3:
NORTHERN BLOT ANALYSIS OF PROSTAPIN EXPRESSION
Northern blot analysis on panels of normal human and prostate tumor xenograft
tissues
S using a labeled PROSTAPIN clone 103 (SEQ ID NO. XX) probe were conducted to
confirm
the prostate specificity of PROSTAPIN expression initially established by RT-
PCR
expression analysis (see Example 1 ). Further Northern blot analysis of
PROSTAPIN
expression is described in Example 4.
Two panels of normal human tissues were evaluated. The results from one of the
panels,
which contained 16 normal human tissues, are shown in FIG. 4 (Panels A & B).
in this first
panel, PROSTAPIN RNA was only detected in prostate, expressed as two distinct
transcripts of about 2.3 and 3.0 kb. To extend this analysis, the clone 103
probe was
used to analyze a second normal tissue panel, comprising an RNA dot blot
matrix of 37
normal human tissues (Clontech, Palo Alto, CA; Human Master BIotT""). The
results,
shown in FIG. 4 (Panel C), show PROSTAPIN expression only in prostate and
trachea. No
expression signal was detected in brain, spinal chord, heart, aorta, skeletal
muscle,
colon, bladder, uterus, stomach, testis, ovary, pancreas, pituitary gland,
adrenal gland,
thyroid gland, salivary gland, mammary gland, kidney, liver, small intestine,
spleen,
thymus, peripheral leukocytes, lymph node, bone marrow, appendix, lung,
placenta, fetal
brain, fetal heart, fetal kidney, fetal liver, fetal spleen, fetal thymus, or
fetal lung.
Northern blot analysis of PROSTAPIN expression in the LAPC-4 AD, LAPC-4 AI and
LAPC-
9 AO prostate tumor xenografts was conducted with the labeled clone 103 probe
(SEQ ID
NO. XX). The results, shown in FIG. 4, Panel D, show detectable expression
only in the
LAPC-9 AD xenograft (as well as a concurrently analyzed normal prostate).
Interestingly,
although two distinct transcripts of about 2.3 and 3.0 kb are detected in
normal prostate
tissue, only the larger transcript is expressed in the LAPC-9 xenograft.
Additionally,
although no PROSTAPIN expression was detected in the LAPC-4 AI subline by
Northern
blotting, RT-PCR analysis of first strand cDNA derived from the LAPC-4 AI
xenograft
showed very low level expression (see Example 1 and FIG. 3).
Further Northern blot analysis of three prostate cancer cell lines derived
from patient's
with advanced stage prostate cancer (LNCaP, PC-3 and DU145) showed no
detectable
expression of PROSTAPIN. The absence of expression in these cell lines was
confirmed
by semi-quantitative RT-PCR (see Example 4, below).
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EXAMPLE 4:
PROSTAPIN EXPRESSION IN CLINICAL TISSUE SAMPLES,
PROSTATE CANCER CELL LINES AND XENOGRAFTS
Further analysis of PROSTAPIN expression in various normal and prostate cancer
clinical
tissue samples was conducted by semi-quantitative RT-PCR in order to further
examine
the question of whether loss of PROSTAPIN expression correlates with advanced
prostate cancer grade and clinical stage. Concurrent analysis of PROSTAPIN
expression
in the LAPC xenografts and several prostate cancer cell lines was conducted.
Graded (Gleason Grade) and staged (according to TMN system) human tissues were
obtained from the Human Tissue Resource Center at the University of California
Los
Angeles. PrEC cells were obtained from Clonetics. The prostate cancer cell
lines were
obtained from readily available sources. For reference, the TMN staging of the
human
prostate tumor tissues used in this analysis was as follows: (1) Stage T2c,
tumor is
confined to the prostate and involves both lobes; (2) Stage T3a, tumor extends
through
the prostate capsule; (3) Stage T3c, extracapsular extension with invasion of
seminal
vesicles.
The human tissue samples included normal prostate, prostate cancer, and 4
examples of
prostate cancer together with their matched normal controls (i.e., from the
same patient).
First strand cDNA was prepared from these tissues as well as the LAPC
xenografts,
prostate cancer cell lines (LNCaP, PC3, DU145) and normal prostate mRNA
(obtained
from Clontech and BioChain). PCR analysis was performed on the normalized
cDNAs
using the primers described in Example 1 (i.e., SEQ ID NOS. XX and XX).
