Note: Descriptions are shown in the official language in which they were submitted.
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NOVEL PROSTATE-SPECIFIC OR TESTIS-SPECIFIC
NUCLEIC ACID MOLECULES, POLYPEPTIDES, AND DIAGNOSTIC
AND THERAPEUTIC METHODS
Field of The Invention
The invention generally relates to the treatment of disorders
associated with prostate and testis dysfunction and cell proliferation, and
specifically relates to the identification and use of novel genes for
diagnosis
and treatment of such disorders.
Bacl~grround of The Invention
Genitourinary disorders are often difficult to diagnose and treat
effectively because they are present non-specifically. Two causes of
genitourinary disorders are disorders of the prostate gland and the testis.
The prostate is a variable sized gland located in the male pelvis, and
is made up of several different cell types, including epithelial cells and
stromal cells. Prostate-associated disorders include prostate cancer, benign
prostatic hyperplasia, and prostatitis. The male hormone testosterone and
other androgen related hormones have major roles in the growth and
function of the prostate. The testis is also subject to many defects,
including developmental anomalies, inflammation, and cancer.
In men, prostate cancer is the most commonly diagnosed cancer and
the second leading cause of cancer mortality following shin cancer. In the
initial stages, prostate cancer is dependent on androgens for growth, and
this dependence is the basis for androgen ablation therapy. In most cases,
however, prostate cancer progresses to an androgen-independent phenotype
for which there is no effective therapy available at present.
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Currently, there is limited information regarding the molecular
details of prostate cancer progression. Several independent approaches
resulted in the identification of a few highly prostate-enriched genes that
may have unique roles in this process. The first such gene discovered was
Prostate Specific Antigen (PSA), the detection of which is currently used as
a diagnostic tool and also as a marker for the progression of prostate cancer,
albeit with significant limitations. More recently, several additional
prostate-enriched genes were identified including prostate-specif c
membrane antigen (PSMA), prostate carcinoma tumor antigen 1 (PCTA-1),
NKX3.1, prostate stem cell antigen (PSCA), DD3, and PCGEM1.
It would be beneficial to provide reagents useful for the diagnosis
and therapy of disorders associated with the prostate and the testis, as well
as other tissues. .
Summar~T of the Invention
The invention provides, in general, a novel prostate-specific or
testis-specific nucleic acid molecules, polypeptides, antibodies, and
modulatory compounds for use in methods of diagnosing, treating, and
preventing diseases and conditions of the prostate and testis, such as cancer.
In a first aspect the invention provides a substantially pure prostate-
specific or testis-specific polypeptide, including a sequence substantially
identical to the sequence of any of SEQ ID NOS: 14, 29, 32, 34, 36, 41, or
53. In a preferred embodiment of the first aspect, the substantially pure
prostate-specific or testis-specific polypeptide includes the sequence of any
of SEQ B7 NOS: 14, 29, 32, 34, 36, 41, or 53. In another preferred
embodiment, the invention provides an isolated nucleic acid molecule
encoding a polypeptide of the first aspect, for example a nucleic acid
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molecule including the sequence of any of SEQ ID NOS: 23, 28, 31, 33, 35,
40, or 52. Preferably, the polypeptide is derived from a mammal, e.g., a
human.
In a second aspect, the invention provides an isolated prostate-
specific or testis-specific nucleic acid molecule including a sequence
substantially identical to SEQ ID NOS: 1-12, 22, 27, 30, and 51.
In a third aspect, the invention provides an isolated prostate-specific
or testis-specific nucleic acid molecule consisting essentially of SEQ ID
NOS: 15-21, 24-26, 42-50, and 54-70.
In preferred embodiments of some of the above aspects, the
invention provides a vector, a cell, a cell including the vector, and a non-
human transgenic animal including the isolated nucleic acid molecules.
In a fourth aspect, the invention provides an isolated nucleic acid
molecule that hybridizes under high stringency conditions to the
complement of any of the sequences set forth in SEQ ID NOS: 1-12, 15-28,
30, 31, 33, 35, 40, 42-50, 51, 52, or 54-70, where the isolated nucleic acid
molecule encodes a prostate-specific or testis-specific polypeptide.
In a fifth aspect, the invention provides an isolated nucleic acid
molecule, where the nucleic acid molecule includes a sequence that is
antisense to the coding strand of any of the prostate-specific or testis-
specific nucleic acid molecules set forth in SEQ ID NOS: 1-12, 15-28, 30,
31, 33, 35, 40, 42-50, 51, 52, or 54-70, or a fragment thereof.
In a sixth aspect, the invention provides a probe for analyzing a
prostate-specific or testis-specific gene or homolog or fragment thereof, the
probe having greater than 55% nucleotide sequence identity to a sequence
encoding any of SEQ ID NOS: 1-12, 15-28, 30, 31, 33, 35, 40, 42-50, 51,
52, or 54-70, or fragment thereof, where the fragment includes at least six
amino acids, and the probe hybridizes under high stringency conditions to
at least a portion of a prostate-specific or testis-specific nucleic acid
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molecule. In a preferred embodiment of this aspect, the probe has 100%
complementarity to a nucleic acid molecule encoding any of SEQ ID NOS:
1-12, 15-28, 30, 31, 33, 35, 40, 42-50, 51, 52, or 54-70, or fragment thereof,
where the fragment comprises at least six amino acids, and said probe
hybridizes under high stringency conditions to at least a portion of a
prostate-specific or testis-specific nucleic acid molecule.
In a seventh aspect, the invention provides an antibody that
specifically binds to a prostate-specific or testis-specific polypeptide that
includes an amino acid sequence that is substantially identical to the amino
acid sequence of any of SEQ ID NOS: 14, 29, 32, 34, 36, 41, or 53.
In an eighth aspect, the invention provides a method of detecting a
prostate-specific or testis-specific gene or fragment thereof in a cell, the
method including contacting the nucleic acid molecule of any of SEQ ID
NOS: 1-12, 15-28, 30, 31, 33, 35, 40, 42-50, 51, 52, or 54-70, or a fragment
thereof, where the fragment is greater than about I 8 nucleotides in length,
with a preparation of genomic DNA from the cell, under high stringency
hybridization conditions, and detectiiZg DNA sequences having about 55%
or greater nucleotide sequence identity to any of SEQ ID NOS: 1-12, 15-28,
30, 31, 33, 35, 40, 42-50, 51, 52, or 54-70, thus identifying a prostate-
specific or testis-specific gene or fragment thereof. Nucleotides encoding
the polypeptides of SEQ ID NOS: 38, 39, or 71-73 can also be used in an
embodiment of this aspect. In a preferred embodiment of this aspect, the
method includes detecting a neoplastic or cancer cell in a patient
predisposed to or at risk for cancer, for example, for prostate cancer.
In a ninth aspect, the invention provides a method for identifying a
test compound that modulates the expression or activity of a prostate-
specific or testis-specific polypeptide, the method including contacting the
prostate-specific or testis-specific polypeptide with the test compound, and
determining the effect of the test compound on the prostate-specific or
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testis-specific polypeptide expression or activity. In a preferred
embodiments of this aspect, the prostate-specific or testis-specific
polypeptide includes an amino acid sequence substantially identical to the
amino acid sequence of SEQ ID NOS: 14, 29, 32, 34, 36, 38, 39, 41, 53, or
71-73, and fragments and analogs thereof.
In a tenth aspect, the invention provides a method of treating a
mammal having a disorder of the prostate or testis, the method including
administering to the mammal a therapeutically effective amount of a
compound that modulates the activity or expression of a prostate-specific or
testis-specific polypeptide, where the compound has a beneficial effect on
the disorder in the mammal. In preferred embodiments of this aspect, the
disorder is prostate cancer, the mammal is a human, or the prostate-specific
or testis-specific polypeptide includes an amino acid sequence substantially
identical to the amino acid sequence of SEQ ID NOS: 14, 29, 32, 34, 36,
38, 39, 41; 53, or 71-73, and fragments and analogs thereof.
In an eleventh aspect, the invention provides a pharmaceutical
composition including at least one dose of a therapeutically effective
amount of a prostate-specific or testis-specific polypeptide or fragment
thereof, in a pharmaceutically acceptable carrier, the composition being
formulated for the treatment of a disorder of the prostate or testis.
In a twelfth aspect, the invention provides a kit for the analysis of a
prostate-specific or testis-specific nucleic acid molecule, the kit including
a
nucleic acid molecule probe for analyzing a prostate-specific or testis-
specific nucleic acid molecule present in a test subject.
In a thirteenth aspect, the invention provides a kit for the analysis of
a prostate-specific or testis-specific polypeptide, the kit including an
antibody for analyzing a prostate-specific or testis-specific polypeptide
present in a test subject.
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As used herein, by "polypeptide," "protein," or "polypeptide
fragment" is meant a chain of two or more amino acids, regardless of any
post-translational modification (e.g., glycosylation or phosphorylation),
constituting all or part of a naturally or non-naturally occurring
polypeptide. By "post-translational modification" is meant any change to a
polypeptide or polypeptide fragment during or after synthesis. Post-
translational modifications can be produced naturally (such as during
synthesis within a cell) or generated artificially (such as by recombinant or
chemical means). A protein can be made up of one or more polypeptides.
By "substantially pure polypeptide" or "substantially pure and
isolated polypeptide" is meant a polypeptide (or a fragment thereof) that
has been separated from components that naturally accompany it.
Typically, the polypeptide is substantially pure when it is at least 60%, by
weight, free from the proteins and naturally occurring organic molecules
with which it is naturally associated. Preferably, the polypeptide is a
prostate-specific or a testis-specific polypeptide that is at least 75%, more
preferably at least 90%, and most preferably at least 99%, by weight, pure.
A substantially pure prostate-specific or a testis-specific polypeptide may
be obtained by standard techniques, for example, by extraction from a
natural source (e.g., prostate or testis tissue or cell lines), by expression
of a
recombinant nucleic acid encoding a prostate-specific or a testis-specific
polypeptide, or by chemically synthesizing the polypeptide. Purity can be
measured by any appropriate method, e.g., by column chromatography,
polyacrylamide gel electrophoresis, or HPLC analysis.
A protein or polypeptide is substantially free of naturally associated
components when it is separated from those contaminants that accompany
it in its natural state. Thus, a protein that is chemically synthesized or
produced in a cellular system different from the cell from which it naturally
originates will be substantially free from its naturally associated
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components. Accordingly, substantially pure polypeptides not only include
those derived from eukaryotic organisms but also those synthesized in E.
coli or other prokaryotes.
The term "identity" is used herein to describe the relationship of the
sequence of a particular nucleic acid molecule or polypeptide to the
sequence of a reference molecule of the same type. For example, if a
polypeptide or nucleic acid molecule has the same amino acid or nucleotide
residue at a given position, compared to a reference molecule to which it is
aligned, there is said to be "identity" at that position.
The level of sequence identity of a nucleic acid molecule or a
polypeptide to a reference molecule is typically measured using sequence
analysis software with the default parameters specified therein, such as the
introduction of gaps to achieve an optimal alignment. The "identity" of
two or more nucleic acid or polypeptide sequences can therefore be readily
calculated by known methods, including but not limited to those described
in Computational Molecular Biology, Lesk, A.M., ed., Oxford University
Press, New York, 1988; Biocomputing: Informatics and Genome Projects,
Smith, D.W., ed., Academic Press, New York, 1993; Computer Analysis of
Sequence Data, Part I, Griffin, A.M., and Griffin, H.G., eds., Humana
Press, New Jersey, 1994; Sequeyace Analysis in Molecular Biology, von
Heinje, Academic Press, 1987; and Sequence Analysis Primer, Gribskov,
and Devereux, eds., M. Stockton Press, New York, 1991; and Carillo and
Lipman, SLAM J. Applied Math. 48:1073, 1988.
Methods to determine identity are available in publicly available
computer programs. Computer program methods to determine identity
between two sequences include, but are not limited to, the GCG program
package (Devereux et al., Nucleic Acids Research 12(1): 387, 1984),
BLASTP, BLASTN, and FASTA (Altschul et al., J. Mol. Biol. 215: 403
(1990). The well known Smith Waterman algorithm may also be used to
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determine identity. The BLAST program is publicly available from NCBI
and other sources (BLAST Manual, Altschul, et al., NCBI NLM NIIi
Bethesda, MD 20894). Searches can be performed in URLs such as the
following htt~//www.ncbi.nhn.nih.~ov/BLAST/unfmished~enome.html; or
http://www.tigr.org/cgi-bin/BlastSearch/blast.cgi. These software programs
match similar sequences by assigning degrees of homology to various
substitutions, deletions, and other modifications. Conservative substitutions
typically include substitutions within the following groups: glycine, alanine;
valine, isoleucine, leucine; aspartic acid, glutamic acid, asparagine,
glutamine; serine, threonine; lysine, arginine; and phenylalanine, tyrosine.
A nucleic acid molecule or polypeptide is said to be "substantially
identical" to a reference molecule if it exhibits, over its. entire length, at
least 50%, 60%, or 70%, preferably at least 80% or 90%, more preferably at
least 95%, and most preferably at least 99% identity to the sequence of the
reference molecule. For polypeptides, the length of comparison sequences
is at least 16 amino acids, preferably at least 20 amino acids or at least
amino acids, more preferably at least 35 amino acids, and most
preferably, the full-length polypeptide. For nucleic acid molecules, the
length of comparison sequences is at least 50 nucleotides, preferably at
20 least 60 nucleotides, more preferably at least 75 nucleotides or at least
110 nucleotides, and most preferably, the full-length nucleic acid molecule.
Alternatively, or additionally, two nucleic acid sequences are "substantially
identical" if they hybridize under high stringency conditions.
By "isolated nucleic acid molecule," "substantially pure nucleic acid
25 molecule," or "substantially pure and isolated nucleic acid molecule" is
meant a nucleic acid molecule (for example, DNA) that is free of the genes
that, in the naturally occurring genome of the organism from which the
nucleic acid molecule of the invention is derived, flank the nucleic acid.
The term iilcludes, for example, a recombinant DNA that is incorporated
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into a vector; into an autonomously replicating plasmid or virus; or into the
genomic DNA of a prokaryote or eukaryote; or that exists as a separate
molecule (e.g., a cDNA or a genomic or cDNA fragment produced by PCR
or restriction endonuclease digestion) independent of other sequences. It
also includes a recombinant DNA that is part of a hybrid gene encoding
additional polypeptide sequence.
