Hypermethylation of the Inhibin
-Subunit Gene in Prostate Carcinoma
Jacqueline F. Schmitt,
Douglas S. Millar,
John S. Pedersen,
Susan L. Clark,
Deon J. Venter,
Mark Frydenberg,
Peter L. Molloy and
Gail P. Risbridger
Monash Institute of Reproduction and Development (J.F.S., M.F.,
G.P.R.), Monash University, Clayton, Victoria 3168, Australia;
Kanematsu Laboratories (D.S.M., J.S.P., S.L.C., G.P.R.), Royal
Prince Alfred Hospital, Camperdown, New South Wales 2050; Melbourne
Pathology (J.S.P.), Collingwood, Victoria 3066; Peter MacCallum Cancer
Institute (D.J.V.), Melbourne 3002; and CSIRO Molecular Science
(P.L.M.), North Ryde, New South Wales 1670, Australia
Address all correspondence and requests for reprints to: Dr. Gail P. Risbridger, Monash Institute of Reproduction and Development, Monash Medical Centre, 246 Clayton Road, Clayton, Victoria 3168, Australia.
 |
ABSTRACT
|
---|
Inhibin is composed of an
- and a ß-subunit. Transgenic
studies assigned a tumor-suppressive role to the inhibin
-subunit,
and in human prostate cancer inhibin
-subunit gene expression was
down-regulated. This study examined the inhibin
-subunit gene
promoter and gene locus to determine whether promoter hypermethylation
or LOH occurred in DNA from prostate cancer. The 5'-untranslated region
of the human inhibin
-subunit gene was sequenced and shown to be
highly homologous to the bovine, rat, and mouse inhibin
-subunit
promoter sequences. A 135-bp region of the human promoter sequence that
continued a cluster of CpG sites was analyzed for hypermethylation.
Significant (P < 0.001) hypermethylation of the
inhibin
-subunit gene promoter occurred in DNA from Gleason pattern
3, 4, and 5 carcinomas compared with nonmalignant tissue samples. A
subset of the carcinomas with a cribriform pattern were unmethylated.
LOH at 2q3236, the chromosomal region harboring the inhibin
-subunit gene, was observed in 42% of prostate carcinomas. These
data provide the first demonstration that promoter hypermethylation and
LOH are associated with the inhibin
-subunit gene and gene locus in
prostate cancer.
 |
INTRODUCTION
|
---|
PROSTATE CARCINOMA IS one of the most
commonly occurring malignancies in Western men and is a leading cause
of carcinoma-related deaths (1, 2, 3). The normal
growth and function of the prostate are dependent on androgens, as well
as growth and differentiation factors, including members of the TGFß
superfamily. Activins are members of the TGFß superfamily and are
composed of homodimers and heterodimers of the
ßA- and ßB-subunits
(4). A unique feature of the activin ß-subunits is their
ability to form dimers with the inhibin
-subunit, resulting in
dimeric inhibin proteins (5, 6, 7). Inhibins and activins are
both implicated in endocrine-related cancers [see review by Risbridger
et al. (8)]. Functional studies using
transgenic mice identified the inhibin
-subunit as a tumor
suppressor gene in the gonads and adrenals (9, 10, 11). Recent
studies identified subsets of patients with ovarian granulosa cell
tumors that showed down- regulation of inhibin
-subunit
expression (12, 13), and in one of these studies there was
a correlation with disease-free survival (12). In the
prostate, inhibin
-subunit protein and mRNA were detected in tissues
from men with benign prostatic hyperplasia (BPH) and regions of
nonmalignant tissue from men with prostate cancer (14, 15). In prostate cancer cells and tissues the expression of
inhibin
-subunit protein and mRNA was down-regulated
(14).
The aim of the present study was to identify the molecular changes to
the inhibin
-subunit gene in prostate carcinoma, i.e.
hypermethylation of the promoter and LOH, because these molecular
changes are often associated with silencing or loss of expression of
tumor suppressor genes. Aberrant methylation of the inhibin
-subunit
gene promoter was reported in human cancers, but hypermethylation
of the promoters of other genes, e.g. the GSTP1 gene and the
ER and PR genes, occurred in prostate cancer (16, 17, 18). The
human inhibin
gene was localized to the q33-q36 region of
chromosome 2 (19), and deletions of 2q were identified in
a number of human tumors, including prostate cancer
(20, 21, 22, 23). Deletions involving the 2q3336 region have not
been reported for prostate carcinoma.
 |
RESULTS
|
---|
Sequence Analysis of the Human 5'-Untranslated Region (UTR) and
Comparison with Bovine, Rat, and Mouse Inhibin
-Subunit Promoter
Regions
The sequence of the 5'-UTR of the human inhibin
-subunit gene
was determined by automated sequencing. Figure 1
shows the alignment of the human
sequence with the promoter sequences of the bovine (24),
rat (25), and mouse (26). Comparison of the
human sequence with the bovine, rat, and mouse sequences revealed
homologies of 73%, 65%, and 70%, respectively. High-sequence
homology (>78% between the human sequence and the other species) was
observed in a region of 297 bp immediately 5' of the ATG translation
start site in the human sequence. Consistent with the mouse, bovine,
and rat promoter regions, the human inhibin
-subunit gene promoter
lacked an obvious TATA box, but conserved the sequence for the
specificity protein (Sp1) upstream promoter element. Inducible promoter
elements included a cAMP response element (CRE) and binding sites for
activator proteins 1, 2, and 3 (AP1, AP2, AP3), corresponding to those
in the rat, mouse, and bovine promoter sequences (24, 25, 26).