The results show that PROSTAPIN is expressed in all normal prostate tissues
and the
prostate tumor specimens derived from patients with localized disease, but
that
PROSTAPIN expression is lost or dramatically attenuated in all tumor specimens
from
patients with extracapsular extension and advanced disease (FIG. 5, lanes 20
and 22). In
addition, the results show complete absence of PROSTAPIN expression in all of
the
prostate cancer cell lines and the LAPC-4 xenografts (FIG. 5, lanes 6, 7, 9,
10, 11 ). All
three of the prostate cancer cell lines originated from advanced prostate
cancer patient
3S metastasis tumors. Specifically, LnCaP was derived from a prostate cancer
lymph node
metastasis, PC-3 was derived from a prostate cancer bone metastasis, and DU-
145 was
derived from a prostate cancer brain metastasis. The LAPC-4 xenograft was
derived
from a lymph node metastasis.
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Consistent with the RT-PCR and Northern blot results obtained in Examples 1
and 3,
respectively, a PROSTAPIN transcript was detected in the LAPC-9 xenograft.
However,
as shown in Example 5, below, the PROSTAPIN transcript expressed in the LAPC-9
xenograft is a substantially mutated, partially unspliced variant which
includes point
mutations, a stop codon in what would be the center of the wild type PROSTAPIN
sequence, and an unspliced intron. Although the biological activity of this
PROSTAPIN
mutant (if any) has not been characterized, it is likely that wild type
PROSTAPIN
functionality is either substantially altered or, more likely, completely
lost. Like the LAPC-
4 xenografts, LAPC-9 also represents advanced stage disease, as it was
generated from
a bone tumor biopsy of a patient with hormone-refractory metastatic prostate
cancer.
The data obtained from this combined analysis indicates that loss of wild type
PROSTAP1N expression andlor expression of a functional mutation correlates
with
advanced stage and metastatic prostate cancer. All prostate cancer cell lines,
xenografts and patient samples derived from advanced stage tumors or
metastasis show
a complete lack or sharp attenuation of wild type PROSTAPIN expression.
Moreover, it
appears that loss of PROSTAPIN expression may coincide with the development of
metastatic disease, since expression is lost even at the lower gradelstage of
extracapsular disease. Loss of functional PROSTAPIN may be one of the first
molecular
events coinciding with andlor leading to the development of metastasis.
EXAMPLE 5:
ISOLATION AND ANALYSIS OF cDNA ENCODING PROSTAPIN MUTATION
As evident from the Northern blot analyses of PROSTAPIN expression described
in
Example 3, only one of the two distinct PROSTAPIN transcripts expressed in
normal
prostate is detectable in the LAPC-9 xenograft. In order to examine the
possibility that
the LAPC-9 xenograft expresses an aberrant version of the PROSTAPIN gene, a
full
length PROSTAPIN cDNA was isolated from LAPC-9. Briefly, an LAPC-9 AD cDNA
library
was constructed in lambda ZAP Express (Stratagene). The library was screened
using
PROSTAPIN clone 103 (SEQ ID NO. XX) as a probe. A positive clone (clone 2) was
identified and sequenced. Clone 2 cONA (SEQ ID NO XX) comprises 2472 by and
has the
nucleotide and deduced amino acid sequences shown in (FIG. 6). Further
analysis
determined that clone 2 represents a partially unspliced version of the
PROSTAPIN
message with an intron of 714 by and point mutations at positions 298 (GfT),
474 (TIC),
572 (AIG), 593 (A/T), 1567 (AIC), 1584 (TIC), 1613 (CIT), 1822 (G!T), 2085
(CIT) in FIG. 6
(clone 2 - SEQ ID NO. XX). In addition, clone 2 contains several point
mutations, one of
which results in a stop codon at amino acid residue 90 in the open reading
frame (FIG. 6).
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All point mutations were confirmed by sequencing of RT-PCR products of first
strand
cDNA derived from LAPC-9 AD.
EXAMPLE 6:
SUBCELLULAR LOCALIZATION OF PROSTAPIN PROTEIN
MATERIALS AND METHODS:
To initially characterize the PROSTAPIN protein, cDNA clone 103 (SEQ ID NO.
