前列腺癌细胞组蛋白H3甲基化的差异

 目的：整体分析雄激素依赖性和非依赖性前列腺癌细胞组蛋白H3甲基化差异,探讨前列腺癌从激素依赖性发展为非激素依赖性的表观遗传机制。方法：应用重甲基细胞培养条件下氨基酸稳定同位素标记(SILAC)技术结合生物质谱分析雄激素依赖性前列腺癌细胞LNCaP和非依赖性前列腺癌细胞DU145组蛋白H3的甲基化修饰谱,寻找差异的修饰位点和模式;通过Western blotting验证所发现的差异修饰;采用real-time PCR检测2株细胞相关甲基化酶和去甲基化酶的表达差异。结果：质谱鉴定发现2株前列腺癌细胞的组蛋白H3存在5个甲基化位点,甲基化模式分别为H3K14me2、H3R17me1、H3K36me1、H3K36me2、H3K36me3、H3R72me2、H3K79me1和H3K79me2。其中,包含甲基化位点H3K36的肽段有2种,分别为“KSAPATGGVKKPHR”和“KSAPSTGGVKKPHR”,前者鉴定到的频数高于后者,两者的区别在于第31位的氨基酸分别为A与S,前者归属于组蛋白H3变异体H31T、H31和H32,主要出现在DU145细胞中,后者归属于组蛋白H3变异体H33,在LNCaP细胞中出现次数稍多;提示2株细胞可能表达不同的组蛋白H3变异体,从而导致甲基化模式的差异。Western blotting检测发现H3K36的一甲基化和二甲基化在2株细胞间没有差异,而其在DU145细胞中的三甲基化程度显著高于LNCaP细胞。Real-time PCR检测显示DU145细胞的H3K36去甲基化酶mRNA表达比LNCaP细胞有所降低。结论：组蛋白H3变异体和H3K36去甲基化酶的差异表达可能导致非激素依赖性前列腺癌细胞H3K36三甲基化增加,成为前列腺癌从激素依赖性发展为非激素依赖性的一种表观遗传转变。     

KAT5 and KAT6B are in positive regulation on cell proliferation of prostate cancer through PI3K-AKT signaling

Histone modifications play important roles in the tumorigenesis and progression of prostate cancer (PCa) and genes involved in histone modifications are seemed as ideal targets for treatment of PCa patients. However, clinical trials have shown that those existing drugs exert the minimal antitumor activity and excess adverse effects on PCa patients. Therefore, it is of great interest to figure out novel specific biomarkers to guide the development of new drugs. In present study, an RNAi screening with 44 genes involved in histone modifications was applied to a PCa cell line, Du145. The results showed that nine genes were in positive regulation of Du145 cell growth. Then four selected genes (KAT2B, KAT5, KAT6B and HDAC1) were found to exert this effect by a gene-specific manner when silenced. And then KAT5 or KAT6B silenced cells were subjected to DNA microarray analysis. The common differentially expressed genes were analyzed by Ingenuity Pathway Analysis (IPA) and found that PDEF signaling, EIF2 signaling and PI3K signaling was suppressed following by KAT5 or KAT6B silencing. Subsequent immunoblotting assay showed that AKT signaling was inhibited, which suggested that KAT5 or KAT6B regulates cancer cell growth through PI3K-AKT signaling. Together with our published data [31] that AURKA inhibitoin increased drug sensitivity of DU145, our work demonstrated the underlying mechanism that how the acetylation enzyme regulates cancer cells survial and might provide potential therapeutic targets for prostate cancer patients in future epigenetic drug development.     

The role of epigenetic modifications in retinoic acid receptor beta2 gene expression in human prostate cancers.

The retinoic acid receptor (RAR) beta gene is a putative tumor suppressor gene on chromosome 3p24, where a high incidence of loss of heterozygosity is detected in many types of tumors. Retinoic acid suppresses cancer cell growth through binding to RARs, especially RARbeta, indicating a critical role in mediating anticancer effects. Selective loss or down-regulation of RARbeta mRNA and protein has been reported in prostate cancers (PCas), although the mechanisms remain unclear. We investigated the role of epigenetic modification in RARbeta2 gene silencing. Aberrant methylation was detected in 11 of 14 (79%) primary PCas, 9 of 10 (90%) hormone-refractory PCas, and 2 of 4 (50%) PCa cell lines, but not in any normal prostate samples. Chromatin immunoprecipitation assay showed that all RARbeta2-negative cells (LNCaP, PC3, and DU145) were hypoacetylated at both histones H3 and H4. After exposure to 5-aza-2prime;-deoxycytidine treatment, Trichostatin A and all-trans retinoic acid induced partial demethylation, increased accumulation of acetylated histones, and markedly restored the expression of RARbeta2 in RARbeta2-negative cells. These data suggest that the RARbeta2 gene may be one of the frequently silenced genes by epigenetic modifications in PCa.     