A potential Smad binding element (SBE) sequence was identified upstream
of the AP1 site (27).

View larger version (73K):
[in this window]
[in a new window]
|
Figure 1. Sequencing of the 5'-UTR of Inhibin -Subunit and
Comparison with Bovine, Rat, and Mouse Sequences
The sequence of the 5'-UTR of the inhibin -subunit gene was
determined and compared with the published sequences for bovine, rat,
and mouse inhibin -subunit gene promoter regions (panel A).
Transcription factor binding sites were identified and include a CRE,
activating factors 1, 2, and 3 (AP1, AP2, and AP3), recognition
sequences, Sp1, and an SBE. CpG sites are marked with an
arrow, and the sequence examined in subsequent
methylation studies is underlined (A) and presented
schematically in panel B.
|
|
Sequencing of the human inhibin
-subunit gene promoter identified a
cluster of seven CpG sites within a 135-bp region from -149 to -284
of the ATG start (Fig. 1
); four of these CpG sites (CpG1, CpG2, CpG5,
CpG6) were unique to the human sequence. CpG5 differed from the bovine,
rat, and mouse sequences by a T-C change and was within a region found
to bind AP2 in the bovine sequence (28). CpG3, CpG4, and
CpG7 were conserved between species and lay within the Sp1, CRE, and
AP3 binding sites, respectively. CpG4 was within an AP1 site and
adjacent to the SBE site.
Methylation Analysis of the Inhibin
-Subunit Gene in
Microdissected Human Prostate Tissues
Methylation was determined for the seven CpG sites in the 135-bp
region from -149 to -284 of the ATG site in the human inhibin
-subunit gene promoter. An overall comparison of DNA from
nonmalignant and malignant prostate samples showed significant
(P < 0.0001) hypermethylation of the inhibin
-subunit gene promoter in prostate cancer (Fig. 2A
). The mean percent methylation in
malignant tissues was 34.26 ± 2.76%, whereas in nonmalignant
tissues it was 14.78 ± 2.17%.

View larger version (14K):
[in this window]
[in a new window]
|
Figure 2. Hypermethylation of the Inhibin -Subunit Gene
Promoter in Prostate Carcinoma
The percentage methylation of each of the seven CpG sites in the
inhibin -subunit gene promoter was determined from DNA isolated from
microdissected nonmalignant epithelium and BPH and from prostate cancer
(Gleason pattern 35). The DNA was bisulfite treated, the inhibin
gene promoter region was PCR amplified, and the PCR products were
cloned. Methylation status was determined by sequence analysis of
1013 clones for each sample and the percentage methylation was
determined over the seven CpG sites. A, The mean percent methylation
was determined for nonmalignant samples (open bars) and
malignant samples (black bars), and the t
test was used to determine statistical significance. ***,
P < 0.0001. B, The mean percent methylation was
determined for nonmalignant samples (NM/BPH, n = 10) and Gleason
pattern 3 (G3, n = 7) and 4/5 (G4/5, n = 11) prostate
carcinoma, and the t test was used to determine
statistical significance. ***, P < 0.0001. C, The
mean percent methylation was determined for nonmalignant samples
(NM/BPH, n = 10) and prostate carcinomas of cribriform pattern and
Gleason pattern 3 or 4 (PCa Cr, n = 8) and of small-gland pattern
and Gleason pattern 35 (PCa SG, n = 10). The t
test was used to determine statistical significance. ***,
P < 0.0001.
|
|
The methylation levels were compared with tumor grade, and
samples were grouped as Gleason pattern 3 (lower grade) or Gleason
pattern 4 and 5 (high grade) prostate carcinoma. Methylation levels in
Gleason pattern 3 (31.90 ± 3.995%) and Gleason pattern 4/5
(30.74 ± 3.63%) tumors where not significantly different,
although both were significantly (P < 0.001)
hypermethylated relative to nonmalignant samples (14.78 ± 2.17%,
Fig. 2B
). Figure 2C
shows a comparison between methylation in prostate
carcinomas of cribriform-pattern (Gleason pattern 3 and 4) and
small-gland pattern (Gleason pattern 35) prostate carcinomas and
nonmalignant prostate tissues. Small-gland prostate carcinomas
(47.43 ± 3.74%) were significantly hypermethylated
(P < 0.0001) relative to nonmalignant samples
(14.78 ± 2.17%) and prostate carcinomas of cribriform cell
arrangement (10.89 ± 1.36%). There was no difference between
cribriform carcinomas and nonmalignant prostate tissues.
Figure 3A
shows a typical pattern of
methylation at the seven individual inhibin
-subunit gene
promoter CpG sites in nonmalignant and malignant tissue samples. The
percent methylation at each CpG site showed variation (Fig. 3B
). Of the
seven CpG sites, CpG5 was rarely methylated in either nonmalignant or
malignant samples. The percent methylation at the remaining sites
varied, but overall analysis showed significant (P <
0.05) hypermethylation at sites CpG14 and CpG7 in malignant compared
with nonmalignant samples. CpG6 had lower methylation levels, and there
was no significant difference between nonmalignant and malignant tissue
samples.
