Affiliations of authors: M. Sasaki, Y. Tanaka, G. Perinchery, A. Dharia, I. Kotcherguina, R. Dahiya, Department of Urology, University of California, San Francisco, and Veterans Affairs Medical Center, San Francisco; S. Fujimoto, Department of Obstetrics and Gynecology, School of Medicine, Hokkaido University, Kitaku, Sapporo, Japan.
Correspondence to: Rajvir Dahiya, Ph.D., Urology Research Center (112F), University of California, San Francisco, and Veterans Affairs Medical Center, 4150 Clement St., San Francisco, CA 94121 (e-mail: Urologylab{at}aol.com).
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ABSTRACT |
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INTRODUCTION |
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We hypothesize that the selective expression or inactivation of steroid receptor isoforms is involved in the pathogenesis of prostate cancer, which is initially dependent on steroid hormones. Consequently, we investigated gene expression and methylation status of promoters for three ER isoforms (ER
-A, ER
-B, and ER
-C), ER
, two PR isoforms (PR-A and PR-B), and AR in five prostate cancer cell lines and in pairs of cancerous and normal prostate tissues from 38 patients. We also investigated the effect of demethylation on steroid receptor expression by treating cells with the demethylating reagent 5-aza-2`-deoxycytidine.
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MATERIALS AND METHODS |
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All five human prostatic cancer cell lines available from cell banks, including the American Type Culture Collection (Manassas, VA)LNCaP, PC3, ND1, DUPro, and DU145were used for these experiments and cultured as described previously (12). Cells were cultured with fresh medium containing 5-aza-2`-deoxycytidine (2 µg/mL) on days 1, 3, and 5 and harvested on day 6. Pairs of cancerous and normal prostate tissues from 38 patients were obtained from the Veterans Affairs Medical Center, San Francisco, CA, and the University of California, San Francisco. All cancer tissues were primary adenocarcinomas, not metastatic tumors. From all specimens of cancerous tissue, 5-µm paraffin-embedded sections were cut and stained with hematoxylineosin. Normal and cancerous regions in these sections were then identified by light microscopy at a magnification of x100, marked, and microdissected as described previously (12). From each specimen, a pool of normal tissue and a pool of cancerous tissue were prepared. Immunohistochemical analysis was not carried out because, to our knowledge, there are no antibodies specific for ER isoforms ER
-A, ER
-B, and ER
-C.
RNA Isolation, Reverse TranscriptionPolymerase Chain Reaction, and 5` Rapid Amplification of Complementary DNA Ends
Total RNA was extracted and complementary DNA (cDNA) was synthesized as described by Sasaki et al. (13). For reverse transcriptionpolymerase chain reaction (RTPCR), regions specific for all three ER isoforms, ER
, PR-A, PR-B, and AR were amplified from the cDNA with specific primers (see Table 1
). A
-actin primer that contained one intron of the
-actin gene was used as a positive control; in the presence of contaminating genomic DNA, additional larger actin bands would be amplified and, in its absence, the larger actin bands were not amplified. Negative control reactions without RNA and without reverse transcriptase were also performed. A modified 5` rapid amplification of cDNA ends (5` RACE) method was used to detect the expression of each PR messenger RNA (mRNA) as described previously (10).
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DNA was isolated from paraffin-embedded sections. Microdissection of normal and cancerous prostate tissues was performed as described previously (13). DNA (approximately 100 ng) was denatured with NaOH and treated with sodium bisulfite for 16 hours (Invitrogen Corp., Purchase, NY) as described previously (13). Before sequencing, all cytosine residues were deaminated and converted to thymine residues by sodium bisulfite modification. 5-Methylcytosine residues were not altered by this treatment.
Methylation-Specific PCR
Fig. 1 shows the schematic diagram of the ER
, ER
, AR, and PR genes. Primers and PCR conditions are summarized in Table 1
. The fragment of DNA to be amplified was intentionally small because DNA fragments isolated from paraffin sections are generally less than 300 base pairs long (10). For methylation-specific PCR (MS-PCR), we used one primer set (U) that anneals to unmethylated DNA and another primer set (M) that anneals to methylated DNA. Unmodified DNA was amplified with the W primer set (wild-type), which serves as a positive control for PCR. PCR was performed with approximately 10 ng of cDNA in 20 µL containing 1.5 mM MgCl2, all four deoxyribonucleoside triphosphates (each at 0.8 mM), and 0.5 U of Taq polymerase (Applied Biosystems, Inc., Foster City, CA). The PCR containing 40 cycles of denaturation (94 °C for 30 seconds), annealing (Table 1
), and extension (72 °C for 45 seconds) was followed by a final incubation at 72 °C for 8 minutes. Eight microliters of each PCR product was mixed with 1 µL of 10x loading dye, and the mixture was subjected to electrophoresis on 3% agarose gels at 180 V and ambient temperature. The bands on the gels were visualized by ethidium bromide staining. Only gels with clear bands were used in this study.
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For confirmation of MS-PCR, PCR products were purified by QIAquick PCR Purification Kit (Qiagen, Valencia, CA), and 30 ng of the product was used as a template for sequencing (10). Double-strand sequence analysis was performed with each primer set by use of an ABI 377 Sequencer and Dye Terminator Cycle sequencing kit (Applied Biosystems, Inc.).
Statistical Analyses
Chi-square analysis with the Yate's correction was used to determine differences in methylation status of these steroid receptor isoforms between endometrial cancerous and normal tissues. All statistical tests were two-sided.