By "antisense," as used herein in reference to nucleic acid
molecules, is meant a molecule having a nucleic acid sequence, regardless
of length, that is complementary to at least 75 nucleotides, and preferably at
least 100, 150, or 200 nucleotides, of the coding strand of a nucleic acid
molecule encoding a prostate-specific or a testis-specific polypeptide, as
described herein. An antisense molecule may also include regulatory
sequences such as transcription enhancers, hormone responsive elements,
ribosomal- and RNA polymerase binding sites, etc., which may be located
upstream or downstream of the coding region, and may have a distance of
several ten base pairs to several ten thousand base pairs. An antisense
nucleic acid molecule can be, for example, capable of preferentially
lowering the production or expression of a prostate-specific or a testis-
specific polypeptide encoded by a prostate-specific or a testis-specific
nucleic acid molecule.
By "prostate-specific" or "testis-specific" nucleic acid molecule is
meant a nucleic acid molecule, such as a genomic DNA, cDNA, or RNA
(e.g., mRNA) molecule, having at least 50, 60, or 75%, more preferably at
least 80, 85, or 95%, and most preferably at least 99% amino acid identity
to the nucleic acid molecules described herein, for example, in Figures 4,
11, and 14. In addition, a nucleic acid molecule having at least 50, 60, or
75%, more preferably at least 80, 85, or 95%, and most preferably at least
99% nucleotide identity to a nucleotide sequence encoding amino acids 1-
200 of STMPl (SEQ ID NO: 14), preferably encoding amino acids 40-150
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of STMP 1, can be considered a prostate-specific or testis-specific nucleic
acid molecule. Specifically excluded from this definition is STEAP
(AF186249) (Hubert, R. S. et al., P~oc Natl Acad Sci USA 96, 14523-
14528, 1999) and nucleic acid molecule sequences set forth in or encoding
ESTs AF 132025, AF 177862, BAB23615, BAA91839, BAB 15559, and
NP 032190.
A preferred prostate-specific nucleic acid molecule may be
preferentially expressed in prostate tissue at a level that is at least 5-fold
higher, preferably at least 10-fold higher, more preferably at least 15-fold
higher, and most preferably at least 20-fold higher than the level of the
same nucleic acid molecule in at least one non-prostate tissue, preferably in
alI other non-prostate tissues. A prostate-specific nucleic acid molecule can
also be expressed at high levels in a non-prostate tissue although, generally,
the Ievel of expression will be the highest in the prostate. Occasionally, as
described herein, a prostate-specific nucleic acid molecule will be
expressed at higher levels in non-prostate tissue (e.g., placenta, lung, or
liver) than in the prostate.
A preferred testis-specific nucleic acid molecule may be
preferentially expressed in testis tissue at a level that is at least 5-fold
higher, preferably at least 10-fold higher, more preferably at least 15-fold
higher, and most preferably at least 20-fold higher than the level of the
same nucleic acid molecule in at least one non- testis tissue, preferably in
all other non- testis tissues. A testis -specific nucleic acid molecule can
also be expressed at high levels in a non- testis tissue although, generally,
the level of expression will be the highest in the testis. Occasionally, as
described herein, a testis -specific nucleic acid molecule will be expressed
at higher levels in non- testis tissue (e.g., placenta, lung, or liver) than
in the
testis.
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By "prostate-specific" or a "testis-specific" polypeptide or "prostate-
specific" or a "testis-specific" protein is meant a polypeptide that is
encoded by a prostate-specific or a testis-specific nucleic acid molecule. A
prostate-specific or testis-specific polypeptide may also be defined as a
polypeptide having at least 50, 60, or 75%, more preferably at least 80, 85,
or 95%, and most preferably at least 99% amino acid identity to the
polypeptides described herein, for example, in Figures 4, 11, and 14.
Specifically excluded from this definition is STEAP (AF 186249) (Hubert,
R. S. et al., PYOC Natl Acad Sci USA 96, 14523-14528, 1999) and
polypeptide sequences set forth in or encoded by ESTs AF132025,
AF177862, BAB23615, BAA91839, BAB15559, andNP_032190. In
addition, a polypeptide having at least 50, 60, or 75%, more preferably at
least 80, 85, or 95%, and most preferably at least 99% amino acid identity
to amino acids 1-200 of STMP1 (SEQ ID NO: 14), preferably amino acids
40-150 of STMP1, can be considered a prostate-specific or testis-specific
polypeptide.
A preferred prostate-specific polypeptide is preferentially expressed
in prostate tissue at a level that is at least 5-fold higher, preferably at
least
10-fold higher, more preferably at least 15-fold higher, and most preferably
at least 20-fold higher than the level of the same polypeptide in at least one
non-prostate tissue, preferably in all other non-prostate tissues. A prostate-
specific polypeptide can also be expressed at high levels in a non-prostate
tissue although, generally, the level of expression will be the highest in the
prostate. Occasionally, as described herein, a prostate-specific polypeptide
will be expressed at higher levels in non-prostate (e.g., placenta, lung,
liver)
than in the prostate.
A preferred testis-specific polypeptide is preferentially expressed in
testis tissue at a level that is at least 5-fold higher, preferably at least
10-
fold higher, more preferably at least 15-fold higher, and most preferably at
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least 20-fold higher than the level of the same polypeptide in at least one
non- testis tissue, preferably in all other non-testis tissues. A testis-
specific
polypeptide can also be expressed at high levels in a non- testis tissue
although, generally, the level of expression will be the highest in the
testis.
Occasionally, as described herein, a testis-specific polypeptide will be
expressed at higher levels in non- testis (e.g., placenta, lung, liver) than
in
the testis.
The term prostate-specific or testis-specific polypeptide includes
homologs, analogs, fragments, and isoforms, e.g., alternatively spliced
isoforms, of the sequences described herein. By "biologically active
fragment" is meant a polypeptide fragment of a prostate-specific or testis-
specific polypeptide that exhibits, for example, extracellular trafficking,
cell
signaling, or other properties that are at least 30%, preferably at least 50%,
more preferably at least 75%, and most preferably at least 100%, compared
with the properties of a full length prostate-specific or testis-specific
polypeptide. By "analog" is meant any substitution, addition, or deletion in
the amino acid sequence of a prostate-specific or testis-specific polypeptide
that exhibits properties that are at least 30%, preferably at least 50%, more
preferably at least 75%, and most preferably at least 100%, compared with
the extracellular trafficking or cell signaling properties of the polypeptide
from which it is derived. Fragments, homologs, and analogs can be
generated using standard techniques, for example, solid phase peptide
synthesis or polymerase chain reaction. For example, point mutations may
arise at any position of the sequence from an apurinic, apyrimidinic, or
otherwise structurally impaired site within the cDNA. Alternatively, point
mutations may be introduced by random or site-directed mutagenesis
procedures (e.g., oligonucleotide assisted or by error prone PCR).
Likewise, deletions and/or insertions may be introduced into the sequences,
and preferred insertions comprise 5'- and/or 3'-fusions with a
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polynucleotide that encodes a reporter moiety or an affinity moiety. Other
preferred insertions comprise a nucleic acid that further includes functional
elements such as a promoter, enhancer, hormone responsive element, origin
of replication, transcription and translation initiation sites, etc. It should
be
appreciated that where insertions with one or more functional elements are
present, the resulting nucleic acid may be linear or circular (e.g.,
transcription or expression cassettes, plasmids, etc:).
For use in the methods of the invention, the terms "prostate-specific"
or "testis-specific" polypeptide further include the polypeptide sequences
set forth in or encoded by ESTs AF132025, AF177862, BAB23615,
BAA91839, BAB 15559, and NP 032190, but does not include STEAD,
and a prostate-specific or testis-specific nucleic acid molecule includes the
nucleotide sequences set forth in or encoding ESTs AF 132025, AF 177862,
BAB23615, BAA91839, BAB 15559, and NP 032190, but does not include
STEAD.
By "prostate-specific or a testis-specific gene or homolog or
fragment thereof' is meant a gene, or homolog of a gene, that encodes a
prostate-specific or testis-specific polypeptide.
By "specifically binds" is meant a compound, e.g., an antibody, that
recognizes and binds a protein or polypeptide, for example, a prostate-
specific or a testis-specific polypeptide, and that when delectably labeled
can be competed away for binding to that protein or polypeptide by an
excess of compound that is not detectably labeled. A compound that non-
specifically binds is not competed away by excess detectably labeled
compound. A preferred antibody binds to any prostate-specific or a testis-
specific polypeptide sequence that is substantially identical to any of the
polypeptide sequences set forth in Figures 4, 11, and 14, or encoded by any
of the nucleotide sequences set forth in Figures 3, 4, 11, and 14, or portions
thereof.
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By a "compound," "test compound," or "candidate compound" is
meant a molecule, be it naturally-occurring or artificially-derived, and
includes, for example, peptides, proteins, synthetic organic molecules,
naturally-occurring organic molecules, nucleic acid molecules, and
components thereof.
By "high stringency conditions" is meant conditions that allow
hybridization comparable with the hybridization that occurs using a DNA
probe of at least 500 nucleotides in length, in a buffer containing 0.5 M
NaHP04, pH 7.2, 7% SDS, 1 mM EDTA, and 1% BSA (fraction V), at a
temperature of 65°C, or a buffer containing 48% formamide, 4.8X SSC,
0.2
M Tris-Cl, pH 7.6, 1X Denhardt's solution, 10% dextran sulfate, and 0.1%
SDS, at a temperature of 42°C (these are typical conditions for
high
stringency Northern or Southern hybridizations). High stringency
hybridization is also relied upon for the success of numerous techniques
routinely performed by molecular biologists, such as high stringency PCR,
DNA sequencing, single strand conformational polymorphism analysis, and
ih situ hybridization. In contrast to Northern and Southern hybridizations,
these techniques are usually performed with relatively short probes (e.g.,
usually 16 nucleotides or longer for PCR or sequencing, and 40 nucleotides
or longer for iu situ hybridization). The high stringency conditions used in
these techniques are well known to those skilled in the art of molecular
biology, and may be found, for example, in Ausubel et al., Currevct
Protocols i~c Molecular Biology, John Wiley & Sons, New York, NY, 1998,
hereby incorporated by reference.
By "probe" or "primer" is meant a single-stranded DNA or RNA
molecule of defined sequence that can base pair to a second DNA or RNA
molecule that contains a complementary sequence ("target"). The stability
of the resulting hybrid depends upon the extent of the base pairing that
occurs. This stability is affected by parameters such as the degree of
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complementarity between the probe and target molecule, and the degree of
stringency of the hybridization conditions. The degree of hybridization
stringency is affected by parameters such as the temperature, salt
concentration, and concentration of organic molecules, such as formamide,
and is determined by methods that are well known to those skilled in the
art. Probes or primers specific for prostate-specific or a testis-specific
nucleic acid molecules, preferably, have greater than 45% sequence
identity, more preferably at least 55-75% sequence identity, still more
preferably at least 75-85% sequence identity, yet more preferably at least
85-99% sequence identity, and most preferably 100% sequence identity to
the nucleic acid sequences encoding the amino acid sequences described
herein. Probes can be detestably-labeled, either radioactively or non-
radioactively, by methods that are well-known to those skilled in the art.
Probes can be used for methods involving nucleic acid hybridization, such
as nucleic acid sequencing, nucleic acid amplification by the polymerase
chain reaction, single stranded conformational polymorphism (SSCP)
analysis, restriction fragment polymorphism (RFLP) analysis, Southern
hybridization, northern hybridization, ih situ hybridization, electrophoretic
mobility shift assay (EMSA), and other methods that are well known to
those skilled in the art.
A molecule, e.g., an oligonucleotide probe or primer, a gene or
fragment thereof, a cDNA molecule, a polypeptide, or an antibody, can be
said to be "detestably-labeled" if it is marked in such a way that its
presence can be directly identified in a sample. Methods for detectably-
labeling molecules are well known in the art and include, without
limitation, radioactive labeling (e.g., with an isotope, such as 32P or 35S)
and
nonradioactive labeling (e.g., with a fluorescent label, such as fluorescein,
or by generating a construct containing green fluorescent protein (GFP)).
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By "transgenic" is meant any cell that includes a DNA sequence or
transgene that is inserted by artifice into a cell and becomes part of the
genome of the organism that develops from that cell. As used herein, the
transgenic organisms are generally transgenic mammals (e.g., mice, rats,
and goats) and the DNA (transgene) is inserted by artifice into the nuclear
genome. By "transgene" is meant any piece of DNA that is inserted by
artifice into a cell, and becomes part of the genome of the organism that
develops from that cell. Such a transgene may include a gene that is partly
or entirely heterologous (i.e., foreign) to the transgenic organism, or may
represent a gene homologous to an endogenous gene of the organism. By
"knockout mutation" is meant an artificially induced alteration in the
nucleic acid sequence (created via recombinant DNA technology or
deliberate exposure to a mutagen) that reduces the biological activity of the
polypeptide normally encoded therefrom by at least 80% relative to the
unmutated gene. The mutation may, without limitation, be an insertion,
deletion, frameshift mutation, or a missense mutation. The knockout
mutation can be in a cell ex vivo (e.g., a tissue culture cell or a primary
cell)
or ih vivo. A "knockout animal" is a mammal, preferably, a mouse,
containing a knockout mutation as defined above.
By "sample" is meant a tissue biopsy, cells, blood, serum, urine,
stool, or other specimen obtained from a patient or test subject. The sample
is analyzed to detect a mutation in a gene encoding a prostate-specific or a
testis-specific polypeptide, or expression levels of a gene encoding a
prostate-specific or a testis-specific polypeptide, as for example, an
indication of the progression of cancer, by methods that are known in the
art or described herein. For example, methods such as sequencing, single-
strand conformational polymorphism (SSCP) analysis, or restriction
fragment length polymorphism (RFLP) analysis of PCR products derived
from a patient sample may be used to detect a mutation in a gene encoding
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a prostate-specific or a testis-specific polypeptide; ELISA may be used to
measure levels of a prostate-specific or a testis-specific polypeptide; and
PCR may be used to measure the level of nucleic acids encoding a prostate-
specific or a testis-specific polypeptide.