LOH at 2q in Human Prostate Carcinoma Biopsies
LOH was determined by microsatellite marker PCR analysis using
tissue obtained by microdissection of needle biopsy samples from 14 men
(AN) with prostate carcinoma (Table 1
).
The quality of the DNA and the suitability of the methodology
for the detection of LOH were validated using PCR primers for the 8p21
microsatellite marker (D8S136). LOH at 8p21 was observed in 60% of
prostate carcinoma samples (Table 1
) and is consistent with that
previously reported (29). Using the same DNA samples,
analysis of the 2q chromosome arm revealed that LOH occurred with at
least one microsatellite marker at 2q3236 in 42% of prostate
carcinomas (Table 1
). Patients A, C, F, G, and L were also analyzed for
methylation. Patient A showed both hypermethylation and LOH, patients
C, F, and G showed only hypermethylation, whereas patient L showed only
LOH.
 |
DISCUSSION
|
---|
This study identified molecular changes to the inhibin
-subunit
gene in prostate carcinoma. Sequence analysis of the inhibin
-subunit promoter region identified a number of potential sites for
methylation. Significant hypermethylation of five of seven of these
sites occurred in DNA from samples of Gleason pattern 3, 4, or 5
prostate carcinoma. In addition, 42% of prostate carcinomas showed LOH
at chromosome 2q3236. These results support the hypothesis that the
inhibin
-subunit is tumor suppressive in the prostate and are
consistent with previous studies using transgenic mice that identified
this subunit as a gonadal and adrenal tumor suppressor (9, 10, 11, 30, 31, 32).
Sequence determination for the human inhibin
-subunit gene promoter
was required to identify putative targets for methylation,
i.e. CpG sites. Comparison of the human sequence with the
bovine, mouse, and rat inhibin
-subunit gene promoter sequences
revealed a high degree of sequence homology, particularly over a region
of 297 bp immediately upstream of the ATG translation start site
(26, 28, 33, 34). Studies with the bovine
(24), mouse (26), and rat (28)
showed that this region had promoter activity in vitro.
Several regulatory elements were conserved between the species and
included a CRE site with an overlapping AP1 site, an Sp1 site, an AP2
site, and an AP3 site. A putative SBE site adjacent to the AP1, similar
to that previously reported to occur within the JunB gene
promoter (27), was also identified. Many of these putative
transcription factor-binding sites had a CpG site within their
sequence.
Hypermethylation of CpG islands within the regulatory regions of tumor
suppressor genes is a common aberration in human cancers
(35, 36, 37) and is often associated with gene silencing
(16, 38, 39, 40, 41). The current study focused on a 135-bp region
(within the 297-bp region discussed above) of the inhibin
-subunit
gene promoter that was highly conserved between the species. This
region contained a cluster of seven CpG sites and housed numerous
potential transcription factor binding sites; by analogy to bovine,
rat, and mouse this region is likely to be essential for promoter
activity (24, 25, 26). Overall, this cluster of CpGs was
hypermethylated in prostate cancer samples relative to nonmalignant
epithelium and BPH samples. Hypermethylation of the inhibin
-subunit promoter was observed in lower grade prostate cancer
(Gleason pattern 3) as well as in high-grade (Gleason pattern 4 and 5)
prostate cancer. Whether or not methylation of this subunit is a cause
or a consequence of malignancy remains to be determined, but loss of
the inhibin
-subunit was believed to initiate gonadal and adrenal
tumor formation in inhibin
-subunit null mice (9, 11, 30).
In the pathological assessment of the samples used for microdissection,
a subset of Gleason pattern 3 and 4 tumors showed a cribriform
arrangement of cells. It was noted that hypermethylation of the inhibin
-subunit gene promoter did not occur in these samples. Thus,
molecular analysis of the methylation status of the inhibin
-subunit
gene promoter provides further evidence for a distinction between small
gland carcinomas and cribriform carcinomas of the prostate
(42). The significance of this finding lies in the report
that cribriform carcinomas have poor prognosis and patient outcome
(43, 44, 45, 46).
The degree of methylation varied between the seven CpG sites examined
in the inhibin
-subunit gene promoter. CpG sites 14 and 5 were
significantly hypermethylated in prostate carcinoma, and CpG6 showed
some hypermethylation, but the difference was not significant. CpG5 was
consistently unmethylated in both nonmalignant and malignant samples.
CpG5 lay within a site shown to bind AP2 in the bovine inhibin
-subunit gene promoter sequence (28). AP2 binding at an
AP2 site within the tau gene promoter prevented access of
DNA methyltransferase (47), and therefore binding of AP2
at CpG5 may account for the consistent observation that this site was
unmethylated.
