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Genos Biosciences Incorporated, La Jolla, California 92037 [L. C. A., C. L., M. L. S., S. K., D. L., V. W., M. L., J. M., N. D., G. M. H.], and Departments of Medicine [D. B. A., W. D. F., B. H., H. I. S.] and Pathology [W. G., C. C-C.], Memorial Sloan-Kettering Cancer Center, New York, New York 10021
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ABSTRACT |
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INTRODUCTION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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To begin to address this question, we used microarray analysis of 9792 genes to obtain a broad picture of the changes in transcription that correlate with androgen-ablation therapy of an androgen-dependent tumor and the emergence of androgen-independence. As a surrogate to the use of human tissue, we used the CWR22 xenograft model, which recapitulates the human condition in that it is dependent on androgens for growth and measured levels of prostate-specific antigen are directly proportional to tumor volume (6 , 7) . s.c. implantation of CWR22 results in exponentially proliferative tumors within approximately 3–4 weeks, which, when deprived of androgens, undergo rapid remission to a nonproliferative tumor mass, as indicated by dramatic decreases in Ki67 immunostaining (8 , 9) . This tumor mass, however, is viable, because readministration of androgens results in an increase in tumor size and an increase in the number of cells in active cell cycle (8) 4 . From androgen-deprived and nonproliferative tumors, rapidly growing androgen-independent derivatives emanate some 3–12 months thereafter.
Here we show that the changes in gene expression following androgen-deprivation in CWR22 were indicative of growth arrest along with decreases in basal cell metabolism. Comparison of expression changes in androgen-deprived tumor cells to androgen-independent cells showed that the evolution of androgen-independent growth requires reentry into active cell cycle. Further comparison of the parent CWR22 tumor to the CWR22-R derivative in which androgens had been readministered for 2 days, led to the discovery of a small subset of androgen-responsive genes which were expressed in the absence of androgens.
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MATERIALS AND METHODS |
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-dihydrotestosterone pellets (12.5 mg; Innovative Research of
America, Sarasota, FL) were placed in the s.c. flanks of the mice prior
to receiving tumor material for the purpose of maintaining constant
amounts of circulating androgens. Tumors of approximately 1.5 x 1.0 x 1.0 cm were evident in 3–4 weeks.
Androgen withdrawal was achieved both by surgical castration under
pentobarbital anesthesia and removal of the testosterone pellets. Tumor
sizes were determined by caliper measurements of height, weight, and
depth. Androgen-independent CWR22-R xenograft tumors were propagated in
nude athymic female BALB/c mice. Addition of androgens to CWR22-R was
accomplished by implantation of sustained-release
5--dihydrotestosterone pellets into the s.c. flanks of mice bearing
viable, rapidly growing tumors. Xenograft tissues from CWR22 tumors in
the presence of androgens, 4-, 8 and 20 days after androgen withdrawal,
and from the CWR22-R tumors in the presence or absence of androgens
were collected after sacrificing the mice and flash frozen in liquid
nitrogen. Tumor fragments were isolated from areas that showed
microscopic evidence of neoplastic enrichment (at least >90%) and a
lack of necrosis. These tumor specimens represent a subset of those
described recently (8)
in which extensive histological and
immunohistochemical analyses were carried out.
Preparation of mRNA and Microarray Hybridization.
Total RNA was isolated from flash-frozen tumor xenograft tissue using
TRIzol reagent (Life Technologies, Inc., Rockville, MD) and treated
with amplification-grade DNaseô reagent (Life Technologies,
Inc.). Poly(A) mRNA was isolated using Oligotex columns (Qiagen,
Valencia, CA) and quantified on a spectrophotometer. Two hundred ng of
each mRNA was visualized on a 0.8% denaturing agarose gel to assess
quantity and quality. Purity as defined by the 260/280-nm
spectrophotometer was typically >1.9. Cohybridizations on the UniGEM
1.0 microarray were performed by Incyte Microarray Operations
(Incyte Pharmaceuticals Incorporated, Palo Alto, CA) using 200 ng of
each test mRNA labeled with Cy3 and 200 ng of mRNA from CWR22 labeled
with Cy5. The UniGEM 1.0 array used in these studies contains 10,000
spotted cDNA elements, 9,704 test elements, and 296 control elements.