XX) was
cloned into the pcDNA 3.1 Myc-His plasmid (Invitrogen) (which encodes a 6His
tag at the
carboxyl-terminus), transfected into 293T cells, and analyzed by subceltular
fractionation. More specifically, the sequence encoding the PROSTAPIN ORF from
PROSTAPIN clone 103 cDNA (SEQ ID NO. XX) was tagged with a 6His tag at the
carboxyl-
terminus and was transfected into 293T cells. Cell lysates were prepared by
dounce
homogenization of cells in a hypotonic buffer. Cellular debris was removed by
low speed
centrifugation (10,000 X g) and the supernatant containing the cytosol and
tight
membrane fraction was separated by a 100,000 X g centrifugation. Equal amounts
of
protein from each fraction were analyzed using an anti-His antibody (Santa
Cruz) that
recognizes recombinant PROSTAPIN or an anti-SV 40 large T antibody (Santa
Cruz). Cell
conditioned media (Cell sup) was also analyzed to identify any secreted
PROSTAPIN
protein.
RESULTS:
Western blot analysis of cytosol and light membrane fractions using an anti-
His antibody
demonstrated that 90% of PROSTAPIN protein was present in the membrane
fraction
(FIG. 7). As a control, SV40 large T antigen, an endogenous cytosolic protein,
was shown
to be largely cytosolic. This suggests that PROSTAPIN is associated with
plasma
membrane andlor endoplasmic reticulum.
EXAMPLE 7s
CHROMOSOMAL LOCALIZATION OF THE PROSTAPIN GENE
The chromosomal localization of PROSTAPIN was determined using the GeneBridge
4
HumanIHamster radiation hybrid (RH) panel (Walter et al., 1994, Nat. Genetics
7:22)
(Research Genetics, Huntsville AI). The following PCR primers, which amplify a
148 by
PROSTAPIN product, were employed:
11 P2A6.9 ata cct gga tgt cag cga aga g (SEQ ID NO. XX)
11 P2A6.10 tag gat cgt gtt ggt atg agt gtg (SEQ ID NO. XX)
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The resulting mapping vector for the 93 radiation hybrid panel DNAs was:
0000101010000012012001000000101001010100110111002010000100000100001010001
01000000101001000010.
This vector and the mapping program at http:llwww-genome.wi.mit.edulcgi-
bin/contiglrhmapper.pl placed PROSTAPIN on chromosome 18q21.3 between D18S983
and D18S537 with an LOD>15. The chromosomal location of PROSTAPIN is
schematically depicted in FIG. 8.
The PROSTAPIN gene co-localizes to the 500 kb 18q21.3 region along with 6
other serpin
family members, including maspin, leupin and bomapin (Bartuski et al., 1997,
Genomics
43:321). The region of 18q21.3 has been associated with loss of heterozygosity
(LOH) in
advanced metastatic prostate cancer and in recurrent disease (Brothman et al.,
1999,
The Prostate 38:303). Loss of expression of PROSTAPIN alone or in combination
with
another serpin may contribute to the growth characteristics and invasiveness
of
aggressive prostate cancer. It is interesting to note that maspin expression
in the
prostate and in the LAPC xenografts (determined by RT-PCR) mimics the
expression of
PROSTAPIN. It may be that some of the genes in this serpin cluster at 18q21
may exhibit
coordinate regulation of expression.
EXAMPLE 8:
IDENTIFICATION OF INTRON-EXON BOUNDARIES OF PROSTAPIN GENE
Genomic PROSTAPIN clones were isolated by screening the human BAC library CID
(Cal.
Tech. library D, purchased from Research Genetics) and a human PAC library
(release I,
Peter deJong, University of New York, Buffalo). Four positive clones were
obtained:
2062H14, 2074J2, 2100K19 from the BAC library; 152122 from the PAC library.
These
clones were confirmed by Southern blot analysis (FIG. 9) using PROSTAPIN clone
103
cDNA probe (SEQ ID NO. XX).
Clone 2074J2 was used to identify the intron-exon boundaries for the PROSTAPIN
gene.
The PROSTAPIN gene was found to contain seven exons and six introns within the
region
of the coding sequence (FIG 10).