CpG4 lay within a CRE and AP1 transcription factor-binding site. The
CRE is required for cAMP-induced up-regulation of inhibin
-subunit
expression (24, 25, 26). Functional studies demonstrated that
CpG methylation blocked transcription factor binding at CRE sites
(48, 49, 50). The AP1 site was adjacent to an SBE recognition
sequence. Both AP1 and SBE are involved in signaling by members of the
TGFß superfamily (27, 51, 52) and located in close
proximity to each other within the promoters of a number of genes
regulated by TGFß (27, 51, 53, 54). The
identification of adjacent AP1 and SBE binding sites in the inhibin
-subunit gene promoter region suggests that the inhibin
-subunit
gene may be another target for regulation by TGFß or other members of
the TGFß superfamily. Methylation of CpG4 in prostate carcinoma could
block both CRE and AP2 transcription factor binding and alter inhibin
-subunit gene expression. This would be consistent with our previous
report that inhibin
-subunit immunoreactivity was down-regulated
in prostate carcinoma (14).
As well as methylation, this study reported LOH at 2q32-q36 in 42% (6
of 14) of prostate carcinomas. Changes at chromosome 2q occur in
prostate carcinoma (55, 56), although allelic loss
involving this specific region was not previously reported. In other
human tumors, deletions at 2q correlated with disease progression and
outcome. For example, in bladder carcinoma (57) and head
and neck squamous cell carcinoma (58), 2q deletions
correlated with advanced disease and poor prognosis.
The functional consequences of the loss of inhibin
-subunit gene
expression are worth consideration. We previously showed the inhibin
ß-subunits were expressed and localized to tumor cells in specimens
from men with prostate cancer. Therefore, in the absence of inhibin
-subunit, the tumor cells retain the capacity to produce activins
but not inhibins. Activins are generally growth inhibitory and induced
apoptosis in the androgen-dependent cell line LNCaP. However, the
androgen-independent cell line PC3 is resistant to the
growth-inhibitory actions of activins. Resistance to activins, like
resistance to TGFß, commonly occurs in tumor cells, and many of the
components of the activin-signaling pathway are tumor suppressive. It
is tempting to speculate whether or not there is a sequence of changes
to the inhibins/activins that contributes to malignancy in the prostate
gland, starting with the loss of inhibin
-subunit expression and
followed by the onset of resistance to activins.
It is not known whether the inhibin
-subunit null mice develop
prostate cancer or other premalignant changes. Prostate cancer
development requires androgens and is normally slow to develop, with
aggressive androgen-independent tumors generally emerging late in life.
The inhibin
null transgenic mouse models develop gonadal tumors,
and the adrenal tumors emerge only upon castration. In the absence of
androgens, these mice will not develop prostate cancer. Furthermore,
the inhibin null mice died by 14 wk of age, and prostate cancer is a
disease with a long latency period. Hence, it is unlikely that prostate
cancer would emerge in the inhibin
null mice in early adulthood,
but these mice should be examined for premalignant changes such as
prostatic intraepithelial neoplasia.
In summary, the data presented in this study demonstrate that
molecular change to the inhibin
-subunit gene occurs in prostate
carcinoma and provides further evidence to support the hypothesis that
the inhibin
-subunit gene is a tumor suppressor. Further molecular
studies that evaluate a larger patient group with known clinical
outcome would identify whether hypermethylation of the inhibin
-subunit gene promoter and/or LOH at 2q32-q36 provide markers of
survival and disease outcome for prostate carcinoma.
 |
MATERIALS AND METHODS
|
---|
Microdissection of Prostate Carcinoma
Formalin-fixed paraffin-embedded needle biopsies from men with
prostate carcinoma were obtained from the archives of Melbourne
Pathology in accordance with the Institutional Ethical Guidelines.
Several regions of Gleason pattern 3, 4, or 5 prostate carcinoma,
nonmalignant epithelium, and BPH tissue were microdissected from a
total of 24 patient tissues, and the DNA was isolated by proteinase K
digestion (50 mM Tris-HCl, pH 8, 1 mM EDTA,
0.5% Tween, 200 ng/ml proteinase K) at 50 C for 72 h. The
digestions were boiled for 10 min and centrifuged, and the supernatant
was used for PCR to detect LOH or for bisulfite conversion to determine
methylation.
Sequence Determination of the Human Inhibin
-Subunit Gene
5'-UTR
The sequence of the inhibin
-subunit 5'-UTR was determined
from a genomic clone and partial sequence supplied by Dr. David Irving
(Biotech Australia Pty. Ltd., Roseville, NSW, Australia). The sequence
was determined using the BigDye Terminator Cycle Sequencing Kit
(PE Applied Biosystems, Foster City, CA) and the automated
ABI PRISM 377 DNA Sequencer (PE Applied Biosystems).