Of the 9,704 test elements, 4,864 represent fragments of known human
transcripts. The remaining 4,840 elements are anonymous
ESTs,5
of which 923 are annotated as weakly, moderately, or highly similar to
known genes.
Northern Hybridization.
Northern blots were prepared according to standard procedures. Briefly,
10 µg of total RNA from each sample was electrophoresed in denaturing
formaldehyde agarose gels. RNA was transferred to Hybond N+ membrane.
Probes were prepared by PCR amplification of the relevant I.M.A.G.E.
cDNA clone, purified on Qiaquick columns (Qiagen) and labeled using the
Prime-It II kit (Stratagene, La Jolla, CA) according to the
manufacturer’s instructions. Blots were hybridized using a sodium
phosphate/SDS buffer, and posthybridization washes were carried out as
described (10)
. Blots were imaged and signals quantified
by a PhosphorImager (Molecular Dynamics, Sunnyvale, CA). Filters were
striped in boiling 0.5% SDS solution. Hybridization signals from an
18S-specific rRNA probe (Ambion, Inc., Austin, TX) were used as a
measure of RNA loading across the blot.
Sequence Verification of cDNA Clones.
I.M.A.G.E. cDNA clones representing EST sequences of interest were
obtained from Genome Systems (St. Louis, MO) and the inserts amplified
by PCR. Sequences were generated using an ABI-377 automated fluorescent
sequencer and queried against the public databases using BLAST (BLASTn
or X, as appropriate). Annotations were compared with those reported by
Incyte, Inc., and UniGene.
Computational Analysis of Microarray Data.
Raw data from the UniGEM microarray hybridizations were received from
Incyte Pharmaceuticals Incorporated (Palo Alto, CA) and exported from
GEMTools (Incyte Pharmaceuticals, Inc.) into a relational database.
These data were subjected to an error-correction procedure to minimize
a systematic bias commonly seen with two-color microarray
hybridizations. Briefly, ratios of all Cy3 and Cy5 signals were
transformed using a moving average correction procedure. The corrected
ratios were then transformed using a power function to minimize
skewness and kurtosis and standardized to yield a distribution with a
mean of zero and a variance of
one6
. After processing, data with a signal-to-background ratio of 3:1 or
greater and a spot area coverage of at least 40% were up-loaded into a
relational database for storage, retrieval, and report generation. Data
were divided up into flat lists of increased and decreased expression
for each experiment and queried across multiple experiments to identify
the behavior of sub-sets of genes relevant to the questions outlined in
the text. Functional classification of differentially expressed genes
was carried out using the "hierarchical" function in GEMTools,
which was constructed from functional information contained in UniGene
(and associated MedLine links) and the LifeSeq database (Incyte
Pharmaceuticals, Inc.).
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RESULTS |
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Incyte Microarray Operations have reported the identification of
differentially expressed genes at ratios as low as
1.75-fold7
However, before the experiments outlined above were performed, we chose
to measure this threshold empirically on the UniGEM 1.0 array.
Identical mRNA populations from CWR22 were co-hybridized to the array.
No differences in gene expression were expected (Fig. 1)
. At 1.75-fold, 106 ESTs were identified as differentially expressed as
compared with 16 differences at >2-fold and only three differences
>2.5-fold. Thus, empirically, at a level of 2.5-fold, we would expect
three differences to occur by chance. We have recently performed
statistical analyses on these and other UniGEM 1.0
experiments6
which show that 2.5-fold is
conservative, corresponding to a P of approximately
10-4 on normalized data. However, because we
hybridized each sample on only a single UniGEM 1.0 array, the degree of
error below 2.5-fold was thought to be too high for reliable for
inter-GEM comparisons (see "Discussion").