By "pharmaceutically acceptable carrier" is meant a carrier that is
physiologically acceptable to the treated mammal while retaining the
therapeutic properties of the compound with which it is administered. One
exemplary pharmaceutically acceptable carrier is physiological saline
solution. Other physiologically acceptable carriers and their formulations
are known to one skilled in the art and described, for example, W
Remiragton: The Science and Pf~actice of Pharmacy, ( 19th edition), ed. A.
Gennaro, 1995, Mack Publishing Company, Easton, PA.
"Therapeutically effective amount" as used herein in reference to
dosage of a medication, refers to the administration of a specific amount of
a pharmacologically active agent (e.g., a prostate-specific or a testis-
specific polypeptide, nucleic acid molecule, or modulatory compound)
tailored to each individual patient manifesting symptoms characteristic of a
specific disorder. For example, a patient receiving the treatment of the
present invention might have prostate cancer. A person skilled in the art
will recognize that the optimal dose of a pharmaceutical agent to be
administered will vary from one individual to another. Dosage in
individual patients should take iilto account the patients height, weight,
rate
of absorption and metabolism of the medication in question, the stage of the
disorder to be treated, and what other pharmacological agents are
administered concurrently.
By "treating " or "treatment" is meant the medical management of a
patient with the intent to cure, ameliorate, stabilize, or prevent a disease,
pathological condition, or disorder. This term includes active treatment,
that is, treatment directed specifically toward the improvement or
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associated with the cure of a disease, pathological condition, or disorder,
and also includes causal treatment, that is, treatment directed toward
removal of the cause of the associated disease, pathological condition, or
disorder. In addition, this term includes palliative treatment, that is,
treatment designed for the relief of symptoms rather than the curing of the
disease, pathological condition, or disorder; preventative treatment, that is,
treatment directed to minimizing or partially or completely inhibiting the
development of the associated disease, pathological condition, or disorder;
and supportive treatment, that is, treatment employed to supplement
another specific therapy directed toward the improvement of the associated
disease, pathological condition, or disorder. The phrase "treatment" also
includes symptomatic treatment, that is, treatment directed toward
constitutional symptoms of the associated disease, pathological condition,
or disorder.
By "disorder of the prostate or testis" is meant a disturbance of
function and/or structure of the prostate or testis in a living organism,
resulting from an external source, a genetic predisposition, a physical or
chemical trauma, or a combination of the above. Such disorders include the
proliferation of prostate or testicular cells. By "cell proliferation" is
meant
the growth or reproduction of similar cells, and the invention provides
reagents for inhibiting proliferation and stimulating proliferation. By
"inhibiting proliferation" is meant the decrease in the number of similar
cells by at least 10%, more preferably by at least 20%, and most preferably
by at least 50%. By "stimulating proliferation" is meant an increase in the
number of similar cells by at least 10%; more preferably by at least 20%,
and most preferably by at least 50%.
The reagents described herein, for example, vectors expressing
antisense, antagonists, or inhibitors of prostate-specific or testis-specific
polypeptides or nucleic acid molecules may be used, for example, to
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suppress the excessive proliferation of prostate or testicular cells. Blocking
prostate-specific or testis-specific polypeptide or nucleic acid molecule
expression or activity in prostate or testicular cells can alter molecular
pathways within cancerous cells and thus trigger apoptosis, i.e., the process
of cell death where a dying cell displays a set of well-characterized
biochemical hallmarks which include cytolemmal blebbing, cell soma
shrinkage, chromatin condensation, and DNA laddering.
Disorders of the prostate or testis include prostate cancer, benign
prostatic hyperplasia, acute prostatitis, testicular cancer, developmental
defects of the prostate or testis (such as cryptorchidism or undescended
testis, and retractile, ascending, or vanished testis).
By "proliferative disease" is meant a disease that is caused by or
results in inappropriately high levels of cell division, inappropriately low
levels of apoptosis, or both. For example, cancers such as prostate cancer,
testicular cancer, lymphoma, leukemia, melanoma, ovarian cancer, breast
cancer, pancreatic cancer, liver cancer, and lung cancer are all examples of
proliferative disease.
By "modulate" or "modulating" is meant changing, either by
decrease or increase, the expression or biological activity of a prostate-
specific or testis-specific nucleic acid molecule or polypeptide, as described
herein. It will be appreciated that the degree of modulation provided by a
modulating compound in a given assay will vary, but that one skilled in the
art can determine the statistically significant change in the level of
biological activity that identifies a compound that modulates a prostate-
specific or testis-specific nucleic acid molecule or polypeptide.
The invention provides several advantages. For example, it provides
methods and reagents that can be used in the diagnosis and treatment of
prostate and testis associated diseases, as well as other disorders and
conditions that are sensitive to the bioactivities of the reagents (e.g.,
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polypeptides, nucleic acid molecules, antibodies) described herein. Since
the prostate-specific or testis-specific polypeptides of the invention have
been found to be highly expressed in the prostate and testis, these
polypeptides can also be used in screens for therapeutics to treat disorders
associated with the prostate and testis. These polypeptides are also
expressed in other tissues, and can be used as therapeutics and diagnostics
for cell proliferative disorders.
Other features and advantages of the invention will be apparent from
the detailed description of the invention, the drawings, and the claims.
Brief Description of The Drawings
Figure 1 shows an exemplary reverse northern analysis of several
clones from a prostate specific cDNA library.
Figure 2 shows an exemplary multiple tissue northern blot.
Figure 3 is a table showing the nucleotide sequences of twelve
clones (SEQ ID NOs: 1-12) isolated from prostate tissue and LNCaP cells .
Figure 4A is a schematic diagram showing the STMPI gene
structure.
Figure 4B shows the nucleotide sequence, including the intron
junction sequences (SEQ 117 NO: 13), and predicted amino acid sequence
(SEQ ID NO: 14) of STMP1..
Figure 4C shows the nucleotide sequences of the exons and 3' UTR
of STMPI (SEQ ID NOs: 15-21).
Figure 4D shows the nucleotide sequence of the ORF of STMPI
(SEQ ID NO: 22).
Figure 4E shows the shows the cDNA sequence (SEQ ID NO: 23),
and predicted amino acid sequence (SEQ ID N0:14) of STMP1.
Figure 4F shows the nucleotide sequences of the exons and 3' UTR
of STMPI ORF2 (SEQ ID NOs: 17-20 and 24-26).
CA 02403637 2002-09-19
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Figure 4G shows the nucleotide sequence of the ORF of STMPI
ORF2 (SEQ ID NO: 27).
Figure 4H shows the cDNA sequence (SEQ ID NO: 28), and
predicted amino acid sequence (SEQ ID NO: 29) of STMP1 ORF2.
Figure 4I shows the nucleotide sequences of the exons and 3' UTR
of STMPI ORF3 (SEQ ID NOs: 17-19 and 24-26).
Figure 4J shows the nucleotide sequence of the ORF of STMPI
ORF3 (SEQ ID NO: 30).
Figure 4K shows the cDNA sequence (SEQ ID NO: 31), and
predicted amino acid sequence (SEQ ID NO: 32) of STMP1 ORF3.
Figure 4L shows the cDNA sequence (SEQ ID NO: 33), and
predicted amino acid sequence (SEQ ID N0:34) of STMP2.
Figure 4M shows the cDNA sequence (SEQ ID NO: 35), and
predicted amino acid sequence (SEQ ID NO: 36) of STMP3.
Figure 5 shows a sequence alignment of STMP 1 (SEQ ID NO: 14),
with STEAP (SEQ ID NO: 37, Accession No. AF186249), and two ESTs
(Accession No. BAA91839 and Accession No. BAB15559; SEQ ID NOs:
38 and 39, respectively).
Figure 6A shows a multiple tissue Northern blot probed with STMPI
or G3Pl~H cDNA.
Figure 6B is a Northern blot probed with STMPI and PSA in the
androgen-responsive prostate cancer cell line LNCaP and in the CWR22
human prostate cancer xenograft model.
Figure 6C is a Northern blot probed with STMPI and NKX3A in
LNCaP, PC-3, and DU-145 cell lines and in the CWR22R human prostate
cancer xenograft model.
Figure 7A shows fluorescence microscopy images of COS-1 cells
transiently transfected with GFP-STMP1.
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Figure 7B shows fluorescence microscopy images of COS-1 cells
transiently transfected with GFP-STMP1 and labeled with antibodies
against Golgi markers.
Figure 8 shows fluorescence microscopy images of COS-1 cells
transiently transfected with GFP-STMP1 and observed by live-cell confocal
microscopy.
Figure 9 shows fluorescence microscopy images of COS-1 cells
transiently transfected with GFP-STMP1 and labeled with an antibody
against an early endosomal marker.
Figure 10 is a schematic diagram showing the SSH9 gene structure
and two mRNA species transcribed from the SSH9 gene.
Figure 1 1A shows the cDNA (SEQ m NO: 40) and predicted amino
acid sequence (SEQ ID NO: 41) for SSH9.
Figure 11B shows the predicted promoter sequence for SSH9 (SEQ
m NO: 42).
Figure 11 C shows the predicted intron-exon boundaries for SSH9
(SEQ ID NOs: 43-50).
Figure 12A is a Northern blot probed with SSH9 in the androgen-
responsive prostate cancer cell line LNCaP cells and in the CWR22 human
prostate cancer xenograft model.
Figure 12B is a Northern blot probed with SSH9 in LNCaP, PC-3,
and DU-145 cell lines, and CWR22R human prostate cancer xenograft
model.
Figure 12C is a multiple tissue Northern blot probed with SSH9 or
GAPDH cDNA.
Figure 13 is a schematic diagram showing the PSL22 gene structure.
Figure 14A shows the nucleotide sequence of the ORF of PSL22
(SEQ ID NO: 51).
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Figure 14B shows the cDNA sequence (SEQ ID NO: 52), and
predicted amino acid sequence (SEQ ID NO: 53) of PSL22.
Figure 14C shows the nucleotide sequences of the TATA promoter
and transcription start site, exons, and 5' and 3' UTRs of PSL22 (SEQ ID
NOs:54-70).
Figure 15 shows a sequence alignment of PSL22 (RhoBP) (SEQ ID
NO: 53), with ESTs NP032190 (mRhoph), AF132025 (dRhoph), and
BAB23615 (SEQ ID Nos:71-73).
Figure 16A is a Northern blot probed with PSL22 in LNCaP, PC-3,
and DU-145 cell lines, and iil the CWR22R human prostate cancer
xenograft model.
Figure 16B is a multiple tissue Northern blot probed with PSL22
cDNA.
Detailed Description of the Invention
The basic biology of the normal prostate and testis, as well as
prostate and testicular cancer initiation and progression is still poorly
understood. It is therefore necessary to delineate the molecular events that
are at the basis of these processes. To achieve this goal, we have
identified, cloned, and characterized highly prostate- and testis-enriched
genes whose gene products have important roles for both the normal
physiology and the pathophysiology of the prostate and the testis. These
gene products also have important roles iil other disorders, for example,
heart, brain, liver, pancreas, kidney, and colon, which are the tissues where
variable low expression, and occasionally, very high expression of specific
gene products, can be detected by Northern analysis.
The invention provides prostate-specific or testis-specific
polypeptides and nucleic acid molecules (see below), and diagnostic and
therapeutic methods employing these polypeptides and nucleic acid
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molecules. The invention also provides methods for identifying
compounds that modulate the biological activities of prostate-specific or
testis-specific polypeptides and nucleic acid molecules, and therapeutic
methods employing these compounds. The diagnostic, therapeutic, and
screening methods of the invention are first described, followed by general
approaches that can be used in carrying out these methods. Finally,'
experimental results supporting the methods of the invention are described.
Bioassays
Prostate-specific and testis-specific polypeptides are expressed in the
prostate and testis, and also in other tissues such as kidney, pancreas,
liver,
lung, and colon. The expression patterns of prostate-specific and testis-
specific polypeptides in specific cells and tissues are used to identify
cellular targets of prostate-specific and testis-specific polypeptide actions,
and to identify bioactivities that are relevant to specific prostate- and
testis-
related diseases, such as prostate cancer, testicular cancer, benign prostatic
hyperplasia, acute prostatitis, and developmental testis defects.
Therapeutic and diagnostic utilities for prostate-specific and testis-
specific polypeptides are identified by, for example, conducting bioassays
ih vitro. Culture systems that reflect prostate-specific and testis-specific
polypeptide expression patterns, along with the distribution of particular
receptors, such as the androgen receptor, are selected. For example,
LNCaP cells express androgen receptors, and respond to one or more
isofonns of prostate-specific and testis-specific polypeptides in a variety of
bioassays. The activities of prostate-specific and testis-specific
polypeptides (e.g., STMP1, SSH9, PSL22) are compared, using sister
cultures, in various dose-response assays, including but not limited to,
inhibition of proliferation, apoptosis, signaling events (e.g. changes in
kinase activity), changes in transcription factor activity (such as that of
the
androgen receptor), intracellular trafficking, or cell signaling. The relative
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potencies of the prostate-specific and testis-specific polypeptides are
determined on the basis of, for example, protein concentration.
Dia~,nostic Methods Employing Prostate-Specific Or Testis-Specific
Nucleic Acid Molecules, Poly~eptides, and Antibodies
Prostate-specific or testis-specific nucleic acid molecules,
polypeptides, and antibodies are used in methods to diagnose or monitor a
variety of diseases and conditions, including those involving mutations in,
or inappropriate expression of, prostate-specific or testis-specific genes.
Prostate-specific or testis-specific expression has been documented in a
variety of tissues, as discussed above. Thus, detection of abnormalities in
prostate-specific or testis-specific genes or their expression is used in
methods to diagnose, or to monitor treatment or development of diseases of
these tissues.
The diagnostic methods of the invention are used, for example, with
patients that have a prostate-related or testis-related disease, for example,
prostate or testicular cancer, in an effort to determine its etiology, and
thus,
to facilitate selection of an appropriate course of treatment. The diagnostic
methods are also used with patients that have not yet developed a prostate-
related or testis-related disease, but who may be at risk of developing such
a disease, or with patients that are at an early stage of developing such a
disease. Many prostate-related or testis-related diseases occur during
development, and thus, the diagnostic methods of the invention are also
carried out on a fetus or embryo during development. Also, the diagnostic
methods of the invention are used in prenatal genetic screening, for
example, to identify parents who may be carriers of a recessive prostate-
related or testis-related mutation.