Detection of Methylation
Methylation was assessed by PCR and sequence analysis of
bisulfite-treated DNA using methodology similar to that previously
described (16, 59). The bisulfite reaction converted
unmethylated cytosines to uracil, whereas methylated cytosines were
unchanged. DNA was isolated from microdissected tissue lysates by
phenol chloroform extraction and ethanol precipitation in the presence
of 10 µg of tRNA. For bisulfite conversion, precipitated DNA was
resuspended in 20 µl PCRTE (10 mM Tris, 0.1
mM EDTA, pH 8.8), 2.2 µl of 3 M NaOH,
and 208 µl of 2 M metabisulfite, and 12 µl 10
mM quinone were added and the reaction incubated at 55 C
for 16 h (59). tRNA (1 µg) was added to each sample
and the DNA was purified using Wizard DNA Clean-Up System desalting
columns (Promega Corp., Madison, WI), eluted in 50 µl of
H2O and incubated with 5.5 µl 3 M
NaOH at 37 C for 15 min. The solutions were neutralized by the addition
of 33.5 µl NH4OAC, pH 7.0, ethanol
precipitated, and resuspended in 10 µl PCRTE. The inhibin
-subunit
5'-UTR region was amplified by nested PCR using primers designed to the
bisulfite converted sequence. Primer sequences 1 (5'-GATAAGAGT-
TTAGATTGGTTTTATTGGTT-3') and 4 (5'-ACACCATAACTCACCTAACCCTACTAATAA-3')
were used for the first round of PCR and primer sequences 3
(5'-ACCCCTTCTACCAA- AATCTACCCAAAA-3') and 7
(5'-GAAGGTGTTGTATGTTTGTATGTGTGAGTT-3') were used for the second round
of PCR. The first round of PCR was performed in 25 µl reactions with
2 µl of bisulfite-converted DNA, PCR buffer (67 mM
Tris/HCl, 16.6 mM ammonium sulfate, 1.7 mg/ml BSA, and 10
mM ß-mercaptoethanol in PCRTE buffer), 1.5 mM
MgCl2, 0.2 mM each of dATP, dCTP,
dGTP, and dTTP, 6 ng/µl of each of the PCR primers 1 and 4, and 1 U
AmpliTaq DNA polymerase (PE Applied Biosystems). PCR
cycles consisted of 95 C for 5 min followed by 5 cycles of 95 C for 1
min, 50 C for 2 min, and 72 C for 3 min and followed by 30 cycles of 95
C for 1 min, 55 C for 2 min, and 72 C for 2 min with a final incubation
step of 72 C for 10 min. A sample of 2 µl from the first PCR was
amplified in a 25 µl reaction as above except that primers 3 and 7
were used. PCR cycling conditions were as for the first reaction, with
the exception that the annealing temperature was increased to 60 C. PCR
products were gel purified, ligated into the pCR 2.1 cloning vector,
and cloned using the TA Cloning Kit according to the manufacturers
instructions (Invitrogen, Carlsbad, CA). For each PCR,
1013 clones were sequenced and the percentage methylation at each of
the seven CpGs was determined. Overall percent methylation for each
sample was determined as the mean of the percent methylation at the
seven individual CpG sites.
LOH Analysis
LOH was determined using microsatellite markers on 2q32-q33
(D2S389), 2q33-q36 (D2S128), and 8p21 (D8S136) and the sequences from
the genome database (http://gdbwww. gdb.org/gdb). Oligonucelotide
primer sequences for each microsatellite marker were: D2S389
5'-TAAAGCCTAGTGG- AAGATCATC-3', 5'-GCTGAGTTAACAGTTATCAACAATT-3'; D2S128
5'-AAACTGAGATTTGTCTAAGGGG-3', 5'-AGCCAGGAATTTTTGCTATT-3' and D8S136
5'-CCTGAGCCC AAAGAGGAGAATAA-3', 5'-TGCTCTGTTTCCACACCGAA- GC-3'. PCR was
performed in 15-µl reactions consisting of 1 µl of tissue lysate
prepared as above, PCR buffer (10 mM Tris-HCl, pH 8.3 and
50 mM KCl), 2.5 mM MgCl2,
0.2 mM each of dATP, dCTP, dGTP, and dTTP, 0.45 µg
forward primer, 0.5 µg reverse primer, 0.05 µg
32P-labeled forward primer, and 0.3 U AmpliTaq
Gold (PE Applied Biosystems). PCR using the 8p12 primers
also included 5% dimethylsulfoxide. PCR cycles consisted of 95 C for 5
min followed by 10 cycles of 95 C for 60 sec, 60 C for 90 sec, and 72 C
for 90 sec followed by 25 cycles in which the annealing temperature was
reduced to 55 C for 90 sec. PCR products were detected by 6% PAGE and
autoradiography. For each patient, several regions of microdissected
tissue were examined individually for LOH. The regions were selected to
include at least two regions of nonmalignant epithelium or stroma and
at least three regions of prostate carcinoma. LOH for a patient was
deemed to be present if at least two regions of carcinoma showed
allelic loss.
 |
ACKNOWLEDGMENTS
|
---|
We thank Biotech Australia Pty. Ltd. for a human inhibin
-subunit genomic clone, Mr. Simon Bardill and the Wellcome Trust
Sequencing Centre for DNA sequence analyses, and Dr. Melissa Southey
and Mr. Leigh Batten (Peter MacCallum Cancer Institute) for helpful
technical advice.
 |
FOOTNOTES
|
---|
This work was supported by the National Health and Medical Research
Council of Australia.
Abbreviations: AP1, -2, -3, Activator proteins 1, 2, 3; BPH,
benign prostatic hyperplasia; CRE, cAMP response element; PCRTE, 10
nM Tris, 0.1 mM EDTA, pH 8.8; SBE, Smad-binding
element; Sp1, specificity protein 1; UTR, untranslated region.
