p21WAF1/CIP1 gene is inactivated in metastatic prostatic cancer cell lines by promoter methylation

INTRODUCTION: p21WAF1/CIP1 may act as a tumour suppressor gene (TSG) and loss of the p21WAF1/CIP1 gene has been reported in several solid tumours. The aim of this study was to see whether p21WAF1/CIP1 was expressed in metastatic prostate cancer cell lines and to determine if there was methylation of the p21WAF1/CIP1 promoter. METHOD: PC3, LNCaP and DU145 metastatic prostate cancer cell lines, 1542NP normal prostate, and RD rhabdomyosarcoma cell lines were cultured in the demethylating agent 5-Aza-2 deoxycytidine (5-Aza-CdR). p21WAF1/CIP1 mRNA expression was analysed by RT-PCR. DNA from untreated cell lines was modified with sodium bisulphite and promoter sequencing was performed. RESULTS: p21WAF1/CIP1 was expressed at low or undetectable levels in metastatic prostate cancer cell lines but expression was reactivated by treatment with 5-Aza-CdR. Sequence analysis of the promoter region revealed several sites of methylation at the 5' end of a CpG island in the PC3, LNCaP and DU145 cell line DNA but not in the normal prostate control DNA. Most notably the Sis-inducible element (SEI)-1-a STAT1-binding site, was methylated. CONCLUSIONS: In this study, we show that p21WAF1/CIP1 expression in metastatic prostate cancer cell lines is enhanced as a result of demethylation of the DNA. Furthermore, several cytosine residues in the promoter region are methylated, including critical binding sites. The inhibition of the STAT1-signalling pathway by methylation of the promoter may inactivate the p21WAF1/CIP1 TSG in prostate cancer.     

Loss of heterozygosity and lack of mutations of the XPG/ERCC5 DNA repair gene at 13q33 in prostate cancer.

BACKGROUND: Three regions of chromosome 13 were previously identified for having loss of heterozygosity (LOH) in human prostate cancer. One of them, at 13q33, was defined by LOH at markers D13S158 and D13S280. The XPG/ERCC5 gene, a DNA repair gene that when mutated in the germline leads to xeroderma pigmentosum, has been mapped to 13q33, within one megabase of D13S158 and D13S280. This paper describes LOH and mutational analysis of the XPG gene in human prostate cancers, in order to determine whether the XPG gene is involved in the development of prostate cancer. METHODS: LOH of the XPG gene was analyzed in 40 primary prostate cancers and 14 metastases by using the microsatellite assay, and its mutations were examined in 5 cell lines, 14 metastases, and 8 tumors with LOH at 13q33 by using the single-strand conformation polymorphism (SSCP)-direct DNA sequencing analysis. RESULTS: Four of the 29 (14%) informative primary tumors and 4 of 8 (50%) metastases showed LOH for the XPG gene. Analysis of the 8 tumors with LOH at the 13q33 region, 14 metastases, and 5 cell lines of prostate cancer revealed two polymorphisms but no mutation of the gene. The polymorphism in exon 2 did not change the amino-acid sequence of the XPG protein, but the exon 15 polymorphism altered codon 1104 from histidine to aspartic acid. The two polymorphisms also occurred in individuals without prostate cancer. CONCLUSIONS: LOH at XPG in prostate cancer supports the conclusion that the 13q33 region contains a gene important in the development of prostate cancer, while lack of mutations of the gene suggests that XPG is not the target gene involved.     

Hypermethylation of the caveolin-1 gene promoter in prostate cancer

BACKGROUND: Hypermethylation of CpG islands in the promoter regions of tumor suppressor genes is one mechanism of tumorigenesis. Caveolin-1 (Cav-1), a gene coding for the structural component of cellular caveolae, is involved in cell signaling and has been proposed to be a tumor suppressor gene in several malignancies. This gene maps to 7q31.1, a site known to be deleted in some prostate tumors. We chose to examine the methylation status of the promoter region of Cav-1 to determine whether this gene could function as a tumor suppressor in prostate cancer METHODS: Genomic DNA from both tumor and normal prostate epithelial cells was obtained from paraffin-embedded prostate sections by laser capture microdissection (LCM). The methylation status of 24 CpG sites at the 5' promoter region of Cav-1 was analyzed by bisulfite-direct-sequencing after amplification by PCR using primers specific for bisulfate modified DNA. Immunohistochemistry staining with a cav-1-specific antibody was also performed to evaluate the expression of the gene RESULTS: Twenty of the 22 (90.9%) informative cases showed promoter hypermethylation in the tumor cell population when compared with adjacent normal prostate cells with an average Methylation Index (potential frequency of total possible methylated Cs) from tumor cells equal to 0.426 vs. 0.186 for normal cells (P = 0.001). While no association with Gleason grade was found, overall increased methylation correlated with PSA failure (P = 0.016), suggestive of clinical recurrence. Elevated immunoreactivity with a Cav-1 antibody was observed in tumor cells from 7 of 26 prostate samples tested; this was associated with a Gleason score but not correlated with PSA failure or Methylation Index CONCLUSIONS: CpG sites at the 5' promoter of Cav-1 are more methylated in tumor than in adjacent normal prostate cells. Hypermethylation of the Cav-1 promoter supports the notion that Cav-1 may function as a tumor suppressor gene in prostate cancer and evidence is presented suggesting that methylation status of this gene is not only a marker for cancer but also may be predictive of outcome.     