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RESULTS |
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We first determined the expression status of steroid receptor isoforms in five prostate cancer cell lines (LNCaP [Fig. 2], PC3, ND1, DU145, and DUPro). In all prostate cancer cell lines, ER
-C was expressed, but ER
-A and ER
-B were not expressed. ER
was not expressed in ND1 and DU145 cells but was weakly expressed in PC3, LNCaP, and DUPro cells. AR was not expressed in ND1, DU145, and DUPro cells but was expressed in PC3 and LNCaP cells. PR-A and PR-B were not expressed in PC3 cells but were expressed in LNCaP, ND1, DU145, and DUPro cells.
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To investigate whether the expression of mRNAs for these steroid isoforms is inactivated by methylation of their promoters, we treated cells with the demethylating agent 5-aza-2`-deoxycytidine and then assessed expression by use of MS-PCR and RTPCR (Fig. 2). Demethylation restored the expression of ER
-A and ER
-B in all cell lines, ER
in ND1 and DU145 cells, and PR-A and PR-B in PC3 cells. Thus, the expression of these steroid receptor isoforms was related to the methylation status of their corresponding promoters.
Pairs of Cancer and Normal Tissues From the Same Patient
To determine whether promoters for these steroid receptor isoforms were methylated in prostate cancer tissues, we analyzed pairs of cancerous and normal tissues from 38 patients (Fig. 3). The promoter for ER
-A was methylated in 36 (95%) of 38 cancerous tissues but was unmethylated in all 38 normal tissues (all P<.001). The promoter for ER
-B was methylated in 35 (92%) of 38 cancerous tissues but was unmethylated in all 38 normal tissues (P<.001). Of interest, the promoter for ER
-C was unmethylated in all 38 pairs of cancerous and normal tissues. The promoter for ER
was methylated in 30 (79%) of 38 cancerous tissues and unmethylated in all normal tissues. The promoter for AR was methylated in three (8%) of 38 cancerous tissues and unmethylated in all normal tissues. Promoters for PR-A and PR-B were unmethylated in all cancerous and normal tissues. We confirmed the results for methylation status of these steroid receptor isoforms by direct DNA sequencing (Fig. 4
). All cytosines were deaminated and converted to thymines after sodium bisulfite modification for these steroid receptors in normal tissues, but 5-methylcytosines were not altered in cancer tissues because methylation protected the residues.
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DISCUSSION |
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Estrogens play an important role in female sexual development and regulation of the menstrual cycle. In males, estrogens affect benign prostatic hyperplasia and the development of prostate cancer (1,23). Loss or inhibition of ER expression in prostate cancer has been documented (23). An inverse association was found between ER expression and histologic grade or pathologic stage of prostate cancer. Estrogen has been used for the treatment of prostate cancer, and its inhibitory effects have been widely acknowledged (1). A low level of ER expression has been associated with a poor response to endocrine therapy (23). Unfortunately, the mechanisms regulating the expression of ER are poorly defined, and no mutation or other gross structural alteration of the ER
gene in prostate cancer tissue has been reported that could clarify the mechanisms (11,24).
The three isoforms of ER are transcribed from three promoters active in a cell- and tissue-specific manner (25). The proximal promoter A was identified as a result of cloning the ER cDNA, followed later by the identification of distal promoters B and C (17). These promoters, which regulate the synthesis of specific transcripts for ER
-A, ER
-B, and ER
-C isoforms, are regulated independently. Although the specific roles of the three ER
isoforms are unclear, the mechanisms regulating their production suggest that the relative levels of ER
-A, ER
-B, and ER
-C in cells are critical for appropriate cellular responses to estrogen.
In this study, we detected the expression of ER-C but not that of ER
-A and ER
-B in all prostate cancer cell lines and tissues tested. In these cell lines, the promoter for ER
-C was unmethylated, whereas those for ER
-A and ER
-B were methylated. In pairs of cancerous and normal prostate tissues, all cancerous tissues had unmethylated ER
-C promoters, but 36 of 38 tumors had methylated ER
-A promoters, and 35 of 38 had methylated ER
-B promoters. Thus, in prostate cancer, the activation status of each ER
isoform appears to be specifically regulated through promoter methylation.
The PR also has two distinct isoforms, PR-A and PR-B, which arise from differential transcription from different promoters within the same gene (4). Because both PR isoforms are detected consistently in both normal and cancerous prostate cells, these receptors should have some role in these cells (26,27). Progesterones have been used to treat certain stages of prostate cancer, although the mechanism of action is unresolved (28). In prostate cancer cell lines, we found that both PR-A and PR-B promoters were methylated and inactivated in PC3 cells but were unmethylated and activated in other cell lines. We found that, in pairs of normal and cancerous prostate tissues, all tissues had unmethylated PR-A and PR-B promoters. Our data suggest that PR-A and PR-B methylation may be a late event in prostate carcinogenesis because PR methylation was observed in only one prostate cancer cell line but not in cancer tissues.
Because the AR protein is a key mediator of growth in the prostate, much research has focused on the role of the AR gene in the development of prostate cancer (29). Although several groups (30) have attempted to find mutations of the AR gene in prostate cancers, only a few mutations have been found, especially in primary prostate cancer. However, in a previous study (13), we reported that the AR promoter was methylated in endometrial cancer during carcinogenesis. When we examined pairs of normal and cancerous prostate tissues, the three prostate cancer tissues that had methylated AR promoters were at a relatively late stage, T3a or T3. Methylation of the AR promoter thus may be a late event in prostate carcinogenesis.
To our knowledge, this is the first report in which the selective methylation and inactivation of promoters for ER-A and ER
-B have been detected in prostate cancers. ER
-C was unmethylated and activated, and ER
-A and ER
-B were methylated and inactivated in all prostate cancer cell lines and in almost all prostate cancer tissues. Our data suggest that each ER
isoform has specific characteristics in prostate carcinogenesis through its methylation and activation status.
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NOTES |
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Manuscript received June 21, 2001; revised December 19, 2001; accepted December 31, 2001.
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