Received for publication August 13, 2001.
Accepted for publication October 10, 2001.
 |
REFERENCES
|
---|
-
Landis SH, Murray T, Bolden S, Wingo PA 1998 Cancer
statistics, 1998 [published errata appear in CA Cancer J Clin
1998 May-Jun;48(3):192 and 1998 Nov-Dec;48(6):329]. CA Cancer J
Clin 48:629[Abstract/Free Full Text]
-
Landis SH, Murray T, Bolden S, Wingo PA 1999 Cancer
statistics, 1999. CA Cancer J Clin 49:831[Abstract/Free Full Text]
-
Pisani P, Parkin DM, Bray F, Ferlay J 1999 Estimates of the
worldwide mortality from 25 cancers in 1990 [published erratum appears
in Int J Cancer 1999 83:870873]. Int J Cancer 83:1829
-
Mason AJ, Niall HD, Seeburg PH 1986 Structure of two human
ovarian inhibins. Biochem Biophys Res Commun 135:957964[Medline]
-
Rivier C, Rivier J, Vale W 1986 Inhibin-mediated feedback
control of follicle-stimulating hormone secretion in the female rat.
Science 234:205208[Medline]
-
Robertson DM, de Vos FL, Foulds LM, McLachlan RI, Burger HG,
Morgan FJ, Hearn MT, de Kretser DM 1986 Isolation of a 31 kDa form of
inhibin from bovine follicular fluid. Mol Cell Endocrinol 44:271277[CrossRef][Medline]
-
Ling N, Ying SY, Ueno N, Shimasaki S, Esch F, Hotta M,
Guillemin R 1986 Pituitary FSH is released by a heterodimer of the
ß-subunits from the two forms of inhibin. Nature 321:779782[Medline]
-
Risbridger GP, Schmitt JF, Robertson DR 2001 Activins and
inhibins and activins in endocrine and other tumors. Endocr Rev 22:836858[Abstract/Free Full Text]
-
Matzuk MM, Finegold MJ, Su JG, Hsueh AJ, Bradley A 1992
-Inhibin is a tumour-suppressor gene with gonadal specificity in
mice. Nature 360:313319[CrossRef][Medline]
-
Matzuk MM, Bradley A 1994 Identification and analysis of tumor
suppressor genes using transgenic mouse models. Semin Cancer Biol 5:3745[Medline]
-
Matzuk MM, Finegold MJ, Mather JP, Krummen L, Lu H, Bradley A 1994 Development of cancer cachexia-like syndrome and adrenal tumors in
inhibin-deficient mice. Proc Natl Acad Sci USA 91:88178821[Abstract]
-
Ala-Fossi SL, Aine R, Punnonen R, Maenpaa J 2000 Is potential
to produce inhibins related to prognosis in ovarian granulosa cell
tumors? Eur J Gynaecol Oncol 21:187189[Medline]
-
Gebhart JB, Roche PC, Keeney GL, Lesnick TG, Podratz KC 2000 Assessment of inhibin and p53 in granulosa cell tumors of the ovary.
Gynecol Oncol 77:232236[CrossRef][Medline]
-
Mellor SL, Richards MG, Pedersen JS, Robertson DM, Risbridger
GP 1998 Loss of the expression and localization of inhibin
-subunit
in high grade prostate cancer. J Clin Endocrinol Metab 83:969975[Abstract/Free Full Text]
-
Thomas TZ, Chapman SM, Hong W, Gurusingfhe C, Mellor SL,
Fletcher R, Pedersen J, Risbridger GP 1998 Inhibins, activins, and
follistatins: expression of mRNAs and cellular localization in tissues
from men with benign prostatic hyperplasia. Prostate 34:3443[CrossRef][Medline]
-
Millar DS, Ow KK, Paul CL, Russell PJ, Molloy PL, Clark SJ 1999 Detailed methylation analysis of the glutathione S-transferase pi
(GSTP1) gene in prostate cancer. Oncogene 18:13131324[CrossRef][Medline]
-
Li LC, Chui R, Nakajima K, Oh BR, Au HC, Dahiya R 2000 Frequent methylation of estrogen receptor in prostate cancer:
correlation with tumor progression. Cancer Res 60:702706[Abstract/Free Full Text]
-
Lau KM, LaSpina M, Long J, Ho SM 2000 Expression of estrogen
receptor (ER)-
and ER-ß in normal and malignant prostatic
epithelial cells: regulation by methylation and involvement in growth
regulation. Cancer Res 60:31753182[Abstract/Free Full Text]
-
Barton DE, Yang-Feng TL, Mason AJ, Seeburg PH, Francke U 1989 Mapping of genes for inhibin subunits
, ßA, and ßB on human and
mouse chromosomes and studies of jsd mice. Genomics 5:9199[CrossRef][Medline]
-
Zhao J, Speel EJ, Muletta-Feurer S, Rutimann K, Saremaslani P,
Roth J, Heitz PU, Komminoth P 1999 Analysis of genomic alterations in
sporadic adrenocortical lesions: gain of chromosome 17 is an early
event in adrenocortical tumorigenesis. Am J Pathol 155:10391045[Abstract/Free Full Text]
-
Saretzki G, Hoffmann U, Rohlke P, Psille R, Gaigal T, Keller
G, Hofler H, Loning T, Petersen I, Dietel M 1997 Identification of
allelic losses in benign, borderline, and invasive epithelial ovarian
tumors and correlation with clinical outcome. Cancer 80:12411249[CrossRef][Medline]
-
Nagai H, Pineau P, Tiollais P, Buendia MA, Dejean A 1997 Comprehensive allelotyping of human hepatocellular carcinoma. Oncogene 14:29272933[CrossRef][Medline]
-