Methylation and mutational analysis of p27(kip1) in prostate carcinoma

BACKGROUND: We have previously identified 12p12-13 as a region of frequent genetic loss in prostate carcinoma. A candidate tumor suppressor gene at this locus is the cyclin dependent kinase inhibitor p27(kip1), which has been implicated as a marker of aggressive prostate carcinoma. Herein, we examine metastatic prostate tumors, xenografts, and cell lines for gene inactivation via mutational inactivation or promoter hypermethylation. METHODS: Mutation analysis was performed on metastatic prostate tumors of 18 patients, eight prostate carcinoma cell lines, and 18 xenografts by PCR amplification of the entire open reading frame of p27(kip1). PCR products were sequenced directly using internal primers. Methylation analysis was performed on four cell lines and nine xenografts using direct sequencing of cloned PCR products of bisulfite treated DNA. Presence of a CpG was consistent with methylation of that cytosine in the original sample. RESULTS: With the exception of the previously reported homozygous deletion, no additional mutations were identified. Methylated CpG residues were identified in three xenografts (LuCAP23, LuCAP35, and PC82) and the methylated residues clustered at six sites; the cytosines 69, 149, 191, 286, 349, and 487 base pairs 5' of the ATG start codon. However, no sample demonstrated promotor methylation in all sequenced clones and the number of methylated base pairs ranged from seven to three, not the level usually associated with gene silencing. CONCLUSIONS: Mutational inactivation of p27(kip1) is a rare event in metastatic prostate carcinoma. While CpG methylation does occur, it is an infrequent event and does not appear to be the mechanism of p27(kip1) down regulation in prostate carcinoma.     

结直肠癌侵袭性与脾酪氨酸激酶基因Syk启动子甲基化的关系

目的　探讨脾酪氨酸激酶 (Syk)基因启动子甲基化与结直肠癌侵袭转移之间的关系。方法　采用巢式双重甲基化特异性聚合酶链反应 (MSP)和逆转录 聚合酶链反应 (RT PCR)的方法检测Syk基因在 12 0例结直肠癌肿瘤组织、癌旁正常组织中的甲基化和表达情况。 结果　 12 0例结直肠癌患者中 ,48例未检测到SykmRNA的表达 ,而癌旁正常组织均有表达。癌旁正常组织未检测到Syk基因启动子甲基化 ,3 7例肿瘤组织发生Syk基因启动子甲基化(3 0 .8% ) ,癌组织Syk基因启动子甲基化率显著增高 (P <0 .0 0 0 5 )。在淋巴结转移的 5 6例中 ,2 4例发生Syk基因启动子甲基化 ,而无淋巴结转移的 64例中 ,13例发生甲基化 ,有淋巴结转移的Syk基因启动子甲基化发生率显著高于无淋巴结转移组 (P =0 .0 0 8)。发生甲基化的肿瘤组织中 ,均无SykmRNA的表达。结论　结直肠癌中 ,SYK基因启动子过甲基化导致其mRNA的失表达 ,从而引起结直肠癌的侵袭性增强 ,它可能是结直肠癌发生侵袭转移的又一种机制。     