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illustrates the two differentially expressed genes (the putative
M-phase phosphoprotein 2, MMP-2, and the
hepatoma transmembrane kinase ligand, ephrinB5 or
lerk-5) for which more than one EST was associated with each
gene. As illustrated, the profiles for each set of two ESTs were
remarkably similar.
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. It should be noted that several genes associated with prostate cancer
progression, particularly the AR, prostate specific
antigen, and human kallikrien 2 genes, among others,
are not represented by I.M.A.G.E clones on the chip. This, however, was
beyond our control, because the microarrays were commercial products
and not fabricated in our laboratories.
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were also significantly decreased at day 20, often with increasing
magnitude. In contrast, genes with increased expression following
androgen deprivation were less uniform over the three time points. Of
the four genes with increased expression at day 4, only one gene,
ceruloplasmin, was elevated at day 8 and day 20. Likewise,
only 4 of 15 genes elevated at day 8 were also elevated at day 20
(Table 1)
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- and ß-tubulin,
M-phase phosphoprotein 2 (two occurrences; see Fig. 2
), and
protein phosphatase type 1. Six of the 51 genes were
annotated as involved in protein degradation and cleavage,
i.e., cathepsin K, ubiquitin-specific
protease 11 and peptidase ß (mitochondrial
processing), and six genes involved in metabolism and respiration,
i.e., glyceraldehyde-3-phosphate dehydrogenase,
lactate dehydrogenase A, -enolase,
phosphogylcerate mutase, NAPDH dehydrogenase and cytochrome c
oxidaseVIic. Other genes with decreased expression were identified as
transcription and translation factors, i.e., the TFIID
component, TAFII20, and the TIF2 component,
TIF2ß. These collectively suggest that CWR22 cells exit
active cell cycle, concomitant with down-regulation of metabolism,
respiration, transcription, and translation. Of the 38 genes that were
elevated after androgen-withdrawal, 22 had annotations associated with
them. No obvious functional theme was apparent. However, we noted two
genes, BTG-1 and EXT1, whose increased expression
suggested a dominant cell-cycle arrest, because both are putative tumor
suppressor genes (11
, 12)
. Other genes, such as
prostate carcinoma tumor antigen, whose expression is
modulated by androgens (13)
, were also identified. We did
not see any clear evidence for the induction of proapoptotic genes.
However, that these cells arrest after androgen starvation does not
presuppose that the tumor mass is then "de-bulked" by apoptosis,
consistent with our observations of little or no increase in terminal
deoxynucleotidyl transferase-mediated nick end labeling-positive
cells following deprivation (8)
.
Differences in Gene Expression Between Androgen-dependent and
-independent Xenografts.
Changes in the levels of transcription between the androgen-dependent
xenograft, CWR22, and the androgen-independent xenograft, CWR22-R, were
measured by cohybridization of mRNA populations derived from each
tumor-type. Above 2.5-fold, 57 genes were identified as differentially
expressed, 13 and 44 with increased and decreased expression,
respectively (Table 1)
. Attempts to functionally classify these genes
were curbed by the large percentage of the genes without functional
annotation (only 4 of 12 that were increased and 3 of 16 unique genes
that were decreased; see below). Nonetheless, of the 13 increased
genes, three were associated with thyroid hormone receptor signaling,
the thyroid hormone receptor, 2
(c-erb-A or TR), the thyroid
hormone receptor-associated cofactor, TRAC1 (Ref.
14
; also known as SMRT), and the
TR corepressor, SUN-CoR (15
, 16)
. We also identified two cell surface receptors,
syndecan I and -integrin-6, whose
dysregulated expression is associated with cancer progression
(17
, 18)
.
Relationship Between Androgen-deprivation and the Emergence of
Androgen Independence.