趋化因子及其受体CXCL12/CXCR4在人前列腺癌转移机制中的作用

目的 探讨趋化因子及其受体CXCL12/CXCR4在人前列腺癌转移机制中的作用.方法 免疫组织化学技术分析CXCL12/CXCR4蛋白在18例前列腺癌组织中的表达;免疫细胞化学技术分析CXCL12/CXCR4蛋白在人前列腺癌细胞株PC3、DU145和LNCap中的表达;迁移、侵袭试验分析外源性CXCL12对PC3、DU145和LNCap体外侵袭能力的调节作用.结果 18例人前列腺癌组织中,17例不同强度表达CXCR4蛋白,1例阴性表达,同时除1例标本弱表达CXCL12蛋白外,其余不表达CXCL12蛋白.3种前列腺癌细胞株均表达CXCR4蛋白,不表达CXCL12蛋白.外源性CXCLl2可明显促进PC3、DU145及LNCap的体外迁移、侵袭,以抗CXCL12或CXCR4抗体预处理PC3、LNCap细胞可以拮抗CXCL12对它们的促迁移、侵袭作用.结论 人前列腺癌组织表达CXCR4蛋白,CXCL12/CXCR4信号通路可能参与前列腺癌的侵袭、转移.     

Gene silencing of TSPYL5 mediated by aberrant promoter methylation in gastric cancers

DNA methylation is crucial for normal development, but gene expression altered by DNA hypermethylation is often associated with human diseases, especially cancers. The gene TSPYL5, encoding testis-specific Y-like protein, was previously identified in microarray screens for genes induced by the inhibition of DNA methylation and histone deacetylation in glioma cell lines. The TSPYL5 showed a high frequency of DNA methylation-mediated silencing in both glioma cell lines and primary glial tumors. We now report that TSPYL5 is also inactivated by DNA methylation and could be a putative epigenetic target gene in gastric cancers. We found that the expression of TSPYL5 mRNA was frequently downregulated and inversely correlated with DNA methylation in seven out of nine gastric cancer cell lines. TSPYL5 mRNA expression was also restored after treating with a DNA methyltransferase inhibitor. In primary gastric tumors, methylation-specific PCR results in 23 of the 36 (63.9%) cases revealed that the hypermethylation at CpG islands of the TSPYL5 was detectable at a high frequency. Furthermore, TSPYL5 suppressed the growth of gastric cancer cells as demonstrated by a colony formation assay. Thus, strong associations between TSPYL5 expression and hypermethylation were observed, and aberrant methylation at a CpG island of TSPYL5 may play an important role in development of gastric cancers.     

Functional analysis of the host defense peptide Human Beta Defensin-1: new insight into its potential role in cancer

Although it is known that innate immunity is key for protecting the body against foreign agents such as bacteria, little is known about elements of the innate immune system that have anti-tumor activity. Human Beta Defensin-1 (hBD-1), an important component of the innate immune response, is lost at high frequencies in malignant prostatic tissue, while high levels of expression are maintained in adjacent benign regions. In prostate carcinoma, frequent genetic alterations occur in the 8p22-23 region and several studies indicate there may be multiple tumor suppressor genes present within this region. The high incidence of loss of hBD-1 expression in prostate cancer, along with its chromosomal location of 8p23.2, raised the possibility that it may play a role in tumor suppression. To gain insight as to its function in prostate cancer, hBD-1 was cloned and ectopically expressed in four prostate cancer cell lines. Induction of hBD-1 expression resulted in a decrease in cellular growth in DU145 and PC3 cells. However, hBD-1 has no effect on the growth of androgen receptor (AR) positive LNCaP prostate cancer cells, but was again growth suppressive to PC3 cells with ectopic AR expression (PC3/AR+). hBD-1 also caused rapid induction of cytolysis and caspase-mediated apoptosis in DU145 and PC3 prostate cancer cells. Although the regulation of hBD-1 was not addressed in this study, our preliminary data demonstrated that the pathways involved may include cMYC and PAX2. Data presented here are the first to provide evidence of its potential role in prostate cancer cell death.     