前列腺癌CRMP4基因启动子甲基化研究

目的分析前列腺癌CRMP4基因启动子区CpG岛甲基化状况及其与CRMP4表达下调的关系,设计较为理想的诊断前列腺癌早期转移的分子诊断探针。方法去甲基化药物5-aza-dC分别以0.5μM、5μM及12.5μM三种不同浓度处理前列腺癌细胞,WesternBlot检测药物处理前后CRMP4表达的变化。硫化测序PCR寻找前列腺癌CRMP4基因启动子区甲基化位点,根据测序结果设计并筛选用于诊断前列腺癌早期转移的甲基化特异性PCR引物。结果去甲基化药物处理后,CRMP4蛋白的表达可以重新上调,并且随着使用的5-aza-dC浓度增加,其表达量也逐渐增加。转移性前列腺癌CRMP4基因启动子区存在2个连续甲基化区域,共有10个CpG位点(-848,-841,-690,-680,-678,-674,-671,-665,-660,-658)其甲基化率极高,而局限性前列腺癌及非肿瘤组织这些CpG位点则未甲基化或有偶发的甲基化。筛选出较为理想的诊断前列腺癌早期转移的甲基化特异性PCR引物,多数转移性前列腺癌及局限性进展期前列腺癌均可扩增出M条带。结论转移性前列腺癌CRMP4表达下调与其基因启动子区CpG岛甲基化相关,筛选出较为理想的诊断前列腺癌早期转移的分子诊断探针,有望为临床早期诊断前列腺癌转移提供新的分子生物学诊断方法。     

前列腺癌细胞株E-钙粘连素基因启动子甲基化与其表达

目的：了解Ｅ－钙粘连素（Ｅ－ｃａｄ）基因启动子ＣｐＧ位点甲基化与前殂腺癌细胞Ｅ－ｃａｄ基因的失活的关系,并探讨Ｅ－ｃａｄ基因异常甲基化机制。方法：采用硫酸氢钠基因组测序法检测良性前列腺上皮和前列腺癌细胞株的Ｅ－ｃａｄ基因甲基化状态,并采用ＲＴ－ＰＣＲ检测各细胞株Ｅ－ｃａｄ和ＤＮＡ甲基转移酶（Ｄｎｍｔｌ）的ｍＲＮＡ表达。结果：有３３％（２／６）的前列腺癌细胞株发生甲基化现象,相应的细胞株中Ｅ－ｃａｄ     

脾酪氨酸激酶基因的再激活对胃癌生成和发展的影响

目的探讨去甲基化酶5-氮-2＇-脱氧胞苷对脾酪氨酸激酶（Syk）基因再表达的影响，以及Syk基因的再表达对胃癌肿瘤生成和发展的影响。方法分别用RT-PCR和MSP法检测SGC7901、MGC803、MKN28和MKN45细胞株中Syk基因表达及甲基化情况；用5-氮-2＇-脱氧胞苷处理人胃癌细胞株5GC7901后，检测此细胞株中Syk基因的甲基化及再表达情况，并用此治疗过的细胞株和未经治疗的细胞株分别接种于裸鼠皮下，对比观察裸鼠的成瘤率。结果SGC7901和MKN45细胞株中没有检测到Syk基因的表达，但可检测到Syk基因的甲基化。检测用5-氮-2＇-脱氧胞苷处理过的人胃癌SGC7901细胞株，Syk基因启动子没有甲基化，RT-PCR可检测到有Syk基因。用无Syk基因表达的SGC7901细胞株接种于10只对照组裸鼠皮下，8周后均有肿块生成；而用有Syk基因再表达的细胞株接种于另10只裸鼠（治疗组）皮下，8周后只有3只有肿块生成；对照组裸鼠的成瘤率显著高于治疗组（χ^2=7．91，P〈0．05）。结论5-氮-2＇-脱氧胞苷通过去甲基化使SGC7901细胞株中Syk基因再表达，Syk基因的再表达抑制了胃癌的生成和发展。     

The use of real-time quantitative polymerase chain reaction to detect hypermethylation of the CpG islands in the promoter region flanking the GSTP1 gene to diagnose prostate carcinoma

PURPOSE: We developed a real-time, quantitative, methylation sensitive polymerase chain reaction (PCR) protocol to analyze hypermethylation of the CpG islands in the promoter region of the pi class glutathione-S-transferase gene GSTP1 in prostate cancer tissue. MATERIALS AND METHODS: A total of 21 prostate cancer and 72 benign prostate hyperplasia (BPH) tissue samples were analyzed. Genomic DNA was digested with restriction enzyme, followed by real-time quantitative PCR amplification. Cycle threshold values were used to determine whether cancer genome was present in these tissues. A cutoff cycle threshold value of 35 was arbitrarily assigned. Samples with a cycle threshold of 35 or less were considered positive for prostate cancer. Conventional nested PCR was also performed for comparison. RESULTS: The mean cycle threshold values plus or minus standard deviation in prostate cancer and BPH cases were 30.12 +/- 2.88 and 37.77 +/- 2.72, respectively. All prostate cancer samples analyzed showed positive results, while 5 of the 72 BPH samples tested positive. Conventional nested PCR data indicated that 19 of 21 prostate cancer cases were positive for the methylation change, while 71 of 72 BPH cases tested negative. The test limitations of real-time PCR and the nested PCR protocols were determined to be 0.048 and 0.64 ng. DNA, respectively. CONCLUSIONS: We established a novel protocol for detecting the methylation change in the 5' regulatory sequence flanking the GSTP1 gene. The sensitivity of this protocol was superior to that of conventional nested PCR. The data also suggest that this novel protocol may accurately discriminate prostate carcinoma from BPH.     