Because androgen-independent tumors are derived from androgen-dependent
cells deprived of androgens, we reasoned that an examination of
expression changes in the androgen-independent derivative, CWR22-R,
should take into account how these genes had behaved during deprivation
and vice versa. Of a total of 34 unique genes that were
found to be elevated >2.5-fold at any time point after
androgen-deprivation, all were found to be expressed at similar levels
in both CWR22 and CWR22-R. This implied that the elevated expression of
all of the genes in androgen-deprived cells was subsequently reduced to
CWR22-like levels in CWR22-R. In contrast, of the 122 genes that were
reduced >2.5-fold in expression following deprivation, 28 remained
significantly decreased in the androgen-independent tumor
(i.e., were reduced in expression in CWR22-R
versus CWR22; Table 2
).
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The decreased expression of the 28 genes seen in both androgen-deprived
CWR22 and androgen-independent CWR22-R cells was not observed in
CWR22-RA, suggesting that, indeed, expression had increased as a result
of androgen exposure (Table 2)
. Eleven of these 28 genes had been
annotated and at least one of them, -tubulin,
has been shown to be androgen-responsive (19)
, suggesting
that the other genes not previously thought to be androgen-responsive
(e.g., HSP40, HSP70-2) may, in fact,
be responsive.
Dysregulated Expression of Androgen-responsive Genes.
We extended this strategy to ask whether any of the genes that were
increased or decreased following androgen-deprivation and subsequently
reexpressed in CWR22-R at similar levels to CWR22 were also
androgen-responsive (i.e., androgen-dependent, but expressed
in the absence of androgens). Of the 94 genes that were decreased
following deprivation and subsequently reexpressed in CWR22-R, only one
gene, the calcium binding protein S100P, was elevated
(12.4-fold) in CWR22-RA as compared with CWR22 (Fig. 3
; Table 3
). We next asked whether any of the 13 genes that were found to be
elevated in CWR22-R as compared with CWR22 (and not changed as a
consequence of androgen-deprivation) responded to androgen exposure,
i.e., whether they were elevated, remained the same, or were
repressed. Seven of 13 genes were elevated in CWR22-RA and CWR22-R,
whereas the expression of the other six genes was reduced in CWR22-RA
as compared with CWR22-R. Although the levels of expression found in
CWR22-RA were suggestive of an additional increase following exposure
of androgens to CWR22-R, Northern analysis showed that only one of the
seven genes, FKBP51/54 was significantly increased.
Expression was elevated from 3.4-fold in CWR22-R (versus
CWR22) to 5.9-fold in CWR22-RA (versus CWR22-R), a composite
increase of approximately 1.73-fold. The other six genes were increased
or decreased <0.7-fold (Fig. 3)
. Reexamination of the
androgen-deprivation data for FKBP51/54 showed decreases of
2.2-, 1.7-, and 2.1-fold at the day-4, -8, and -20 time points,
respectively, suggesting in fact that this gene might exhibit the same
behavior as the calcium binding protein, S100P, during
androgen-deprivation, which we confirmed by Northern analysis (Fig. 3)
.
Although similar in behavior to S100P, FKBP51/54 was unique
in that it was the only gene of 122 unique genes that was decreased
following androgen-withdrawal and reexpressed at higher levels in the
androgen-independent derivative, CWR22-R.
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. Providing that the levels of expression are
low or absent in CWR22 as well as in androgen-deprived cells, these
genes represent candidates for the molecular diagnosis of
androgen-independent cells even in the presence of androgen-dependent
cells. However, because the microarray experiments described here
only measure differential gene expression, one cannot predict the
absolute levels of expression in androgen-dependent tumors. Thus,
Northern analysis was carried out to assess expression in CWR22 and 20
days after castration. These genes showed variable but generally low
levels of expression in the parent tumor and during
androgen-deprivation, but they were increased in expression in CWR22-R.