Watson RH, Roy Jr WJ, Davis M, Hitchcock A, Campbell IG 1997 Loss of heterozygosity at the
-inhibin locus on chromosome 2q is not
a feature of human granulosa cell tumors. Gynecol Oncol 65:387390[CrossRef][Medline]
-
Ungefroren H, Wathes DC, Walther N, Ivell R 1994 Structure of
the
-inhibin gene and its regulation in the ruminant gonad: inverse
relationship to oxytocin gene expression. Biol Reprod 50:401412[Abstract]
-
Pei L, Dodson R, Schoderbek WE, Maurer RA, Mayo KE 1991 Regulation of the
inhibin gene by cyclic adenosine
3',5'-monophosphate after transfection into rat granulosa cells. Mol
Endocrinol 5:521534[Abstract]
-
Su JG, Hsueh AJ 1992 Characterization of mouse inhibin
gene and its promoter. Biochem Biophys Res Commun 186:293300[Medline]
-
Jonk LJ, Itoh S, Heldin CH, ten Dijke P, Kruijer W 1998 Identification and functional characterization of a Smad binding
element (SBE) in the JunB promoter that acts as a transforming growth
factor-ß, activin, and bone morphogenetic protein-inducible enhancer.
J Biol Chem 273:2114521152[Abstract/Free Full Text]
-
Thompson DA, Cronin CN, Martin F 1994 Genomic cloning and
sequence analyses of the bovine
-, ßA- and ßB-inhibin/activin
genes. Identification of transcription factor AP-2-binding sites in the
5'-flanking regions by DNase I footprinting. Eur J Biochem 226:751764[Abstract]
-
Vocke CD, Pozzatti RO, Bostwick DG, Florence CD, Jennings SB,
Strup SE, Duray PH, Liotta LA, Emmert-Buck MR, Linehan WM 1996 Analysis
of 99 microdissected prostate carcinomas reveals a high frequency of
allelic loss on chromosome 8p1221. Cancer Res 56:24112416[Abstract]
-
Matzuk MM, Kumar TR, Shou W, Coerver KA, Lau AL, Behringer RR,
Finegold MJ 1996 Transgenic models to study the roles of inhibins and
activins in reproduction, oncogenesis, and development. Recent Prog
Horm Res 51:123154[Medline]
-
Cipriano SC, Chen L, Kumar TR, Matzuk MM 2000 Follistatin is a
modulator of gonadal tumor progression and the activin-induced wasting
syndrome in inhibin-deficient mice. Endocrinology 141:23192327[Abstract/Free Full Text]
-
Lopez P, Vidal F, Rassoulzadegan M, Cuzin F 1999 A role of
inhibin as a tumor suppressor in Sertoli cells: down-regulation upon
aging and repression by a viral oncogene. Oncogene 18:73037309[CrossRef][Medline]
-
Feng ZM, Li YP, Chen CL 1989 Analysis of the 5'-flanking
regions of rat inhibin
- and ß-B-subunit genes suggests two
different regulatory mechanisms. Mol Endocrinol 3:19141925[Abstract]
-
Albiston AL, Lock P, Burger HG, Krozowski ZS 1990 Cloning and
characterization of the rat
-inhibin gene. Mol Cell Endocrinol 68:121128[CrossRef][Medline]
-
Baylin SB, Herman JG 2000 DNA hypermethylation in
tumorigenesis: epigenetics joins genetics. Trends Genet 16:168174[CrossRef][Medline]
-
Baylin SB, Herman JG, Graff JR, Vertino PM, Issa JP 1998 Alterations in DNA methylation: a fundamental aspect of neoplasia. Adv
Cancer Res 72:141196[Medline]
-
Melki JR, Vincent PC, Clark SJ 1999 Concurrent DNA
hypermethylation of multiple genes in acute myeloid leukemia. Cancer
Res 59:37303740[Abstract/Free Full Text]
-
Lee WH, Morton RA, Epstein JI, Brooks JD, Campbell PA, Bova
GS, Hsieh WS, Isaacs WB, Nelson WG 1994 Cytidine methylation of
regulatory sequences near the pi-class glutathione
S-transferase gene accompanies human prostatic
carcinogenesis. Proc Natl Acad Sci USA 91:1173311737[Abstract/Free Full Text]
-
Nelson JB, Chan-Tack K, Hedican SP, Magnuson SR, Opgenorth TJ,
Bova GS, Simons JW 1996 Endothelin-1 production and decreased
endothelin B receptor expression in advanced prostate cancer. Cancer
Res 56:663668[Abstract]
-
Nelson JB, Lee WH, Nguyen SH, Jarrard DF, Brooks JD, Magnuson
SR, Opgenorth TJ, Nelson WG, Bova GS 1997 Methylation of the 5' CpG
island of the endothelin B receptor gene is common in human prostate
cancer. Cancer Res 57:3537[Abstract]
-
Graff JR, Herman JG, Lapidus RG, Chopra H, Xu R, Jarrard DF,
Isaacs WB, Pitha PM, Davidson NE, Baylin SB 1995 E-cadherin expression
is silenced by DNA hypermethylation in human breast and prostate
carcinomas. Cancer Res 55:51955199[Abstract]
-
McNeal JE, Reese JH, Redwine EA, Freiha FS, Stamey TA 1986 Cribriform adenocarcinoma of the prostate. Cancer 58:17141719[Medline]
-
Cohen RJ, McNeal JE, Baillie T 2000 Patterns of
differentiation and proliferation in intraductal carcinoma of the
prostate: significance for cancer progression. Prostate 43:1119[Medline]
-
McNeal JE, Yemoto CE 1996 Spread of adenocarcinoma within
prostatic ducts and acini. Morphologic and clinical correlations.