Hypermethylation of MCAM gene is associated with advanced tumor stage in prostate cancer

BACKGROUND: DNA methylation has emerged as a promising biomarker for prostate cancer detection. In this report, we screened 36 candidate genes generated by a bioinformatic analysis of the human genome, and found that the melanoma cell adhesion molecule (MCAM) was an excellent candidate for cancer-specific methylation in prostate cancer. METHODS: Direct sequencing of bisulfite-treated genomic DNA, conventional methylation-specific PCR (MSP), real-time quantitative methylation-specific PCR, immunohistochemistry, colony formation assay, and statistical analysis. RESULTS: We found that the melanoma cell adhesion molecule (MCAM) gene promoter was specifically methylated in prostate cancer cell lines and primary prostate cancer (PCa) but not in non-neoplastic prostate (BPH) tissues by direct sequencing of bisulfite-treated genomic DNA and conventional methylation-specific PCR (MSP). Further analysis with quantitative MSP showed greater hypermethylation of the MCAM promoter (80%, 70/88) in primary prostate cancer compared to 12.5% (3/24) in BPH. Prostatic intraepithelial neoplasias (PIN), potential precursors of prostate carcinoma, showed an intermediate methylation rate of 23% (7/30). We further observed that MCAM promoter methylation was directly correlated with tumor stage (pT3+pT4) (P = 0.001) and Gleason score (P = 0.018) in primary prostate carcinoma. CONCLUSIONS: Our results suggest that MCAM promoter hypermethylation deserves further attention as a potential diagnostic prostatic DNA marker in human prostate cancer.     

Silencing of pi-class glutathione S-transferase in MDA PCa 2a and MDA PCa 2b cells

BACKGROUND: Loss of expression of the glutathione S-transferase-pi (GSTP1) is the most common genetic alteration described in human prostate cancer, occurring in virtually all tumors regardless of grade or stage. Of the available human prostate cancer cell lines, only LNCaP mirrors this phenotype. We investigated whether the prostate cancer cell lines MDA PCa 2a and MDA PCa 2b share this phenotype. METHODS: GSTP1 protein and mRNA levels were assessed in the MDA PCa 2a and MDA PCa 2b cell lines by Western and Northern blot. DNA methylation was evaluated by Southern blot analysis of genomic DNA digested with the methylation-sensitive restriction enzymes BssHII, NotI, and SacII. Re-expression of GSTP1 was determined by RT-PCR following treatment with 5-azacytidine, a DNA methyltransferase inhibitor, and/or the histone deacetylase inhibitor trichostatin A (TSA). RESULTS: Like all human prostatic carcinomas in vivo, both the MDA PCa 2a and 2b cell lines lack protein and mRNA expression of GSTP1. This lack of expression is associated with methylation in the GSTP1 gene promoter. Treatment with the methyltransferase inhibitor 5-azacytidine resulted in re-expression of GSTP1. By itself, TSA did not result in re-expression of GSTP1, nor did it augment expression induced by 5-azacytidine. CONCLUSIONS: MDA PCa 2a and 2b appear to be useful models of human prostatic carcinoma in that they lack expression of GSTP1 due to gene silencing via promoter methylation. Inhibition of histone acetylation does not appear to affect GSTP1 expression.     

survivin启动子与PSMA启动子/增强子在前列腺癌细胞系中的转录活性比较

目的:通过研究并比较前列腺特异性膜抗原(PSMA)基因启动子、增强子和survivin基因启动子在不同前列腺癌细胞系(LNCaP和PC-3细胞)中的转录活性,为前列腺癌的靶向性基因治疗提供依据。方法:采用PCR扩增PSMA基因的启动子、增强子和survivin基因启动子,分别克隆入pGL3-Basic,脂质体转染前列腺癌细胞和张氏肝细胞,检测各启动子在细胞中的转录活性。结果:survivin基因启动子在前列腺癌细胞中均具有较强活性,均明显高于PSMA启动子/增强子,其中S2pro启动活性最强,达到CMV启动子活性的1/3,然而,survivin启动子及PSMA启动子/增强子在肝细胞系中几乎不表达。结论:survivin启动子在前列腺癌细胞中具有较强启动活性,可望成为新的前列腺癌靶向性基因治疗工具。