Prostate-specific or testis-specific abnormalities that are detected
using the diagnostic methods of the invention include those characterized
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by, for example, (i) abnormal prostate-specific or testis-specific
polypeptides, (ii) prostate-specific or testis-specific genes containing
mutations that result in the production of such polypeptides, and (iii)
mutations that result in production of abnormal amounts of prostate-
s specific or testis-specific polypeptides.
Levels of prostate-specific or testis-specific expression in a patient
sample are determined by using any of a number of standard techniques
that are well known in the art. For example, prostate-specific or testis-
specific expression in a biological sample (e.g., a blood, prostate or testis
tissue sample, or amniotic fluid) from a patient is monitored by standard
northern blot analysis or by quantitative PCR (see, e.g., Ausubel et al.,
Current Protocols in Molecular Biology, John Wiley & Sons, New York,
NY, 1998; PCR Technology: Principles and Applications for DNA
Amplification, H.A. Ehrlich, Ed., Stockton Press, NY; Yap et al. Nucl.
Acids. Res. 19:4294, 1991).
A biological sample obtained from a patient can be analyzed for one
or more mutations in prostate-specific or testis-specific nucleic acid
molecules using a mismatch detection approach. Generally, this approach
involves PCR amplification of nucleic acid molecules from a patient
sample, followed by identification of a mutation (i. e., a mismatch) by
detection of altered hybridization, aberrant electrophoretic gel migration,
binding, or cleavage mediated by mismatch binding proteins, or by direct
nucleic acid molecule sequencing. Any of these techniques can be used to
facilitate detection of mutant prostate-specific or testis-specific genes, and
each is well known in the art. Examples of these techniques are described,
for example, by Orita et al. (Proc. Natl. Acad. Sci. USA 86:2766-2770,
1989) and Sheffield et al. (Proc. Natl. Acad. Sci. USA 86:232-236, 1989).
Mismatch detection assays also provide an opportunity to diagnose a
prostate-specific or testis-specific gene-mediated predisposition to a disease
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before the onset of symptoms. For example, a patient heterozygous for a
prostate-specific or testis-specific mutation that suppresses normal prostate-
specific or testis-specific biological activity or expression may show no
clinical symptoms of a prostate-specific or testis-specific gene-related
disease, and yet possess a higher than normal probability of developing a
prostate or testicular disease. Given such a diagnosis, patients can take
precautions to minimize their exposure their exposure to adverse
environmental factors and to carefully monitor their medical condition (for
example, through frequent physical examinations). As mentioned above,
this type of diagnostic approach can also be used to detect prostate-specific
or testis-specific mutations in prenatal screens.
The prostate-specific or testis-specific diagnostic assays described
above can be carried out using any biological sample (for example, a blood,
prostate, or testis tissue sample, or amniotic fluid) in which a prostate-
specific or testis-specific polypeptide or nucleic acid molecule is normally
expressed. A mutant prostate-specific or testis-specific gene can also be
identified using these sources as test samples. Alternatively, a prostate-
specific or testis-specific mutation, as part of a diagnosis for
predisposition
to a prostate-specific or testis-specific gene-associated disease, can be
tested for using a DNA sample from airy cell, for example, by mismatch
detection techniques. Preferably, the DNA sample is subjected to PCR
amplification prior to analysis.
In yet another diagnostic approach of the invention, an immunoassay
is used to detect or monitor prostate-specific or testis-specific protein
expression in a biological sample. Anti-prostate-specific or testis-specific--
polypeptide polyclonal or monoclonal antibodies (as described below) can
be used in any standaxd immunoassay format (e.g., ELISA, Western blot, or
RIA; see, e.g., Ausubel et al., supra) to measure prostate-specific or testis-
specific polypeptide levels. These levels are compared to wild-type
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prostate-specific or testis-specific levels. For example, an increase in
prostate-specific or testis-specific polypeptide production may be indicative
of a condition or a predisposition to a condition involving overexpression
of prostate-specific or testis-specific polypeptide biological activity, such
as
late stage prostate cancer.
Immunohistochemical techniques can also be utilized for prostate-
specific or testis-specific polypeptide detection. For example, a tissue
sample can be obtained from a patient, sectioned, and stained for the
presence of prostate-specific or testis-specific polypeptide using an anti-
prostate-specific or testis-specific antibody (see below) and any standard
detection system (e.g., one that includes a secondary antibody conjugated to
horseradish peroxidase). General guidance regarding such techniques can
be found in, e.g., Bancroft et al., Theory ahd Practice ofHistological
TeclZyaiques, Churchill Livingstone, 1982, and Ausubel et al., supra.
In a preferred example, a combined diagnostic method can be
employed that includes an evaluation of prostate-specific or testis-specific
protein production (for example, by immunological techniques or the
protein truncation test (Hogerrorst et al., Nature Genetics 10:208-212,
1995), and a nucleic acid molecule-based detection technique designed to
identify more subtle prostate-specific or testis-specific mutations (for
example, point mutations). As described above, a number of mismatch
detection assays are available to those skilled in the art, and any preferred
technique can be used. Mutations in prostate-specific or testis-specific
genes can be detected that either result in loss or gain of prostate-specific
or
testis-specific polypeptide or nucleic acid molecule expression or loss or
gain of normal prostate-specific or testis-specific polypeptide or nucleic
acid molecule biological activity.
Prostate-specific or testis-specific polypeptides or nucleic acid
molecules can be used to correlate the course of prostate cancer to a marker
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other than PSA, to monitor the course of an anticancer therapy, or to detect
a neoplastic cell in a system. For example, a predetermined quantity of an
RNA encoding a prostate-specific or testis-specific polypeptide is
correlated with the presence of a neoplastic cell, for example, from a
biopsy. The total RNA is extracted from the biopsy specimen, and a real
tune quantitative rt-PCR employing individual reactions with primer pairs
specific to prostate-specific or testis-specific sequences is performed in
parallel with a biopsy specimen known to be free of cancer cells. Biopsy
specimens are determined to have a cancer cell, where the detected
prostate-specific or testis-specific mRNA quantity is at least 5 times higher
than in the control specimen. An exemplary extraction of total RNA
utilizes the Quiagen BioRobot kit in conjunction with the BioRobot 9600
system, and the real time rtPCR is performed in a Perkin Ehner ABI Prism
7700.
In alternative aspects of the inventive subject matter, the method of
detecting a neoplastic cell need not be limited to biopsy tissues from
prostate or testis tissue, but may employ various alternative tissues,
iilcluding lymphoma tumor cells, and various solid tumor cells, so long as
such tumor cells overproduce mRNA of prostate-specific or testis-specific
polypeptides. Appropriate alternative tumor cells can readily be identified
by the above described method. Likewise, the system need not be restricted
to a mammal, but may also include cell-, and tissue cultures grown in vitro,
and tumor cells and specimens from animals other than mammals. For
example, tumor cell and tissue grown ih vitro may advantageously be
utilized to investigate drug action on such cells, and sequences encoding
prostate-specific or testis-specific polypeptides may conveniently be
employed as tumor marker. Alternatively, body fluids (e.g., serum, saliva,
etc.) that may or may not contain tumor cells are also contemplated a
suitable substrate for the method presented herein, so long as they contain
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to at least some extent mRNA encoding a prostate-specific or testis-specific
polypeptide.
In still other aspects of contemplated methods, the polypeptide
quantity need not necessarily be limited to at least 5 times more than the
control specimen in order to establish that the tissue has a cancer cell. For
example, where the concentration of the polypeptide is hormone dependent,
amounts between 3-8 fold and more may. be appropriate. In contrast, where
the concentration of cancer cells in the biopsy specimen is relatively low,
amounts of less than 5-fold, including 1.5 to 4.9-fold and less are
contemplated.
The detection process may include fluorescence detection,
luminescence detection, scintigraphy, autoradiography, and formation of a
dye. For example, for microscopic analysis of biopsy specimens, luciferase
labeled probes are particularly advantageous in conjunction with a
luminescence substrate (e.g., luciferin). Luminescence quantification may
then be performed utilizW g a CCD-camera and image analysis system.
Similarly, radioactivity may be detected via autoradiographic or
scintigraphic procedures on a tissue section, ill a fluid or on a solid
support.
Where the probe is a natural or synthetic ligand of a prostate-specific or
testis-specific polypeptide, the ligand may include molecules with a chemi-
cal modification that increase the affinity to the polypeptide and/or induce
irreversible binding to the polypeptide. For example, transition state
analogs or suicide inhibitors for a particular reaction catalyzed by the
polypeptide are especially contemplated. Labeling of antibodies, antibody
fragments, small molecules, and binding of the labeled entity is a technique
that is well known in the art, and all known methods are generally suitable
for use in conjunction with methods contemplated herein. Furthermore, the
probe need not be limited to a fluorescein labeled antibody, and alternative
probes include antibody fragments (e.g., Fab, Fab', scFab, etc.).
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Still further contemplated variations include substitution of one or
more atoms or chemical groups in the sequence with a radioactive atom or
group. For example, where cDNAs are employed as a hybridization-
specific probes, a fluorophor or enzyme (e.g., j3-galactosidase for
generation of a dye, or luciferase for generation of luminescence) may be
coupled to the sequence to identify position and/or quantity of a comple-
mentary sequence. Alternatively, where contemplated cDNA molecules are
utilized for affinity isolation procedures, the cDNA may be coupled to a
molecule that is known to have a high-affinity (i.e., I~<10-4mol-I) partner,
such as biotin, or an oligo-histidyl tag. In another example, one or more
phosphate groups may be exchanged for a radioactive phosphate group with
a 32P or 33P isotope to assist in detection and quantification, where the
radiolabeled cDNA is employed as a hybridization probe.
Therabeutic Methods Emplovin~ Prostate-Specific Or Testis-Specific
Nucleic Acid Molecules, Polypeptides, and Antibodies
The invention includes methods of treating or preventing prostate-
specific or testis-specific diseases. Therapies are designed to circumvent or
overcome a prostate-specific or testis-specific gene defect, or inadequate or
excessive prostate-specific or testis-specific gene expression, and thus
modulate and possibly alleviate conditions involving defects in prostate-
specific or testis-specific genes or proteins. In considering various
therapies, it is understood that such therapies axe, preferably, targeted to
the
affected or potentially affected organs, for example, the prostate or the
testis. Reagents that are used to modulate prostate-specific or testis-
specific biological activity can include, without limitation, full length
prostate-specific or testis-specific polypeptides; prostate-specific or testis-
specific cDNA, mRNA, or antisense RNA; prostate-specific or testis-
specific antibodies; and any compound that modulates prostate-specific or
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testis-specific polypeptide or nucleic acid molecule biological activity,
expression, or stability.
Treatment or prevention of diseases resulting from a mutated
prostate-specific or testis-specific gene is accomplished, for example, by
replacing a mutant prostate-specific or testis-specific gene with a normal
prostate-specific or testis-specific gene, administering a normal prostate-
specific or testis-specific gene, modulating the function of a mutant
prostate-specific or testis-specific protein, delivering normal prostate-
specific or testis-specific protein to the appropriate cells, or altering the
levels of normal or mutant. prostate-specific or testis-specific protein. It
is
also possible to correct a prostate-specific or testis-specific gene defect to
modify the physiological pathway (e.g., an intracellular trafficking
pathway) in which the prostate-specific or testis-specific protein
participates.
To replace a mutant protein with normal protein, or to add protein to
cells that do not express sufficient or normal prostate-specific or testis-
specific protein, it may be necessary to obtain large amounts of pure
prostate-specific or testis-specific proteiiz from cultured cell systems in
which the protein is expressed (see, e.g., below). Delivery of the protein to
the affected tissue can then be accomplished using appropriate packaging or
administrating systems. Alternatively, small molecule analogs that act as
prostate-specific or testis-specific molecule agonists or antagonists can be
administered to produce a desired physiological effect (see below).
Gene therapy is another therapeutic approach for preventing or
ameliorating diseases caused by prostate-specific or testis-specific gene
defects. Nucleic acid molecules encoding wild type prostate-specific or
testis-specific proteins can be delivered to cells that lack sufficient,
normal
prostate-specific or testis-specific biological activity (e.g., cells carrying
mutations in prostate-specific or testis-specific genes). The nucleic acid
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molecules must be delivered to those cells in a form in which they can be
taken up by the cells and so that sufficient levels of protein, to provide
effective prostate-specific or testis-specific function, call be produced.
Alternatively, for some prostate-specific or testis-specific mutations, it may
be possible slow the progression of the resulting disease or to modulate
prostate-specific or testis-specific activity by introducing another copy of a
homologous gene bearing a second mutation iiz that gene, to alter the
mutation, or to use another gene to block any negative effect.
Transducing retroviral, adenoviral, and adeno-associated viral
vectors can be used for somatic cell gene therapy, especially because of
their high efficiency of infection and stable integration and expression (see,
e.g., Cayouette et al., Human Gene Therapy 8:423-430, 1997; Kido et al.,
Current Eye Researcla 15:833-844, 1996; Bloomer et al., Journal of
Irirology 71:6641-6649, 1997; Naldini et al., Science 272:263-267, 1996;
and Miyoshi et al., PYOC. Natl. Acad. Sci., USA 94:10319-1032, 1997). For
example, the full length prostate-specific or testis-specific gene, or a
portion thereof, can be cloned into a retroviral vector and expression can be
driven from its endogenous promoter, from the retroviral long terminal
repeat, or from a promoter specific for a target cell type of interest (such
as
aortic or other vascular cells). Other viral vectors that can be used include,
for example, vaccinia virus, bovine papilloma virus, or a herpes virus, such
as Epstein-Barn Virus (also see, for example, the vectors of Miller, Human
Gene Therapy 15-14, 1990; Friedman, Science 244:1275-1281, 1989;
Eglitis et al., BioTechniques 6:608-614, 1988; Tolstoshev et al., Current
Opinion in Biotechnology 1:55-61, 1990; Sharp, The Lancet 337:1277-
1278, 1991; Cornetta et al., Nucleic Acid Research and Molecular Biology
36:311-322, 1987; Anderson, Science 226:401-409, 1984; Moen, Blood
Cells 17:407-416, 1991; or Miller et al., Biotechnology 7:980-990, 1989).