Am J Surg Pathol 20:802814[CrossRef][Medline]
-
Rubin MA, de La Taille A, Bagiella E, Olsson CA, OToole KM 1998 Cribriform carcinoma of the prostate and cribriform prostatic
intraepithelial neoplasia: incidence and clinical implications. Am
J Surg Pathol 22:840848[CrossRef][Medline]
-
Wilcox G, Soh S, Chakraborty S, Scardino PT, Wheeler TM 1998 Patterns of high-grade prostatic intraepithelial neoplasia associated
with clinically aggressive prostate cancer. Hum Pathol 29:11191123[Medline]
-
Tohgi H, Utsugisawa K, Nagane Y, Yoshimura M, Ukitsu M, Genda
Y 1999 The methylation status of cytosines in a
gene promoter
region alters with age to downregulate transcriptional activity in
human cerebral cortex. Neurosci Lett 275:8992[CrossRef][Medline]
-
Iguchi-Ariga SM, Schaffner W 1989 CpG methylation of the
cAMP-responsive enhancer/promoter sequence TGACGTCA abolishes specific
factor binding as well as transcriptional activation. Genes Dev 3:612619[Abstract]
-
Iannello RC, Gould JA, Young JC, Giudice A, Medcalf R, Kola I 2000 Methylation-dependent silencing of the testis-specific Pdha-2
basal promoter occurs through selective targeting of an activating
transcription factor/cAMP-responsive element-binding site. J Biol
Chem 275:1960319608[Abstract/Free Full Text]
-
Iannello RC, Young J, Sumarsono S, Tymms MJ, Dahl HH, Gould J,
Hedger M, Kola I 1997 Regulation of Pdha-2 expression is mediated by
proximal promoter sequences and CpG methylation. Mol Cell Biol 17:612619[Abstract]
-
Mulder KM 2000 Role of Ras and Mapks in TGFß signaling.
Cytokine Growth Factor Rev 11:2335[CrossRef][Medline]
-
Kim SJ, Angel P, Lafyatis R, Hattori K, Kim KY, Sporn MB,
Karin M, Roberts AB 1990 Autoinduction of transforming growth factor
ß1 is mediated by the AP-1 complex. Mol Cell Biol 10:14921497[Medline]
-
Greenwel P, Inagaki Y, Hu W, Walsh M, Ramirez F 1997 Sp1 is
required for the early response of
2(1) collagen to transforming
growth factor-ß1. J Biol Chem 272:1973819745[Abstract/Free Full Text]
-
Yingling JM, Datto MB, Wong C, Frederick JP, Liberati NT, Wang
XF 1997 Tumor suppressor Smad4 is a transforming growth factor
ß-inducible DNA binding protein. Mol Cell Biol 17:70197028[Abstract]
-
Alers JC, Rochat J, Krijtenburg PJ, Hop WC, Kranse R,
Rosenberg C, Tanke HJ, Schroder FH, van Dekken H 2000 Identification of
genetic markers for prostatic cancer progression. Lab Invest 80:931942[Medline]
-
Suarez BK, Lin J, Burmester JK, Broman KW, Weber JL, Banerjee
TK, Goddard KA, Witte JS, Elston RC, Catalona WJ 2000 A genome screen
of multiplex sibships with prostate cancer. Am J Hum Genet 66:933944[CrossRef][Medline]
-
Zhao J, Richter J, Wagner U, Roth B, Schraml P, Zellweger T,
Ackermann D, Schmid U, Moch H, Mihatsch MJ, Gasser TC, Sauter G 1999 Chromosomal imbalances in noninvasive papillary bladder neoplasms
(pTa). Cancer Res 59:46584661[Abstract/Free Full Text]
-
Ransom DT, Barnett TC, Bot J, de Boer B, Metcalf C, Davidson
JA, Turbett GR 1998 Loss of heterozygosity on chromosome 2q: possibly a
poor prognostic factor in head and neck cancer. Head Neck 20:404410[CrossRef][Medline]
-
Clark SJ, Harrison J, Paul CL, Frommer M 1994 High sensitivity
mapping of methylated cytosines. Nucleic Acids Res 22:29902997[Abstract]