Examples of some of these genes are shown in Fig. 3
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DISCUSSION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The parallel expression analysis of thousands of genes affords unique opportunities to visualize such biological processes, and a growing number of formats exist with which one can achieve these goals (20, 21, 22) . Microarray hybridization is perhaps the most straightforward approach, but for laboratories without an "in-house" array system, "hybridization-by-contract" (e.g., through Incyte Pharmaceuticals, Incorporated) is expensive. Thus, our approach in this study was to use a minimum number of UniGEM arrays to gain insight into the process under study. Empirical determination of differential expression was facilitated through a "self-versus-self" cohybridization where a fold difference of >2.5 did not detect a significant number of changes. Clearly, rehybridization of the samples two to three times each would help identify cognate differences at lower fold thresholds (e.g., 1.8–2.0-fold), but would have been prohibitively expensive. Thus, we consider the current data to be indicative, rather than definitive, for the process under study, and that follow-up by other methods such as Northern analysis or quantitative real-time reverse transcription-PCR essential for those genes that can be defined as interesting in the present context.
Attempts to functionally classify differentially expressed genes were successful for sets of genes in which the majority has associated annotations. In this regard, we were able to characterize the sets of genes depressed following androgen deprivation. Collectively, the decrease in expression of multiple cell cycle components, as well decreases in metabolism, respiration, and transcription/translation suggest that androgen-deprivation elicits growth arrest in CWR22 cells. Because readministration of androgens can elicit tumor regrowth, we consider the androgen-deprived cells to be in an arrested, but viable, state. We also identified increases in the expression of several genes following androgen-deprivation that have not been associated with prostate cancer progression, but which may act to suppress to tumor growth or whose expression is positively correlated with growth inhibitory signals (e.g., androgen-deprivation). These included BTG-1, which can suppress the growth of NIH3T3 cells, and which may act directly or indirectly to modulate the transcriptional regulation of cell cycle genes (11) and EXT-1, a putative tumor suppressor gene identified through mutation analysis in hereditary multiple exostoses (12) . Between 2–2.5-fold, we also identified several other genes whose functions were provocative in this regard: (a) mac25, whose expression is decreased in many cancer cells, including breast, from an initially high level in senescent epithelial cells (23 , 24) , (b) tumor growth inhibitory factor, which has been identified as a smad-2-binding protein and repressor of transcription (25) and, (c) TSC-22, which is thought to act as a tumor suppressor and repressor of transcription and whose down-regulated expression is correlated with tumor cell line growth (26 , 27) .
Simple comparison of CWR22 relative to androgen-deprived cells and CWR22-R showed that the vast majority of genes modulated between CWR22 and androgen-deprived cells returned to CWR22-like levels in CWR22-R. Because all of the cell cycle components (positive and negative) returned to "normal," we conclude that evolution to androgen-independence in CWR22-R required reentry into active cell cycle.
The genes that we identified in androgen-starved and androgen-independent cells did not show any evidence of expression expected from infiltrating immune cells, consistent with our histological analysis that the tumor fragments used for expression analysis were highly enriched for the neoplastic cell populations.
We and others have shown previously that the levels of AR mRNA (for which ESTs were not represented on the UniGEM 1.0 chip) and AR protein in CWR22 decrease following androgen-deprivation, but are subsequently reexpressed at some point before the emergence of hormone-refractory disease (8 , 19) . It should follow, then, that the compendium of AR-responsive genes would subsequently increase in expression in CWR22-R. However, we identified at least 28 genes that were not reexpressed and two, S100P and FKBP51, that were. These results suggest that the AR-signaling pathway is partly reactivated in the absence of androgens. However, the mechanism of AR gene reactivation (i.e., whether by ligand-independent means or other mechanisms) is not clear from the present study. It should be noted that administration of androgens to CWR22-R (i.e., CWR22-RA) may only have resulted in the up- or down-regulation of genes that were most strongly androgen-dependent, and that other genes with the characteristics of FKBP51 and S100P may yet exist. However, the relevance of identifying androgen-responsive genes that are reexpressed in the absence of androgens is supported by the observation that one of the two elevated genes, S100P, is androgen-responsive and has been shown to be dysregulated in androgen-independent derivatives of LNCaP cells and in androgen-independent PC-3 and DU145 cells (28) . This underscores the usefulness of the strategy documented here, which sought to identify such genes by comparing proliferative tumors under different androgen-stimulated conditions (i.e., CWR22 versus CWR22-R versus CWR22-R replete with androgens). It is worth noting that had we simply compared CWR22 versus CWR22-R, we would not have identified S100P as an interesting candidate gene in this context.