Retroviral vectors are particularly well developed and have been used in
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clinical settings (Rosenberg et al., N. Ehgl. J. Med 323:370, 1990;
Anderson et al., U.S. Patent No. 5,399,346).
Gene transfer can also be achieved using non-viral means involving
transfection ih vitro, by means of any standard technique, including but not
limited to, calcium phosphate, DEAF dextran, electroporation, protoplast
fusion, and liposomes. Transplantation of normal genes into the affected
tissues of a patient can also be accomplished by transferring a normal
prostate-specific or testis-specific gene into a cultivatable cell type ex
vivo,
after which the cell (or its descendants) is injected into a taxgeted tissue.
Another strategy for inhibiting prostate-specific or testis-specific function
using gene therapy involves intracellular expression of an anti-prostate-
specific or testis-specific antibody or a portion of an prostate-specific or
testis-specific antibody. For example, the gene (or gene fragment)
encoding a monoclonal antibody that specifically binds to prostate-specific
or testis-specific polypeptide and inhibits its biological activity is placed
under the transcriptional control of a tissue-specific gene regulatory
sequence. Another therapeutic approach involves administration of
recombinant prostate-specific or testis-specific polypeptide, either directly
to the site of a potential or actual disease-affected tissue (for example, by
injection) or systemically (for example, by any conventional recombinant
protein administration technique). The dosage of a prostate-specific or
testis-specific polypeptide depends on a number of factors, including the
size and health of the individual patient but, generally, between about 0.006
mg/kg to about 0.6 mg/kg, inclusive, is administered per day to an adult in
any pharmaceutically acceptable formulation.
Non-viral approaches can also be employed for the introduction of
therapeutic DNA into cells predicted to be subject to diseases involving a
prostate-specific or testis-specific disorder. For example, a prostate-
specific or testis-specific nucleic acid molecule or an antisense nucleic acid
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molecule can be introduced into a cell by lipofection (Felgner et al., Proc.
Natl. Acad. Sci. USA 84:7413, 1987; Ono et al., Neuroscience Letters
17:259, 1990; Brigham et al., Am. J. Med. Sci. 298:278, 1989; Staubinger et
al., Methods in Enzymology 101:512, 1983), asialoorosomucoid-polylysine
conjugation (Wu et al., Journal ofBiological Chemistry 263:14621, 1988;
Wu et al., Journal of Biological Chemistry 264:16985, 1989), or, less
preferably, micro-injection under surgical conditions (Wolff et al., Science
247:1465, 1990).
Prostate-specific or testis-specific cDNA expression for use in gene
therapy methods can be directed from any suitable promoter (e.g., the
human cytomegalovirus (CMV), simian virus 40 (SV40), or
metallothionein promoters), and regulated by any appropriate mammalian
regulatory element. For example, if desired, enhancers known to
preferentially direct gene expression in specific cell types can be used to
direct prostate-specific or testis-specific expression. The enhancers used
can include, without limitation, those that are characterized as tissue- or
cell-specific enhancers. Alternatively, if a prostate-specific or testis-
specific genomic clone is used as a therapeutic construct (such clones can
be identified by hybridization with prostate-specific or testis-specific
cDNA, described above), regulation can be mediated by the cognate
regulatory sequences, or, if desired, by regulatory sequences derived from a
heterologous source, including any of the promoters or regulatory elements
described above.
Antisense-based strategies can be employed to explore prostate-
specific or testis-specific gene function and as a basis for therapeutic drug
design. These strategies are based on the principle that sequence-specific
suppression of gene expression (via transcription or translation) can be
achieved by intracellular hybridization between genomic DNA or mRNA
and a complementary antisense species. The formation of a hybrid RNA
CA 02403637 2002-09-19
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duplex interferes with transcription of the target prostate-specific or testis-
specific-encoding genomic DNA molecule, or processing, transport,
translation, or stability of the target prostate-specific or testis-specific
mRNA molecule.
Antisense strategies can be delivered by a variety of approaches.
For example, antisense oligonucleotides or antisense RNA can be directly
administered (e.g., by intravenous injection) to a subject in a form that
allows uptake into cells. Alternatively, viral or plasmid vectors that encode
antisense RNA (or antisense RNA fragments) can be introduced into a cell
i~c vivo or ex vivo. Antisense effects can be induced by control (sense)
sequences; however, the extent of phenotypic changes are highly variable.
Phenotypic effects induced by antisense effects are based on changes in
criteria such as protein levels, protein activity measurement, and target
mRNA levels.
For example, prostate-specific or testis-specific gene therapy can
also be accomplished by direct administration of antisense prostate-specific
or testis-specific mRNA to a cell that is expected to be adversely affected
by the expression of wild-type or mutant prostate-specific or testis-specific
polypeptides. The antisense prostate-specific or testis-specific mRNA can
be produced and isolated by any standard technique, but is most readily
produced by ih vitro transcription using an antisense prostate-specific or
testis-specific cDNA under the control of a high efficiency promoter (e.g.,
the T7 promoter). Administration of antisense prostate-specific or testis-
specific mRNA to cells can be carried out by any of the methods fox direct
nucleic acid molecule administration described above.
An alternative strategy for inhibiting prostate-specific or testis-
specific function using gene therapy iilvolves intracellular expression of an
anti-prostate-specific or testis-specific antibody or a portion of an anti-
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prostate-specific or testis-specific antibody. For example, the gene (or gene
fragment) encoding a monoclonal antibody that specifically binds to
prostate-specific or testis-specific and inhibits its biological activity can
be
placed under the transcriptional control of a tissue-specific gene regulatory
sequence.
Another therapeutic approach within the invention involves
administration of recombinant prostate-specific or testis-specific
polypeptide, either directly to the site of a potential or actual disease-
affected tissue (for example, by injection) or systemically (for example, by
any conventional recombinant protein administration technique). The
dosage of prostate-specific or testis-specific depends on a number of
factors, including the size and health of the individual patient, but,
generally, between 0.1 mg and 100 mg, inclusive, axe administered per day
to an adult in any pharmaceutically acceptable formulation.
In a patient diagnosed as having a prostate-specific or testis-specific
mutation gene or a prostate-specific or testis-specific disease, or as
susceptible to prostate-specific or testis-specific gene mutations, aberrant
prostate-specific or testis-specific polypeptide or nucleic acid molecule
expression (even if those mutations or expression patterns do not yet result
in alterations in prostate-specific or testis-specific expression or
biological
activity), or to a prostate-specific or testis-specific disease, any of the
above-described therapies are administered before the occurrence of the
disease phenotype. Also, compounds shown to modulate prostate-specific
or testis-specific polypeptide or nucleic acid molecule expression or
prostate-specific or testis-specific polypeptide or nucleic acid molecule
biological activity are administered to patients diagnosed with potential or
actual diseases by any standard dosage and route of administration.
Alternatively, gene therapy using an antisense prostate-specific or testis-
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specific mRNA expression construct is undertaken to reverse or prevent the
gene defect prior to the development of the full course of the disease.
The therapeutic methods of the invention are, in some cases, targeted
to prenatal treatment. For example, a fetus found to have a prostate-
s specific or testis-specific mutation is administered a gene therapy vector
W cluding a normal prostate-specific or testis-specific gene, or administered
a normal prostate-specific or testis-specific protein. Such treatment may be
required only for a short period of time, or may, in some form, be required
throughout such a patient's lifetime. Any continued need for treatment,
however, is determined using, for example, the diagnostic methods
described above. Also as discussed above, prostate-specific or testis-
specific polypeptide or nucleic acid molecule abnormalities may be
associated with diseases in adults, and thus, adults are subject to the
therapeutic methods of the invention as well.
Additionally, prostate-specific or testis-specific polypeptides may be
used to stimulate an immune system to assist in generating immunity
against, for example, prostate cancer cells.
The methods of the present invention can be used to diagnose or
treat the disorders described herein in any mammal, for example, humans,
domestic pets, or livestock. Where a non-human mammal is treated or
diagnosed, the prostate-specific or testis-specific polypeptide, nucleic acid
molecule, or antibody employed is preferably specific for that species.
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Identification of Molecules that Modulate Prostate-Specific Or Testis-
Specific Polypeptide or Nucleic Acid Molecule Biological Activity or
Whose Biological Activity is Modulated by Prostate-Specific Or Testis-
Specific Polypeptides or Nucleic Acid Molecules
Isolation of prostate-specific or testis-specific cDNAs (as described
herein) also facilitates the identification of molecules that increase or
decrease prostate-specific or testis-specific polypeptide or nucleic acid
molecule biological activity. Similarly, molecules whose activity is
modulated by prostate-specific or testis-specific polypeptide or nucleic acid
molecule biological activity can be identified. According to one approach,
candidate molecules are added at varying concentrations to the culture
medium of cells expressing prostate-specific or testis-specific mRNA.
Prostate-specific or testis-specific biological activity is then measured
using
standard techniques. The measurement of biological activity can include,
without limitation, the measurement of prostate-specific or testis-specific
protein and nucleic acid molecule expression levels, response to androgens,
or intracellular localization and trafficking.
If desired, the effect of candidate modulators on expression can also
be measured at the level of prostate-specific or testis-specific protein
production using the same general approach and standard immunological
detection techniques, such as western blotting or immunoprecipitation with
a prostate-specific or testis-specific-specific antibody (see below).
A test compound that is screened in the methods described above
can be a chemical, be it naturally-occurring or artificially-derived. Such
compounds can include, for example, polypeptides, synthesized organic
molecules, naturally occurring organic molecules, nucleic acid molecules,
and components thereof. Candidate prostate-specific or testis-specific
modulators include peptide as well as non-peptide molecules (e.g., peptide
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or non-peptide molecules found, e.g., in a cell extract, mammalian serum,
or growth medium in which mammalian cells have been cultured).
Administration of Prostate-Specific Or Testis-Specific Polypeptides,
Prostate-Specific Or Testis-Specific Nucleic Acid Molecules, and
Modulators of Prostate-Specific Or Testis-Specific Polypeptide or Nucleic
Acid Molecule Synthesis or Function
A prostate-specific or testis-specific protein, nucleic acid molecule,
or modulator is administered within a pharmaceutically-acceptable diluent,
carrier, or excipient, in unit dosage form to patients or experimental
animals. Also, conventional pharmaceutical practice is employed to
provide suitable formulations or compositions in which to administer
neutralizing prostate-specific or testis-specific antibodies or prostate-
specific or testis-specific-inhibiting compounds (e.g., a prostate-specific or
testis-specific antisense molecule or a prostate-specific or testis-specific
dominant negative mutant) to patients suffering from a prostate-specific or
testis-specific disease, such as prostate cancer, testicular cancer, benign
hyperplasia of the prostate, or developmental defects of the prostate or
testis. Administration can begin before or after the patient is symptomatic.
Any appropriate route of administration can be employed, for
example, administration can be parenteral, intravenous, infra-arterial,
subcutaneous, intramuscular, intracranial, intraorbital, ophthalmic,
intraventricular, intracapsular, intraspinal, intracisternal, intraperitoneal,
intranasal, inhalation to deep lung, aerosol, by suppositories, oral, or
topical
(e.g. by applying an adhesive patch carrying a formulation capable of
crossing the dermis and entering the bloodstream). Preferably, the
admiilistration is local to the afflicted tissue, such as prostate or testis
tissue. Therapeutic formulations can be in the form of liquid solutions or
suspensions; for oral administration, formulations can be in the form of
CA 02403637 2002-09-19
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tablets or capsules; and for intranasal formulations, in the form of powders,
nasal drops, or aerosols. Any of the above formulations may be a
sustained-release formulation.
Methods that are well known in the art for making formulations are
found, for example, in Remington's Pharmaceutical Sciences, (1 gtn
edition), ed. A. Gennaro, 1990, Mack Publishing Company, Easton, PA.
Formulations for parenteral administration can, for example, contain
excipients; sterile water; or salW e; polyalkylene glycols, such as
polyethylene glycol; oils of vegetable origin; or hydrogenated napthalenes.
Sustained-release, biocompatible, biodegradable lactide polymer,
lactide/glycolide copolymer, or polyoxyethylene-polyoxypropylene
copolymers can be used to control the release of the compounds. Other
potentially useful parenteral delivery systems for prostate-specific or testis-
specific modulatory compounds include ethylene-vinyl acetate copolymer
particles, osmotic pumps, implantable infusion systems, and liposomes.
Formulations for inhalation can contain excipients, for example, lactose, or
can be aqueous solutions containing, for example, polyoxyethylene-9-lauryl
ether, glycocholate, and deoxycholate, or can be oily solutions for
administration in the form of nasal drops, or as a gel.
Prostate-Specific Or Testis-Specific Fra rnents
Polypeptide fragments that include various portions of prostate-
specific or testis-specific proteins are useful in identifying the domains
important for their biological activities, such as protein-protein
interactions
and transcription. Methods for generating such fragments are well known
in the art (see, for example, Ausubel et al., supra), using the nucleotide
sequences provided herein. For example, a prostate-specific or testis-
specific protein fragment can be generated by PCR amplifying a desired
prostate-specific or testis-specific nucleic acid molecule fragment using
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oligonucleotide primers designed based upon the prostate-specific or testis-
specific nucleic acid sequences. Preferably, the oligonucleotide primers
include unique restriction enzyme sites that facilitate insertion of the
amplified fragment into the cloning site of an expression vector (e.g., a
mammalian expression vecoor, see above). This vector can then be
introduced into a cell (e.g., a marrnnalian cell; see above) by artifice,
using
any of the various techniques known in the art such as those described
herein, resulting in the production of a prostate-specific or testis-specific
polypeptide fragment in the cell containing the expression vector.
Prostate-specific or testis-specific polypeptide fragments (e.g.,
chimeric fusion proteins) can also be used to raise antibodies specific for
various regions of prostate-specific or testis-specific polypeptides.
Preferred prostate-specific or testis-specific fragments include, without
limitation, fragments including the N-terminal domain of STMP 1 (amino
acids 1-200), the PSCR domain, and fragments thereof.
