Hypermethylation of the Inhibin {alpha}-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
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Inhibin is composed of an {alpha}- and a ß-subunit. Transgenic studies assigned a tumor-suppressive role to the inhibin {alpha}-subunit, and in human prostate cancer inhibin {alpha}-subunit gene expression was down-regulated. This study examined the inhibin {alpha}-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 {alpha}-subunit gene was sequenced and shown to be highly homologous to the bovine, rat, and mouse inhibin {alpha}-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 {alpha}-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 2q32–36, the chromosomal region harboring the inhibin {alpha}-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 {alpha}-subunit gene and gene locus in prostate cancer.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
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 {alpha}-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 {alpha}-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 {alpha}-subunit expression (12, 13), and in one of these studies there was a correlation with disease-free survival (12). In the prostate, inhibin {alpha}-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 {alpha}-subunit protein and mRNA was down-regulated (14).

The aim of the present study was to identify the molecular changes to the inhibin {alpha}-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 {alpha}-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 {alpha} 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 2q33–36 region have not been reported for prostate carcinoma.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Sequence Analysis of the Human 5'-Untranslated Region (UTR) and Comparison with Bovine, Rat, and Mouse Inhibin {alpha}-Subunit Promoter Regions
The sequence of the 5'-UTR of the human inhibin {alpha}-subunit gene was determined by automated sequencing. Figure 1Go 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 {alpha}-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).



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Figure 1. Sequencing of the 5'-UTR of Inhibin {alpha}-Subunit and Comparison with Bovine, Rat, and Mouse Sequences

The sequence of the 5'-UTR of the inhibin {alpha}-subunit gene was determined and compared with the published sequences for bovine, rat, and mouse inhibin {alpha}-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 {alpha}-subunit gene promoter identified a cluster of seven CpG sites within a 135-bp region from -149 to -284 of the ATG start (Fig. 1Go); 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 {alpha}-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 {alpha}-subunit gene promoter. An overall comparison of DNA from nonmalignant and malignant prostate samples showed significant (P < 0.0001) hypermethylation of the inhibin {alpha}-subunit gene promoter in prostate cancer (Fig. 2AGo). The mean percent methylation in malignant tissues was 34.26 ± 2.76%, whereas in nonmalignant tissues it was 14.78 ± 2.17%.



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Figure 2. Hypermethylation of the Inhibin {alpha}-Subunit Gene Promoter in Prostate Carcinoma

The percentage methylation of each of the seven CpG sites in the inhibin {alpha}-subunit gene promoter was determined from DNA isolated from microdissected nonmalignant epithelium and BPH and from prostate cancer (Gleason pattern 3–5). The DNA was bisulfite treated, the inhibin {alpha} gene promoter region was PCR amplified, and the PCR products were cloned. Methylation status was determined by sequence analysis of 10–13 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 3–5 (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. 2BGo). Figure 2CGo shows a comparison between methylation in prostate carcinomas of cribriform-pattern (Gleason pattern 3 and 4) and small-gland pattern (Gleason pattern 3–5) 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 3AGo shows a typical pattern of methylation at the seven individual inhibin {alpha}-subunit gene promoter CpG sites in nonmalignant and malignant tissue samples. The percent methylation at each CpG site showed variation (Fig. 3BGo). 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 CpG1–4 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.



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Figure 3. Methylation of the Seven CpG Sites in the Inhibin {alpha}-Subunit Gene Promoter in Prostate Carcinoma

The percentage methylation of each of the seven CpG sites in the inhibin {alpha}-subunit gene promoter was determined from DNA isolated from microdissected nonmalignant epithelium and BPH and from prostate cancer (Gleason pattern 3–5). The DNA was bisulfite treated, the inhibin {alpha} gene promoter region was PCR amplified, and the PCR products were cloned. Methylation status was determined by sequence analysis of 10–13 clones for each sample. The percentage methylation was determined for each CpG site and panel A shows typical examples of the percentage methylation at each CpG site represented as a pie charts with black shading indicative of methylation. Panel B shows the mean percent methylation was determined for nonmalignant samples (n = 10, open bars) and malignant samples (n = 18, black bars). The t test was used to determine statistical significance. *, P < 0.05.

 
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 (A–N) with prostate carcinoma (Table 1Go). 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 1Go) 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 2q32–36 in 42% of prostate carcinomas (Table 1Go). 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.


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Table 1. LOH in Prostate Carcinoma

 

    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
This study identified molecular changes to the inhibin {alpha}-subunit gene in prostate carcinoma. Sequence analysis of the inhibin {alpha}-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 2q32–36. These results support the hypothesis that the inhibin {alpha}-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 {alpha}-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 {alpha}-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 {alpha}-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 {alpha}-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 {alpha}-subunit was believed to initiate gonadal and adrenal tumor formation in inhibin {alpha}-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 {alpha}-subunit gene promoter did not occur in these samples. Thus, molecular analysis of the methylation status of the inhibin {alpha}-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 {alpha}-subunit gene promoter. CpG sites 1–4 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 {alpha}-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 {alpha}-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 {alpha}-subunit gene promoter region suggests that the inhibin {alpha}-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 {alpha}-subunit gene expression. This would be consistent with our previous report that inhibin {alpha}-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 {alpha}-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 {alpha}-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 {alpha}-subunit expression and followed by the onset of resistance to activins.

It is not known whether the inhibin {alpha}-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 {alpha} 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 {alpha} 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 {alpha}-subunit gene occurs in prostate carcinoma and provides further evidence to support the hypothesis that the inhibin {alpha}-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 {alpha}-subunit gene promoter and/or LOH at 2q32-q36 provide markers of survival and disease outcome for prostate carcinoma.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
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 {alpha}-Subunit Gene 5'-UTR
The sequence of the inhibin {alpha}-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 {alpha}-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 manufacturer’s instructions (Invitrogen, Carlsbad, CA). For each PCR, 10–13 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 {alpha}-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.


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 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 

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