One of the other genes identified by this approach, FKBP51, an FK-506 binding protein with isopropyl peptidyl isomerase activity (29) , was unique in being the only gene in which expression levels were changed after androgen-deprivation and subsequently expressed at higher levels in CWR22-R versus CWR22. It is of note that this gene was also overexpressed 2.3-fold in another androgen-independent xenograft, MSKPC-6, which some of us have derived from CWR22.10Although this gene has not been reported as androgen-responsive, it is known to be a component part of the protein complex (which includes the heat shock proteins HSP90, HSP70, and HSP40), responsible for binding and folding native nuclear steroid hormone receptor proteins (e.g., estrogen, progesterone, and mineralocorticoid receptors; Ref. 29 ). As far as we are aware, an association with native AR has not been explored nor reported.
Androgen-exposure of CWR22-R also led to the identification of a set of
genes whose expression was not influenced by androgens, which we refer
to as "stable up-regulated." Northern analysis of some of these
genes showed low or undetectable expression in CWR22 and CWR22 starved
of androgens, but increased expression in CWR22-R. Strikingly, three of
the up-regulated genes were found to be involved in thyroid hormone
receptor signaling: (a) the thyroid hormone receptor
(TR); (b) the
ligand-independent corepressor, SMRT or TRAC1;
and (c) the unliganded corepressor, SUN-CoR. One
of these, SUN-CoR, whose expression was elevated in CWR22-R,
was found to be repressed following readditions of androgens, showing
that expression was increased only in the absence of androgens. These
results suggest that imbalances of T3 signaling components might play a
role in androgen-independent growth, which is plausible based on
reports showing that T3 can potentiate, albeit indirectly, the
transcription of some, but not other, androgen-responsive genes
(30
, 31)
.
Aside from the mechanisms responsible for independent growth, these up-regulated genes represent putative molecular diagnostics for androgen-independent disease, providing that these observations are recapitulated at the protein level. In this regard, Bubendorf et al. (32) have recently reported the combined use of cDNA microarrays and tissue multiblocks for the identification of markers in CWR22-R derivatives, which were then tested by immunohistochemistry on appropriate tumor samples. Their work identified two genes, IGFBP2 and HSP27, which were over-expressed in 100% and 31% of hormone-refractory tumors, respectively. Presumably, the application of the methods outlined by Bubendorf et al. (32) to the sets of genes identified here would identify those relevant to human prostate cancer. That we did not identify either of these genes as overexpressed in the CWR22-R derivative used in the current study underscores the potential tumor heterogeneity and, perhaps, multiple mechanisms through which prostate tumors may escape the growth arrest induced by androgen-deprivation.
It is important to note that CWR22 and CWR22-R represents a single
model of the evolution of hormone-autonomous tumor growth derived from
a single patient, and that other models exist [e.g., LNCaP
and LNCaP-C4 (33)
; LAPC-4 and LAPC-9 (34
, 35)
]. We further recognize that androgen-independent tumors are
heterogeneous, whether growing as xenografts or in humans, and probably
include both androgen-dependent as well as androgen-independent tumor
clones (35)
. Nevertheless, the identification of changes
in gene expression that are consistent with the results of other model
systems and humans is encouraging (e.g., BTG-1,
PCTA-1, S100P and the human selenium binding protein in
LNCaP cells; -tubulin and
-enolase in CWR22 and prostate specific
membrane antigen in man, among many others) and suggest that the
genes identified in the current study provide a useful guide-post for
the molecular analysis of hormone-refractory prostate cancer.