Synthesis of Prostate-Specific Or Testis-Specific Proteins, Polypeptides,
and Po~eptide Fragmments
Those skilled in the art of molecular biology will understand that a
wide variety of expression systems can be used to produce recombinant
prostate-specific or testis-specific proteins. The precise host cell used is
not
critical to the invention. The prostate-specific or testis-specific proteins
can
be produced in a prokaryotic host (e.g., E. cola) or in a eukaryotic host
(e.g.,
S. ce~evisiae, insect cells such as Sf9 cells, or mammalian cells such as
COS, NIH 3T3, CHO, or HeLa cells). These cells are commercially
available from, for example, the American Type Culture Collection,
Rockville, MD (see also Ausubel et al., Currefat Protocols iu Molecular
Biology, John Wiley & Sons, New York, NY, 1998). The method of
transformation and the choice of expression vehicle (e.g., expression
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vector) will depend on the host system selected. Transformation and
transfection methods are described, e.g., in Ausubel et al., Current
Protocols in Molecular Biology, John Wiley & Sons, New York, NY, 1998,
and expression vehicles can be chosen from those provided, e.g. in Pouwels
et al., Clohiug hectors: A Laboratory Manual, 1985, Supp. 1987).
The characteristics of prostate-specific or testis-specific nucleic acid
molecules are analyzed by introducing such genes into various cell types or
using in vitro extracellular systems. The function of prostate-specific or
testis-specific proteins produced in such cells or systems are then examined
under different physiological conditions. Also, cell lines can be produced
that over-express the prostate-specific or testis-specific gene product,
allowing purification of prostate-specific or testis-specific proteins for
biochemical characterization, large-scale production, antibody production,
and patient therapy.
The polypeptides of the invention may be produced in vivo or in
vitro, and may be chemically and/or enzymatically modified. The
polypeptides can be isolated from prostate tissue or prostate cancer cells
that may or may not be in a hormone dependent state. Alternatively, and
especially where larger amounts (i.e., >l0mg) are desirable, recombinant
production (e.g., in a bacterial, yeast, insect cell, or mammalian cell
system)
may advantageously be employed to generate significant quantities of
prostate-specific or testis-specific polypeptides.
Furthermore, recombinant production not only offers a more eco-
nomical strategy to produce the polypeptides of the iilvention, but also
allows specific modification in the amino acid sequence and composition to
tailor particular biochemical, catalytic and physical properties: For
example, where increased solubility of is desirable, one or more
hydrophobic amino acids may be replaced with hydrophilic amino acids.
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Alternatively, where reduced or increased catalytic activity is required, one
or more amino acids may be replaced or eliminated.
In still another example, the polypeptides of the invention can be
synthesized as fusion proteins including, for example, fusions with en-
zymatically active partners (e.g., for dye formation or substrate conversion)
and fluorescent partners such as GFP, EGFB, BFP, etc.
With respect to chemical and enzymatic modifications of
contemplated polypeptides, it is many modifications are appropriate,
including addition of mono-, and bifunctional linkers, coupling with
protein- and non-protein macromolecules, and glycosylation. For example,
mono- and bifunctional linkers are especially advantageous where poly-
peptides are immobilized to a solid support, or covalently coupled to a
molecule that enhances immunogenicity of contemplated polypeptides
(e.g., KLH, or BSA conjugation). Alternatively, the polypeptides may be
coupled to antibodies or antibody fragments to allow rapid retrieval of the
polypeptide from a mixture of molecules. Further couplings include
covalent and non-covalent coupling of polypeptides with molecules that
prolong the serum half life and/or reduce immunogenicity such as
cyclodextranes and polyethylene glycols.
Use of Prostate-Specific Or Testis-Specific Antibodies
Antibodies to prostate-specific or testis-specific proteins are used to
detect prostate-specific or testis-specific proteins or to inhibit the
biological
activities of prostate-specific or testis-specific proteins. For example, a
nucleic acid molecule encoding an antibody or portion of an antibody can
be expressed within a cell to inhibit prostate-specific or testis-specific
function. In addition, the antibodies can be coupled to compounds, such as
radionuclides and liposomes for diagnostic or therapeutic uses. Antibodies
that inhibit the activity of a prostate-specific or testis-specific
polypeptide
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can also be useful in preventing or slowing the development of a disease
caused by inappropriate expression of a wild type or mutant prostate-
specific or testis-specific gene. For example, the antibodies of the
invention may be utilized to localize and locally quantify disease-specific
markers in prostate or testis tissue sections, e.g, in prostate or testicular
cancer.
Detection Of Prostate-Specific Or Testis-Specific Gene Expression
As noted, the antibodies described above can be used to monitor
prostate-specific or testis-specific protein expression. In situ hybridization
of RNA can be used to detect the expression of prostate-specific or testis-
specific genes. RNA ifZ situ hybridization techniques rely upon the
hybridization of 'specifically labeled nucleic acid probe to the cellular
RNA in individual cells or tissues. Therefore, RNA ih situ hybridization is
a powerful approach for studying tissue- and temporal-specific gene
expression. In this method, oligonucleotides, cloned DNA fragments, or
antisense RNA transcripts of cloned DNA fragments corresponding to
unique portions of prostate-specific or testis-specific genes are used to
detect specific mRNA species, e.g., in the tissues of animals, such as mice,
at various developmental stages, or to monitor tumor progression. Other
gene expression detection techniques are known to those of skill in the art
and can be employed for detection of prostate-specific or testis-specific
gene expression.
Identification of Additional Prostate-Specific Or Testis-Specific Genes
Standard techniques, such as the polymerase chain reaction (PCR)
and DNA hybridization, as well as the SSH and other techniques described
herein, can be used to clone prostate-specific or testis-specific homologues
in other species and other prostate-specific or testis-specific genes in
CA 02403637 2002-09-19
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humans. Prostate-specific or testis-specific genes and homologues can be
readily identified using low-stringency DNA hybridization or low-
stringency PCR with human prostate-specific or testis-specific probes or
primers. Degenerate primers encoding human prostate-specific or testis-
specific or human prostate-specific or testis-specific amino acid sequences
can be used to clone additional prostate-specific or testis-specific genes and
homologues by RT-PCR.
Additional prostate-specific or testis-specific genes include genes
expressed during various growth and developmental phases of the diseased
prostate or testis, e.g., those involved in prostate cancer, benign prostatic
hyperplasia, or testicular cancer, and genes expressed as a result of a drug
regimen.
Construction of Trans,~enic Animals and Knockout Animals
Characterization of prostate-specific or testis-specific genes provides
information that allows prostate-specific or testis-specific knockout animal
models to be developed by homologous recombination. Preferably, a
prostate-specific or testis-specific lmockout animal is a maxmnal, most
preferably a mouse. Similarly, animal models of prostate-specific or testis-
specific overproduction can be generated by integrating one or more
prostate-specific or testis-specific sequences into the genome of an animal,
according to standard transgenic techniques. Moreover, the effect of
prostate-specific or testis-specific gene mutations (e.g., dominant gene
mutations) can be studied using transgenic mice carrying mutated prostate-
specific or testis-specific transgenes or by introducing such mutations into
the endogenous prostate-specific or testis-specific gene, using standard
homologous recombination techniques.
A replacement-type targeting vector, which can be used to create a
knockout model, can be constructed using an isogenic genomic clone, for
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example, from a mouse strain such as 1291Sv (Stratagene Inc., LaJolla,
CA). The targetiilg vector can be introduced into a suitably-derived line of
embryonic stem (ES) cells by electroporation to generate ES cell lines that
carry a profoundly truncated form of a prostate-specific or testis-specific
gene. To generate chimeric founder mice, the targeted cell lines are
injected into a mouse blastula-stage embryo. Heterozygous offspring can
be interbred to homozygosity. Prostate-specific or testis-specific knockout
mice provide a tool for studying the role of prostate-specific or testis-
specific polypeptides and nucleic acid molecules in embryonic
development and in disease. Moreover, such mice provide the means, ih
vivo, for testing therapeutic compounds for amelioration of diseases or
conditions involving a prostate-specific or testis-specific polypeptide or
nucleic acid molecule-dependent or prostate-specific or testis-specific
polypeptide or nucleic acid molecule-affected pathway.
Animal Models
The prostate-specific and testis-specific polypeptides, antisense
compounds, etc., of the invention can also be used in conjunction with
animal models of prostate or testis disorders, to test the therapeutic,
diagnostic, and screening methods of the invention. An exemplary prostate
cancer model in transgenic mice is called TRAMP, in which the SV40 large
T antigen is targeted to the prostate (Greenberg et al., PNAS 92, 3439-
3443, 1995). Another test system is the CWR22 (androgen-dependent) and
CWR22R (androgen-independent) xenografts, as known in the art and as
described herein. Growth, PSA secretion, metastasis, etc. of these
xenografts could be monitored in the presence and absence of the prostate-
specific or testis-specific polypeptides, nucleic acid molecules, and other
compounds of the invention. Other animal models, for example, animal
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models of other forms of cancer, or immunocompromised animals, e.g.,
nude mice, may also be used.
The following Examples will assist those skilled in the art to better
understand the invention and its principles and advantages. It is intended
that these Examples be illustrative of the invention and not limit the scope
thereof.
EXAMPLE 1
Suppression Subtraction Of Prostate- And Testes-Specific Genes And
Subclonin~ Into Pzero
cDNA derived from poly(A)+ RNA of 10 different normal human
tissues were subtracted against normal human prostate cDNA using
suppression subtraction hybridization (SSH) (Diatchenlco, L. et al., P~oc.
Natl. Acad. Sci. USA 93, 6025-6030, 1996) and the resulting cDNA
fragments were cloned into an appropriate vector. SSH was performed as
described (Clontech PCR-Select Cloning Kit) using prostate poly(A)+ RNA
against a pool of poly(A)+ RNA obtained from ten normal human tissues
(heart, brain, placenta, lung, liver, skeletal muscle, kidney, spleen, thymus,
and ovary). Upon secondary PCR amplification (12 cycles), the reactions
were extracted with phenol/chloroform, the DNA with ethanol, and the
pellets washed once with 70% ethanol. After dryilig, the DNA pellet was
dissolved in 0.2XTE or MQ dH20 and cut with RsaI in a 20 ~1 reaction for
2 hrs at 37°C to excise adaptors. After digestion, the reactions were
run on
a 1.5% agarose gel, with molecular size markers on one side, at 5 V/cm, 40
min. Care was taken not to expose the gel to short wavelength UV light.
The adapter bands were excised, and the gel was run at 5 V/cm for 15 min
in a reversed electric field to concentrate the cDNA bands.
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The gel was visualized (long wave W light) and the amplified
cDNAs, ranging in size between 100 bp-lkB, were excised. The DNA was
purified using the QAIEX gel DNA purification kit. The purified DNA
was cloned into EcoRV-cut, dephosphorylated pZERO (Invitrogen).
Ligation reactions were performed in 10 ~.l final volume in the presence of
5% PEG, 1X T4 Ligase buffer at 37°C overnight and a 1/5 dilution of
1~,1
of the ligation mix (PSL) was transformed into DH10B electrocompetent
cells (>101° efficiency) or equivalent. Colonies were picked and the
presence of cDNA inserts was confirmed. To that end, PCR was performed
with T7 and SP6 primers directly from the colonies. 10% of the reactions
were run on a 1.5% agarose gel to visualize amplified products. The
colonies with inserts were grown and glycerol stocks (15%) were prepared
and stored at -80°C.
EXAMPLE 2
Reverse Northern Blot And Sequence Analyses
To clone androgen-responsive genes represented in the PSL, the
reverse northenl technique was used (Hedrick, S.M. et al., Nature 308, 149-
153, 1984; Sakaguchi, N. et al., EMBO J 5: 2139-2147, 1986). In this
procedure, RNA made from two populations of cells that are to be
compared is used to make cDNA probes that are then hybridized to two
identical arrays of clones. To that end, PSL clones were amplified by PCR
and spotted on nylon filters in 96-well format to generate two identical
blots for each set of 92 clones (the remaining four spots were used for
positive and negative controls). To malce the probes, the androgen-
responsive prostate cancer cell line LNCaP was used (Horoszewicz, J.S. et
al., Cafacer Res. 43, 1809-1818, 1983) and was either left untreated (the (-)
probe) or treated with the synthetic androgen 81881 for 24 hours (the (+)
probe). Poly(A)+ RNA was isolated from these cells and was used to make
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the 32P-labeled probes. After hybridization with the (-) and (+) probes,
clones that showed differential hybridization were selected for further
analysis, i.e., confirmation by a secondary reverse northern blot, and
northern blotting.
Reverse northern screening on the cDNA clones was done essentially
as described previously (Hedrick, S.M. et al., supYa; Sakaguchi, N. et al.,
supra) with some modifications. DNA (approximately 400 ng) from PCR
amplification in step 6 was diluted in 200 ~1 of 0.4M NaOH, 10 mM EDTA
and mixed well by pipetting. After incubation at 95°C for 5-10 minutes,
the tubes were chilled on ice. Denatured DNA was blotted on two separate
pieces of Zeta Probe GT+ membrane (Bio-Rad) using a dot-blot apparatus
(Bio-Rad). Positive (Prostate specific antigen (PSA) cDNA) and negative
(glyceraldehyde 3-phosphate dehydrogenase (G3PDH) cDNA) controls
were included on each blot (bottom right) in duplicate. The membranes
were rinsed with 2XSSC, air dried, and then baked at 80°C for 30
minutes.
An exemplary reverse northern analysis is shown in Figure 1. Note that
there was a substantial increase in PSA hybridization in the (+) blot (probe
prepared from cells that have been stimulated by androgens) compared with
the (-) blot (probe prepared from unstimulated cells), whereas there was no
significant change in hybridization of G3PDH between the two blots.
Arrowheads indicate the positive clones identified in this experiment.
To verify the tissue-specific nature of the isolated sequences,
positive clones were tested in a standard northern blot against RNA
preparations of multiple non-prostate tissue samples. Figure 2 shows a
multiple tissue northern blot using NKX3A as a probe, to show an
exemplary tissue expression pattern seen in the positive clones. Lanes 1-
10, and 12-16 are RNA preparations from non-prostate tissues, lane 11 is a
RNA preparation from prostate, lane 12 is a RNA preparation from testis.