The following seven pairs of primers were designed within introns to amplify
exonic
sequences from genomic DNA for sequencing or for single-stranded
conformational
polymorphism (SSCP) analysis:
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99/58560
Exon
1:
ProsEi atgggttctctcagcacagctaacg(SEQ ID NO. XX)
(within exon, begins at
start site)
Pros2 aattaattttgctgacccagagcg (SEQ ID NO. XX)
Exon
2:
Pros3 gttagctatcactactgatcttgatc(SEQ 10 NO. XX)
Pros4 atgggcaaaagaaggagcttttctac(SEQ ID NO. XX)
Exon
3:
Pros5 tttattcagaggcaaacaccttgct(SEQ ID NO. XX)
Pros6 atgtcatgtgactcttctcactcttc(SEQ ID NO. XX)
Exon
4:
Pros? gaattttagaatacattgagctgtag(SEQ 10 NO. XX)
Pros8 atctgcctatgtcaggtgcagacttc(SEQ ID NO. XX)
Exon
5:
Pros9 taaatttctcatgactcttcacct (SEQ ID NO. XX)
Pros10 tatcctccaacatttgtcatgagtctg(SEQ ID NO. XX)
Exon
6:
Prosl1 tagagtgttcatgcagatatccgtgt(SEQ 10 NO. XX)
Prosl2 aatcaatgactacgctaatgtcatgag(SEQ ID NO. XX)
Exon
7:
Prosl3 gaagttgaaccactcacactgagaatt(SEQ ID NO. XX)
ProsEi6 attgtctctgcacctcatctgcaa (SEQ ID NO. XX)
(5'untranslated region)
An example of genomic amplification
of PROSTAPIN exonic sequences
using these
primer
sets
is shown
in FIG.11.
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EXAMPLE 9:
GENERATION OF PROSTAPIN POLYCLONAL ANTIBODY
Sheep polyclonal anti-PROSTAPIN antibodies were generated using a purified GST-
S PROSTAPIN fusion protein. The fusion protein contains amino acid residues 1-
106 from
the PROSTAPIN sequence and was generated by PCR using the following primers:
GST-pros5': gtggatccatgggttctctcagca (underlined sequence: BamHl site)
(SEQ lD NO. XX)
GST-pros3': ataccc tggcaatgctgagg (underlined sequence: Smal site)
(SEQ ID NO. XX)
The PCR product was inserted directionally into a pGEX-4T (GST-fusion) vector
(Pharmacia). Fusion protein was produced in bacteria and purified using a
Glutathione-
Sepharose column (Phanmacia).
Sheep were immunized with 200 ~glinjection every two weeks for an eight week
period.
Immune serum was affinity purified using a GST-PROSTAPIN column. Purified
antibody
was used to probe western blots of lysates from several cell lines including:
293T cells
transfected with pcDNA vector with or without His-tagged PROSTAPIN, TsuPr1
cells
infected with retrovirus generated with the retrovirai expression vector
pSRatkneo with
or without PROSTAPIN (FIG. 12). The results show that the anti-PROSTAPIN
antibody
recognizes His-tagged PROSTAPIN with similar intensity as an anti-His
antibody. In
addition, untagged PROSTAPIN expressed in TsuPr1 cells is also recognized with
high
efficiency and specificity.
EXAMPLE 10
WESTERN BLOT ANALYSIS OF PROSTAPIN EXPRESSION IN PROSTATE
CANCER
Western blotting of lysates derived from LAPC xenografts (LAPC-4 AD, 9AD, and
9Ai), prostate cancer cell lines (TsuPr1, LNCaP, PC-3) and a prostate tumor-
normal matched patient sample using the purified antibody described in Example
9 showed PROSTAPIN expression only in the normal prostate tissue (FIG. 13).
Significantly lower levels of prostapin were detected in the matched prostate
cancer sample. PROSTAPIN expression was undetectable in the xenografts and
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the prostate cancer cell lines. These results confirm loss or down-regulation
of
PROSTAPIN in prostate cancer.
10 Throughout this application, various publications are referenced within
parentheses.
The disclosures of these publications are hereby incorporated by reference
herein in
their entireties.
The present invention is not to be limited in scope by the embodiments
disclosed herein,
which are intended as single illustrations of individual aspects of the
invention, and any
which are functionally equivalent are within the scope of the invention.
Various
modifications to the models and methods of the invention, in addition to those
described
herein, will become apparent to those skilled in the art from the foregoing
description
and teachings, and are similarly intended to fall within the scope of the
invention. Such
modifications or other embodiments can be practiced without departing from the
true
scope and spirit of the invention.
41