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FOOTNOTES |
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1 Supported by CaPCURE (D. B. A. and
H. I. S.), the American Cancer Society (D. B. A.), The PepsiCo
Foundation, and the Eleanor and Paul Stevens Foundation. D. B. A. is
supported by a CaPCURE Young Investigator Award. 
2 These authors contributed equally to this
work.
3 To whom requests for reprints should be
addressed, at Genomics Institute of the Novartis Research Foundation,
3115 Merryfield Road, San Diego, CA 92121. Phone: (858) 812-1522; Fax:
(619) 812-1584; E-mail: garret_hampton{at}yahoo.com
5 The abbreviations used are: EST, expressed
sequence tag; AR, androgen receptor; FKBP51/54, 54-kDa
progesterone receptor associated protein; BTG-1, B-cell
translocation gene-1.
7 Incyte Technical Survey, "GEM Microarray
reproducibility study," 1999. Internet address: www.incyte.com.
9 D. B. A., W. D. F., and B. H.
Received 2/ 8/00. Accepted 9/ 1/00.
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 S. E. Dowen, T. Crnogorac-Jurcevic, R. Gangeswaran, M. Hansen, J. J. Eloranta, V. Bhakta, T. A. Brentnall, J. Luttges, G. Kloppel, and N. R. Lemoine Expression of S100P and Its Novel Binding Partner S100PBPR in Early Pancreatic Cancer Am. J. Pathol., January 1, 2005; 166(1): 81 - 92. [Abstract] [Full Text] [PDF]
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K. V. Desai, A. M. Michalowska, P. Kondaiah, J. M. Ward, J. H. Shih, and J. E. Green Gene Expression Profiling Identifies a Unique Androgen-Mediated Inflammatory/Immune Signature and a PTEN (Phosphatase and Tensin Homolog Deleted on Chromosome 10)-Mediated Apoptotic Response Specific to the Rat Ventral Prostate Mol. Endocrinol., December 1, 2004; 18(12): 2895 - 2907. [Abstract] [Full Text] [PDF]
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H.-K. Lin, Y.-C. Hu, D. K. Lee, and C. Chang Regulation of Androgen Receptor Signaling by PTEN (Phosphatase and Tensin Homolog Deleted on Chromosome 10) Tumor Suppressor through Distinct Mechanisms in Prostate Cancer Cells Mol. Endocrinol., October 1, 2004; 18(10): 2409 - 2423. [Abstract] [Full Text] [PDF]
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 H. I Scher, G. Buchanan, W. Gerald, L. M Butler, and W. D Tilley Targeting the androgen receptor: improving outcomes for castration-resistant prostate cancer Endocr. Relat. Cancer, September 1, 2004; 11(3): 459 - 476. [Abstract] [Full Text] [PDF]
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A. M. Velasco, K. A. Gillis, Y. Li, E. L. Brown, T. M. Sadler, M. Achilleos, L. M. Greenberger, P. Frost, W. Bai, and Y. Zhang Identification and Validation of Novel Androgen-Regulated Genes in Prostate Cancer Endocrinology, August 1, 2004; 145(8): 3913 - 3924. [Abstract] [Full Text] [PDF]
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 M. J. Linja, K. P. Porkka, Z. Kang, K. J. Savinainen, O. A. Janne, T. L. J. Tammela, R. L. Vessella, J. J. Palvimo, and T. Visakorpi Expression of Androgen Receptor Coregulators in Prostate Cancer Clin. Cancer Res., February 1, 2004; 10(3): 1032 - 1040. [Abstract] [Full Text] [PDF]
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 S. Chauhan, S. Kunz, K. Davis, J. Roberts, G. Martin, M. C. Demetriou, T. C. Sroka, A. E. Cress, and R. L. Miesfeld Androgen Control of Cell Proliferation and Cytoskeletal Reorganization in Human Fibrosarcoma Cells: ROLE OF RhoB SIGNALING J. Biol. Chem., January 9, 2004; 279(2): 937 - 944. [Abstract] [Full Text] [PDF]