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Twelve clones with no significant homology to known sequences (by
BLAST analysis) were isolated from prostate tissue and LNCaP cells. SEQ
ID NOs: 1-9 were identified as androgen-responsive differentially-
expressed genes in the prostate, while SEQ ID NOs: 10-12 were identified
as androgen-responsive differentially-expressed genes in LNCaP cells.
EXAMPLE 3
Isolation And Characterization Of The STMP 1 Gene And mRNA
A normal prostate cDNA library was screened by 5'- and 3'-RACE
analysis, and resulted in the full-length cDNA for L74. Since computer-
aided secondary structure prediction of the deduced amino acid sequence of
L74 suggested the presence of a six-transmembrane domain in its C-
terminal half, L74 was renamed Six-Transmembrane Protein of Prostate 1
(STMP 1).
When the full-length STMPl cDNA was used in BLAST analysis, it
was found to match a BAC clone (GenBank accession # AC002064) except
for a 313 by repetitive unit in the 3' UTR region, thereby identifying it as
the STMPI gene and localizing it to Chr7q21. The repetitive region is likely
to be a cloning or sequencing artifact of the BAC clone. Computational
exon/intron junction analysis and alignment of the full-length cDNA
sequence with the BAC clone revealed that STMPI gene is composed of six
exons and five iritrons (Figure 4A). The transcription start site, the
location
and size of the exons and introns, and the location of the partial cDNA
clone L74 (black box) are indicated. The start (atg) and stop codons (tga),
as well as the putative polyadenylation signal (pA) are also indicated. The
first two exons are short, non-coding exons of 83 and 61 bp, whereas exons
3-6 encode the open reading frame (ORF) and are 525, 528, 165, and 3281
by long, respectively (Figure 4C). The STMPl gene spans around 26 kb,
which is in part due to the extremely large size of intron 2 (12713 bp).
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There are three different predicted promoters within 4 kb upstream of the
STMPI initiation codon, none of which has any significant TATA or
CAAT box consensus sequences, suggesting that STMPI is transcribed
from a TATA-less promoter.
The STMPI cDNA (GenBank accession # AY008445) has a predicted
5' untranslated region (5'UTR) of approximately 1 kb (deduced by RACE
analysis) and an unusually long 3'UTR of approximately 4 kb that
comprises ~77% of the total cDNA sequence. The ORF starts within the
3rd exon and is predicted to encode a 490 amino-acid protein (Figure 4B).
A search for protein motifs identified six predicted transmembrane domains
in the C-terminal half of STMP 1 starting at F209 (Figures 4B and 4E).
Only the cDNA sequence surrounding the ORF is indicated. The exon-
intron junctions are indicated and the location of the predicted
transmembrane domains are highlighted (TM 1-6) (Figure 4B). The stop
codon is indicated with an asterisk. STMP1 has two alternatively spliced
forms, shown in Figures 4F-4K, which lead to two predicted isoforms of
the protein.
EXAMPLE 4
STMP 1 Belongs To A New Subfamily Of Six-Transmembrane Domain
Proteins
BLAST analysis of GenBank with the predicted STMP1 amino acid
sequence identified two independent ESTs and STEAP, a recently
discovered cell membrane protein enriched in prostate for expression. An
alignment of these sequences, obtained by Clustal and GenDoc programs, is
shown in Figure 5. Completely conserved residues are shaded in blaclc;
residues that are conserved in two or three of the sequences are shaded light
and dark gray, respectively. This alignment suggested that while the EST
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BAA91S39 cDNA may be close to full-length, BAB15559 cDNA may
represent a partial sequence.
The sequences of two proteins related to STMP 1 were determined
(Figures 4L and 4M, STMP2 and STMP3, respectively). The STMP2 and
STMP3 sequences contain the EST sequences. The GFP-fusion of STMP2
gives similar localization as STMPl. Both STMP2 and STMP3 are more
widely distributed and have higher levels in some tissues other than the
prostate. For example, STMP2 has the highest expression in the placenta
and the lung, and is also highly expressed in the heart, liver, prostate, and
testis, while STMP3 has the highest expression in the liver, and is also
highly expressed in the heart, placenta, lung, kidney, pancreas, prostate,
testis, small intestine, and colon.
The sequence similarity between STMP1 and STEAP is limited and
not significant before residue 210 of STMP1 where the predicted six-
transmembrane coding domain starts. This suggests that the N-terminal
region is structurally and functionally related among STMP proteins,
forming a six-transmembrane protein subfamily that is distinct from
STEAP.
EXAMPLE 5
STMP1 Expression Is Hi-~hly Enriched In Prostate
The expression profile of STMPl was then determined in various
human tissues by Northern analysis, in which a multiple tissue Northern
blot was hybridized to the STMPI probe (see Materials and Methods). As
shown in Figure 6A, STMPI hybridized to a major mRNA species of 6.5
kb, and three minor mRNA species of 2.2, 4.0, and 4.5 kb in the prostate
tissue. The stronger hybridization that is observed with G3PDH in the
heart and skeletal muscle samples is due to its higher expression in these
tissues. The lanes represent: l.Heart, 2. Brain, 3. Placenta, 4. Lung, 5.
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Liver, 6. Skeletal Muscle, 7. Kidney, 8. Pancreas, 9. Spleen, 10. Thymus,
11. Prostate, 12. Testis, 13. Ovary, 14. Small Intestine, 15. Colon, 16.
Peripheral blood leukocyte. The location of the full-length 6.5 kb mRNA,
as well as the lower molecular weight STMPI species are indicated by
arrows to the left of the figure. There was 15-20-fold lower mRNA
expression of the 6.5 kb band in the heart, brain, kidney, pancreas, and
ovary, compared to prostate, and no detectable expression in other tissues.
In contrast, the three lower molecular weight species, encoded by
alternatively spliced forms of STMPl, were only detectable in the prostate.
Hybridization with a glyceraldehyde 3-phosphate dehydrogenase (G3PDH)
cDNA probe resulted in approximately similar signals in all lanes, except
for the heart and skeletal muscle where G3PDH is known to be more
abundant compared with other tissues. These data show that STMPI
expression is high in the prostate, although expression can be seen in other
tissues, and that STMPI has isoforms that are restricted to the prostate for
expression.
EXAMPLE 6
Characterization Of STMP 1 Expression
Since androgen is a major hormonal stimulus for the nornzal prostate
gland and for early stage prostate cancer, the possible androgen regulation
of STMPI was assessed by Northern analysis in the androgen-responsive
prostate cancer cell 1i11e LNCaP. Cells were either left untreated or treated
with the synthetic androgen 81881 (10-8 M) with increasing amounts of
time (hours) as indicated (Figure 6B), harvested, and total RNA isolated
and used in Northern analysis with STMPI cDNA as probe. The same
membrane was also probed for the androgen-dependent gene PSA.
Relative induction of mRNA accumulation is indicated at the bottom of the
lanes, as determined by phosphorimager analysis (Molecular Dynamics).
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The CWR22 xenograft was grown in nude mice and tumor samples were
collected either before (t=0) or l, 2, or 4 weeks after castration. Total RNA
was isolated and was then used in Northern analysis with the same probes.
Ethidium bromide-stained 18S RNA is shown as a control for RNA
integrity and loading. At 6 h, there was an approximately 25% increase in
STMPI expression, which was lost by 24 h, with a final 20% decrease
observed at 48 h compared with basal levels. In contrast, the mRNA
accumulation of the androgen-regulated gene PSA dramatically increased
upon androgen stimulation in a time-dependent manner, as expected,
reaching approximately 22-fold higher levels by 48 hours. Relative
ilzduction of STMPI mRNA accumulation is indicated at the bottom of the
lanes determined by phosphorimager analysis. As is shown in Figure 6B,
STMP1 displayed similar expression levels in untreated and 81881-treated
LNCaP cells, indicating that STMPl expression is not significantly
regulated by androgens in LNCaP cells.
To determine the possible androgenic regulation of STMPI expression
in an ih vivo setting, the androgen-dependent xenograft model CWR22,
which is derived from a primary human prostate tumor was used
(Wainstein, M. A. et al., Cancer Res 54, 6049-6052, 1994). Since they are
androgen-dependent for growth, the CWR22 tumors in nude mice display
marked regression upon castration and may regress completely. CWR22
xenografts were grown in nude mice in the presence of a sustained release
testosterone pellet. After the tumors had grown, the mice were castrated,
the testosterone pellets were removed, and the regressing tumors were
collected at 1, 2, or 4 weeks post-castration. Total RNA was prepared from
these tumor samples and used in Northern analysis. As shown in Figure
6B, similar to the obsevations in LNCaP cells, STMPI mRNA
accumulation in the CWR22 tumors showed no significant change upon
castration and was not affected by the presence of androgens (note that
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there is underloading of RNA for CWR22 2wk sample). In contrast, the
mRNA accumulation of the androgen-regulated gene PSA was dramatically
decreased upon castration, dropping to approximately 16% of pre-castrate
levels by two weeks post-castration. These results are consistent with the
findings in LNCaP cells and suggest that STMPI expression is not
significantly regulated by androgens in prostate cancer cells. STMPI
expression was substantially lower in the CWR22 tumors compared with
LNCaP cells.
The expression profile of STMPI was also analyzed in the androgen-
independent prostate cancer cell lines PC3 and DU145, as well as in four
independent, relapsed derivatives of CWR22 tumors, named CWR22R
(Nagabhushan, M. et al., Ca~ce~ Res 56, 3042-3046, 1996), representative
of advanced prostate cancer (Figure 6C). LNCaP (in the presence (+) or
absence (-) of 81881 (10-$ M)), PC-3, or DU-145 cells were grown and
total RNA was isolated. Four independent lines of the androgen
independent human prostate cancer xenograft CWR22R, were grown in
nude mice, tumors were collected, and total RNA was isolated and used in
Northern analysis with STMPI or the androgen target gene NKX3.1 cDNAs
as probes. Ethidium bromide-stained 18S RNA is shown as a control for
RNA integrity and loading. The relative induction of STMPI and NKX3.1
mRNA accumulation is indicated at the bottom of the lanes deternzined by
phosphorimager analysis (Molecular Dynamics). As is shown in Figure
6C, STMPl expression was high in LNCaP cells and did not significantly
change in response to 81881 treahnent compared with a ~9-fold induction
of the androgen target gene NKX3.1. There was no STMPI expression in
the androgen-independent prostate cancer cell lines PC-3 or DU-145, as
was the case for NK~Y3.1. In contrast, there was significant STMPI
expression in tumors from all four independent CWR22R xenograft lines
tested, ranging between ~30-60% of that observed in LNCaP cells. A
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similar overexpression pattern was also observed for NKX3.1 (Figure 6C)
consistent with previous findings (Korkmaz, K. S. et al., Gehe 260, 25-36,
2000).
An interesting property of STMPI expression profile is that even
though it is expressed at low levels in the androgen dependent CWR22
xenograft, it is highly expressed in the relapsed CWR22R which is
androgen receptor (AR) positive, but is not responsive to androgens. This
indicates that STMP1 expression is deregulated once the prostate tumor
progresses from an androgen-dependent to an androgen-independent phase.
In addition, STMPl is not expressed in the AR-negative prostate cancer cell
lines PC-3 and DU-145, but is expressed at high levels in the AR-positive
cell line LNCaP and the CWR22 and CWR22R xenografts. Thus,
expression of STMPl is correlated with the presence of a functional AR in
the cell.
It has been known for over 50 years that androgens play a key role
both in the development and maintenance of the normal prostate and the
initiation and progression of prostate cancer. Androgen withdrawal results
in involution of both the normal prostate gland as well as a prostate tumor
in the early stages of the disease that is still androgen dependent.
Consequently, androgen withdrawal is commonly used as treatment to
reverse tumor growth. However, in the case of the prostate tumor, after a
few months or years, the tumor recurs in almost all cases in an androgen-
independent state. At this point there is no effective therapy and prognosis
for survival is extremely poor. Siizce STMPl is overexpressed during this
later androgen-insensitive state, it will be a useful tool iil diagnostic and
therapeutic applications for prostate cancer.
These data indicate that STMPI expression is deregulated once
prostate cancer progresses from an androgen-dependent to an androgen-
independent state.
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EXAMPLE 7
Intracellular Localization Of STMP 1
To gain insight into the intracellular localization pattern of STMPl, a
green fluorescent protein (GFP)-STMP 1 fusion protein was generated. The
use of such GFP chimeric proteins has recently become a standard method
to assess intracellular localization and dynamics of proteins. COS-1 cells
were transiently transfected with GFP-STMP1, fixed and processed for
confocal microscopy as described in Materials and Methods.
A series of 11 confocal sections along the z-axis were collected
through a single cell at nominal 100 nm intervals. Three of the consecutive
sections and the projection of all 11 sections are shown in Figure 7A.
Arrows indicate tubular-vesicular structures (VTS) in different sizes,
shapes, and locations (Bar=S~.m). In all 11 z-plane sections, GFP-STMP1
showed bright juxtanuclear distribution pattern, characteristic of the Golgi
complex. Additionally, GFP-STMP1 was dispersed in spots of vaxiable size
throughout the cytoplasm and at the cell periphery (z-7, projection). Some
of these bright fluorescent spots were tubular (z-6, arrow and Figure 8) or
vesicular (z-5, arrow) in morphology.
To determine more directly whether GFP-STMP 1 was localized to
the Golgi complex, we compared its intracellular distribution with those of
two well characterized Golgi markers, the medial Golgi enzyme
mannosidase II (ManII) (Rabouille, C. et al., J Cell Sci 108, 1617-1627,
1995) and the coat protein (3-COP (Pepperkok, R. et al., Cell 74, 71-82,
1993). COS-1 cells were transfected with GFP-STMP1, fixed, labeled with
the appropriate primary and secondary antibodies and imaged by confocal
laser scanning microscopy. Green GFP-STMP1 fluorescence and red
(Texas Red-labeled secondary antisera) [3-COP and ManII fluorescence
were detected by confocal laser microscopy. Panels to the right show the
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overlay images with yellow/orange staining indicating the regions of
colocalization. Bars=S~m. As shown in Figure 7B, the distribution of
GFP-STMP 1 extended throughout the Golgi complex, as evidenced by
significant colocalization with both ManII and (3-COP. However, some
areas of non-overlap between the GFP-STMP1 and both Golgi markers
were observed suggesting that STMP1, at least in part, is differentially
localized within the Golgi complex compared with these two markers.