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J. Holzbeierlein, P. Lal, E. LaTulippe, A. Smith, J. Satagopan, L. Zhang, C. Ryan, S. Smith, H. Scher, P. Scardino, et al. Gene Expression Analysis of Human Prostate Carcinoma during Hormonal Therapy Identifies Androgen-Responsive Genes and Mechanisms of Therapy Resistance Am. J. Pathol., January 1, 2004; 164(1): 217 - 227. [Abstract] [Full Text] [PDF]
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 W. G. Nelson, A. M. De Marzo, and W. B. Isaacs Prostate Cancer N. Engl. J. Med., July 24, 2003; 349(4): 366 - 381. [Full Text] [PDF]
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T. R. Hubler, W. B. Denny, D. L. Valentine, J. Cheung-Flynn, D. F. Smith, and J. G. Scammell The FK506-Binding Immunophilin FKBP51 Is Transcriptionally Regulated by Progestin and Attenuates Progestin Responsiveness Endocrinology, June 1, 2003; 144(6): 2380 - 2387. [Abstract] [Full Text] [PDF]
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F. Jiang and Z. Wang Identification of Androgen-Responsive Genes in the Rat Ventral Prostate by Complementary Deoxyribonucleic Acid Subtraction and Microarray Endocrinology, April 1, 2003; 144(4): 1257 - 1265. [Abstract] [Full Text] [PDF]
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C. R. Sinars, J. Cheung-Flynn, R. A. Rimerman, J. G. Scammell, D. F. Smith, and J. Clardy Structure of the large FK506-binding protein FKBP51, an Hsp90-binding protein and a component of steroid receptor complexes PNAS, February 4, 2003; 100(3): 868 - 873. [Abstract] [Full Text] [PDF]
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C. G. Tepper, D. L. Boucher, P. E. Ryan, A.-H. Ma, L. Xia, L.-F. Lee, T. G. Pretlow, and H.-J. Kung Characterization of a Novel Androgen Receptor Mutation in a Relapsed CWR22 Prostate Cancer Xenograft and Cell Line Cancer Res., November 15, 2002; 62(22): 6606 - 6614. [Abstract] [Full Text] [PDF]
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P. S. Nelson, N. Clegg, H. Arnold, C. Ferguson, M. Bonham, J. White, L. Hood, and B. Lin The program of androgen-responsive genes in neoplastic prostate epithelium PNAS, September 3, 2002; 99(18): 11890 - 11895. [Abstract] [Full Text] [PDF]
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S. Mousses, L. Bubendorf, U. Wagner, G. Hostetter, J. Kononen, R. Cornelison, N. Goldberger, A. G. Elkahloun, N. Willi, P. Koivisto, et al. Clinical Validation of Candidate Genes Associated with Prostate Cancer Progression in the CWR22 Model System using Tissue Microarrays Cancer Res., March 1, 2002; 62(5): 1256 - 1260. [Abstract] [Full Text] [PDF]
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D. Kim, C. W. Gregory, F. S. French, G. J. Smith, and J. L. Mohler Androgen Receptor Expression and Cellular Proliferation During Transition from Androgen-Dependent to Recurrent Growth after Castration in the CWR22 Prostate Cancer Xenograft Am. J. Pathol., January 1, 2002; 160(1): 219 - 226. [Abstract] [Full Text] [PDF]
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 M. Ladanyi, W. C. Chan, T. J. Triche, and W. L. Gerald Expression Profiling of Human Tumors: The End of Surgical Pathology? J. Mol. Diagn., August 1, 2001; 3(3): 92 - 97. [Full Text] [PDF]
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0.4 is
equivalent to a fold-change of -2.5.