Since GFP-STMP1 was associated with VTS (Figure 7A and Figure 8),
more specific localization of GFP-STMP 1 to the trans-Golgi network
(TGN), an important site for the sorting of proteins destined to the plasma
membrane, secretory vesicles, or lysosomes (Farquhar, M. G. & Palade, G.
E. Trends Cell Biol 8, 2-10, 1998; Mellman, I. & Warren, G., Cell 100, 99-
112, 2000; Lemmon, S. K. & Traub, L. M., Curr OpifZ Cell Biol 12, 457-
466, 2000) was assessed. An antibody against TGN46, a TGN resident
protein that shuttles between the TGN and the plasma membrane (Prescott
AR, et al., Eur J Cell Biol 72, 238-246, 1997; Ponnambalam, S. et al., J
Cell Sci. 109, 675-685, 1996), was used in immunoflourescence
microscopy experiments as above. As shown in Figure 7B, GFP-STMP1
extensively colocalized with TGN46, greater than that observed with ManII
and (3-COP, suggesting that in the Golgi complex, STMP1 is primarily
localized to.the TGN. Note that the images with TGN46 were obtained
with lower objective power.
F.X A 1VTPT .R R
STMP 1 Shuttles Between The Gobi And The Plasma Membrane And
Colocalizes To The Early Endosomes
The dynamic properties and intracellular trafficking of GFP-STMP 1
were studied using confocal time-lapse imaging in living cells. COS-1 cells
were transiently transfected with GFP-STMP1 and, 16 h after transfection,
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12 consecutive images were collected from live cells every 20s at 37°C
by
confocal laser scanning microscope (Figure 8). The upper panel shows a
VTS extending out and retracting back to the Golgi body (white arrows).
In the middle panel and the first image in the lower panel (160s), red
arrows indicate the translocation of a VTS from the Golgi body to the cell
periphery. hi the lower panel, yellow arrows point to the movement of a
VTS from the edge of the cell towards the Golgi body. Note that the results
shown are representative of multiple time-lapse analyses and the changes in
the images are not due to movement from the plain of focus. Bar=S~m.
As shown in Figure 8, some VTS were found to be detaching and some
to be associating with the Golgi complex. The VTS were highly dynamic
and pleiomorphic ili size. Some of the VTS followed straight or curvilinear
paths, some moved ili a stop-and-go fashion, and some showed saltatory
movements. The VTS indicated at the top panel (white arrows) extended
away from and then retracted back to the Golgi. The VTS in the middle
panel and the first image in the lower panel (red arrows) detached from the
Golgi complex, paused, and then moved towards the cell periphery until it
disappeared at the cell edge suggesting that STMP1 is associated with the
secretory pathway. The VTS in the lower panel (yellow arrow) moved
from the cell periphery towards the Golgi body suggesting that STMP1 is
localized to the endocytic pathway.
EXAMPLE 9
Colocalization Of GFP-STMP1 With The Early Endosomal Marker EEA1
To probe whether GFP-STMP1 was associated with the endocytic
pathway, the intracellular distribution of GFP-STMP1 was compared with
that of the early endosome protein EEA1 (Stenmark, H. et al., JBiol Clzem
271, 204048-204054, 1996). COS-1 cells were transfected with GFP-
STMP1, fixed, immunostained with EEA1 antibodies and observed by
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confocal laser scanning microscopy. Green GFP-STMP 1 fluorescence and
red (Texas Red-labeled secondary antiserum) EEA1 fluorescence were
detected by confocal laser microscopy. The panel to the right shows the
overlay images with yellow/orange staining indicating the regions of
colocalization. Arrows indicate examples of the VTS in the cell periphery
which contain both EEA1 and STMP1. Bar=Spm. As shown in Figure 9,
EEA1 manifested a similar intracellular distribution in both transfected and
untransfected cells. Furthermore, GFP-STMP1 significantly colocalized
with EEA1 both in the cell periphery and also in the perinuclear area
(Figure 9, arrows) suggesting that STMP1 is associated with early
endosomes and the endocytic pathway.
EXAMPLE 10
Isolation And Characterization of the SSH9 Gene And mRNA
The SSH9 gene was identified and mapped (Figure 10). The
predicted promoter site, the transcription start site, and the location and
size
of the exons and introns are indicated. The start and stop codons, as well as
two polyadenylation signals, leading to two alternatively spliced transcripts,
are also indicated. Figures 11A-C show the nucleotide and predicted amino
acid sequence of SSH9, as well as the predicted promoter sequence and
exon-intron boundaries.
The expression profile of SSH9, determined in various human tissues
by Northern analysis (Figure 12C), revealed that the 0.7 kb splice variant of
SSH9 was highly testis-specific, while the 1.4 kb transcript was expressed
in both prostate and testis.
The androgen regulation of SSH9 was examined in LNCaP cells and
in CWR22 xenografts (Figure 12A) revealed that SSH9 is not regulated in
LNCaP cells, but is regulated in CWR22 xenografts. The expression
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profile of SSH9 was also examined in the androgen-independent prostate
cancer cell lines PC3 and DU145, and in CWR22R cells (Figure 12B).
EXAMPLE 11
Isolation And Characterization of the PSL22 Gene And mRNA
The PSL22 gene was identified and mapped (Figure 13). The
location and size of the exons and introns, the location of the partial cDNA
clone (black box), as well as the alignment of the full-length cDNA clone
with GenBank Accession Nos. AC00~551 and AC011449, are indicated.
Figures 14A-C show the nucleotide sequence of the ORF, cDNA and
predicted amino acid sequence, as well as the predicted promoter, exon, and
UTR sequences of PSL22.
BLAST analysis of GenBank with the predicted PSL22 amino acid
sequence identified PSL22 as a Rho binding protein. Figure 15 shows a
multiple sequence alignment of PSL2~ with related proteins. Completely
conserved residues are shown in black; residues found in three sequences
are shaded.
The expression profile of PS'L22, determined in various human
tissues by Northern analysis (Figure 116B), revealed that while the highest
expression was seen in the prostate, high expression was seen in the kidney,
pancreas, and colon.
The androgen regulation of PSL22 was examined iii LNCaP cells, in
the androgen-independent prostate cancer cell lines PC3 and DU145, and in
CWR22R cells (Figure 16A). The results showed that PSL22 is androgen
regulated in LNCaP cells, where it is highly expressed, but is not androgen
regulated in the PC3 and DU145 cells.
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EXAMPLE 12
Materials And Methods
The following materials and methods were used in performing the
exemplary experiments shown herein. It is understood that these materials
and methods are subject to modifications that do not change the nature of
the invention, as will be understood by those of ordinary skill in the art.
Probes
Poly (A)+ RNA 1 ~g [(-) or (+)]
Random primer (N7) 200 ng
RNAse-free sterile H20 to 20 u1
Heat at 70°C for 10 min, and chill on ice.
While heatiilg the RNA samples, the following solution was
prepared:
SX 1st strand buffer 10 ~,1
0.1 mM DTT 5 ~l
10 mM each dTTP+dGTP 2 ~ 1
3aP alpha dATP 5 ~l
3zP alpha dCTP 5 ~,1
Superscript II (200 LT /~.I, BRL) 2~,1
The solution was mixed by pipetting, spun briefly, incubated at
25°C
for 5 min, and then for an additional 1 hour at 37°C. 2 ~1 of lOmM dCTP
+
dATP was added and the mixture was incubated for 30 min at 37°C and
then heat inactivated at 70°C for 10 min. Unincorporated nucleotides
were
removed using prespun G25 columns (Bio-Rad). Specific activity (which
should be over Sx108cpm/~g) was calculated.
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Hybridization
Freshly prepared 25 ml Hybridization mix (7% SDS, 0.5 M
NaHP04, 1mM EDTA) was pre-warmed at 65°C and 12.5 ml was used for
prehybridization of each membrane, 5-10 min at 65 °C. The probe was
heat denatured at 95°C for 3-5 min and transferred to the
prehybridization
mix at 65°C. Hybridization was carried out at 65°C overnight.
Washing
Wash solution I (2xSSC and 1% SDS) and II (O.IxSSC and 0.5%
SDS) were prewarmed, and the membrane were washed once with Solution
I and then with Solution II for 30 min at 65°C. The membranes were
covered with plastic wrap and exposed to a phoshorimager screen.
Selection
Clones that showed differences between the (-) and (+) blots were
picked (usually 1-8 on each blot pair). A secondary round of reverse
northern analysis for confirmation was performed, this time spotting each
clone in duplicate on each blot. After phosphorimager analysis, the blots
were stripped in O.IxSSC and 0.5% SDS for 2x15 min at 95°C and
hybridized with a PSA probe (or depending on the hormone that is being
used, with a probe for any abundant target genes in the tissue under study).
For the clones that were confirmed to be different from PSA, for
differential expression in the secondary reverse northern, northern analysis
was performed using established protocols. A time course of 81881
induction of LNCaP cells, as well as the CWR22 xenograft model upon
androgen ablation (Wainstein, M. A. et al., Cafacer Res. 54, 6049-6052,
1994) and the androgen-independent CWR22R relapsed xenograft
(Nagabhushan, M. et al., Cahce~ Res. 56, 3042-6, 1996), was used.
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Seguence analysis
Sequence analysis was perfomed by the dideoxy chain termination
methods using an ABI automated sequencer. Homology search was done
ushzg a basic BLAST algorithm. Figure 3 shows a table of results obtained
from the BLAST analysis of isolated clones and their homology to known
genes (The cutoff for significant homology was 50% identity).
Isolation of prostate cancer related genes from LNCaP cells
The prostate cancer cell line LNCaP was cultured in two batches in
culture conditions similar to those previously described (Horoszewicz JS et
al., CahceY Res. 43: 1809-1818, 1983). The first batch was left untreated,
while the second batch was treated with the synthetic androgen 81881 for
24 hrs. Cells from both batches were harvested and total RNA was then
isolated from each batch. From the total RNA, polyA+ RNA was obtained
using standard procedures, and was used in the Suppression Subtraction
Hybridization (SSH; Diatchenko et al., supra) procedure to identify hor-
mone regulated genes. The tester in the SSH procedure was cDNA from
untreated cells and the driver was cDNA from 81881-treated cells. The
suppression subtraction protocol was performed according to the original
description of the method (Diatchenko et al., supra).
Cell culture
LNCaP, PC-3 and DII-145 cells were routinely maintained and
treated as described previously (Korkmaz, K. S. et al., DNA Cell Biol 19,
499-506, 2000; Korkmaz, K. S. et al., Gene 260, 25-36, 2000).
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Xenograft studies
Transplantation, growth, and harvesting of tumors from mice
bearing the CWR22 and CWR22R xenografts were as previously described
(Wainstein, M. A., supra; Nagabhushan, M., supra).
Cloniil-g-and-plasmid construction
A 262 by cDNA fragment was originally obtained from a screen of a
prostate specific library (Ausubel, F. M., et al. (1997) Current Protocols if2
Molecular Biology (John Wiley and Sons, New York) and termed L74. 5'
Rapid Amplification of cDNA Ends (R.ACE) was performed
(oligonucleotide sequences available upon request) using the Marathon-
Ready cDNA that was prepared from normal prostate tissue (Clontech)
andlor SMART-RACE LNCaP cDNA library (Clontech) that was
generated according to the manufacturer's recommendations. RACE
products were cloned into pCRII-TOPO (Invitrogen), positive clones were
confirmed by Southern analysis, and sequenced. In parallel, a ~,gtl0 cDNA
library made from a pool of normal human prostates (Clontech) was
screened by established procedures to obtain additional clones. Overlapping
clones were used to deduce the full-length STMPI cDNA sequence.
The full-length STMPl ORF was amplified by using primers centered
around the start and stop codons (sequences available upon request) and
fused in frame to the C-terminus of green flourescent protein (GFP) using
the vector pcDNA3.1-NT-GFP-TOPO (Invitrogen) to generate GFP-
STMP 1.
Northern analysis
Total RNA was prepared by the single step guanidine thiocyanate
procedure and used in Northern analysis (18). 15 ~g of total RNA was used
per lane. Probes were generated by random priming and had a specific
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activity of >3x10$ dpm/~.g. A cDNA fragment of ,STMPl spanning residues
145-2202 by was used as probe. Bands were visualized and quantitated by
phosphorimager analysis (Molecular Dynamics).
Confocal microscopy
COS-1 cells were transiently transfected by electroporation using a
BTX square-wave pulser at 150 V, 1 ms duration. Cells were grown either
on cover slips placed in 6-well tissue culture plates for indirect
immunofluorescence or on Lab-Tek Chambered Coverglass (Nalge Nunc
International) for live-cell microscopy. Transiently transfected cells were
observed 16 h after transfection by Leica TCS-SP confocal microscope. All
live-cell experiments were done at 37°C.
Indirect immunofluorescence
The indirect immunofluorescence was carried out as previously
described (Misteli, T. & Spector, D. L. Mol Cell 3, 697-705, 1999). The
following antibodies were used: anti-~3-coat proteW ((3-COP) antiserum
(kindly provided by J. Lippincott-Schwartz), anti-mannosidase II (kindly
provided by T. Misteli), anti-TGN46 (Serotec, kindly provided by J.S.
Bonifacino), and anti-EEA1 (Affinity Biotechnologies). Texas Red-
conjugated secondary antibodies specific for mouse and rabbit were
purchased from ICN Biomedicals (Costa Mesa, CA).
Other Embodiments
All publications and patent applications mentioned in this
specification are herein incorporated by reference to the same extent as if
each independent publication or patent application was specifically and
individually indicated to be incorporated by reference.
67
CA 02403637 2002-09-19
WO 01/72962 PCT/USO1/09410
While the invention has been described in connection with specific
embodiments thereof, it will be understood that it is capable of further
modifications and this application is intended to cover any variations, uses,
or adaptations of the invention following, in general, the principles of the
invention and including such departures from the present disclosure that
come within known or customary practice within the art to which the
invention pertains and may be applied to the essential features hereinbefore
set forth, and follow in the scope of the appended claims.
68