The role of RASSF1A methylation in cancer

Tumour suppressor gene inactivation is critical to the pathogenesis of cancers; such loss of function may be mediated by irreversible processes such as gene deletion or mutation. Alternatively tumour suppressor genes may be inactivated via epigenetic processes a reversible mechanism that promises to be more amenable to treatment by therapeutic agents. The CpG dinucleotide is under-represented in the genome, but it is found in clusters within the promoters of some genes, and methylation of these CpG islands play a critical role in the control of gene expression. Inhibitors of the DNA methyltransferases DNMT1 and DNMT3b have been used in a clinical setting, these nucleotide analogues lack specificity but the side effects of low dose treatments were minimal and in 2004 Vidaza (5-azacitidine) was licensed for use in myelodysplastic syndrome. Methylation inhibitors are also entering trials in conjunction with another class of epigenetic modifiers, the histone deacetylase inhibitors and this epigenetic double bullet offers hope of improved treatment regimes. Recently there has been a plethora of reports demonstrating epigenetic inactivation of genes that play important roles in development of cancer, including Ras-association domain family of genes. Epigenetic inactivation of RASSF1A (Ras-association domain family 1, isoform A) is one of the most common molecular changes in cancer. Hypermethylation of the RASSF1A promoter CpG island silences expression of the gene in many cancers including lung, breast, prostate, glioma, neuroblastoma and kidney cancer. Several recent studies have illustrated the diagnostic and prognostic potential of RASSF1A methylation. This presents RASSF1A methylation as an attractive biomarker for early cancer detection which, for most cancers, results in improved clinical outcome. DNA methylation analysis is applicable to a range of body fluids including serum, urine, bronchioalveolar lavage and sputum. The ease with which these body fluids can be acquired negates the need for invasive procedures to obtain biopsy material. This review will discuss the feasibility of using RASSF1A methylation as a diagnostic and prognostic marker in cancer management.     

The use of multicolor fluorescence technologies in the characterization of prostate carcinoma cell lines: a comparison of multiplex fluorescence in situ hybridization and spectral karyotyping data

Recent studies have identified several chromosome regions that are altered in primary prostate cancer and prostatic carcinoma cell lines. These targeted regions may harbor genes involved in tumor suppression. We used multiplex fluorescence in situ hybridization (M-FISH) to screen for genetic rearrangements in four prostate cancer cell lines, LNCaP, LNCaP.FCG, DU145, and PC3, and compared our results with those recently obtained using spectral karyotyping (SKY). A number of differences was noted between abnormalities characterized by SKY and M-FISH, suggesting variation in karyotype evolution and characterization by these two methodologies. M-FISH analysis showed that hormone-resistant cell lines (DU145 and PC3) contained many genetic alterations (> or =15 per cell), suggesting high levels of genetic instability in hormone-refractory prostate cancer. Most chromosome regions previously implicated in prostate cancer were altered in one or more of these cell lines. Several specific chromosome aberrations were also detected, including a del(4)(p14) and a del(6)(q21) in the hormone-insensitive cell lines, a t(1;15)(p?;q?) in LNCaP, LNCaP, and PC3, and a i(5p) in LNCaP.FCG, DU145, and PC3. These clonal chromosome abnormalities may pinpoint gene loci associated with prostate tumourigenesis, cancer progression, and hormone sensitivity.     

ENDOD1在前列腺癌组织及细胞中的表达及意义

目的:观察比较核酸内切酶结构域内含蛋白1(endonuclease domain containing1,ENDOD1)在良性前列腺增生和前列腺癌组织中的表达差异;筛选存在ENDOD1特异性低表达的前列腺癌细胞系,继而通过调控该细胞ENDOD1蛋白表达,研究其在前列腺癌细胞中的生物学功能,初步探索ENDOD1基因与前列腺癌发生、进展的联系。方法:利用免疫组化SP法检测20例良性前列腺增生和21例前列腺癌术后标本组织中ENDOD1表达情况;利用RT-qPCR和Western blot方法观察ENDOD1的mRNA和蛋白在前列腺正常上皮细胞和不同类型前列腺癌细胞中的表达差异,筛选出特异性低表达细胞系;构建pCMV-N-Flag-ENDOD1重组质粒,转染前列腺癌细胞株,过表达ENDOD1蛋白,通过MTT法测定调控前后前列腺癌细胞活力的变化,流式细胞术检测细胞周期和凋亡,Transwell实验评价肿瘤细胞迁移和侵袭能力的改变。结果:免疫组化评分的方差分析结果显示ENDOD1表达与前列腺癌Gleason评分呈负性关联;RT-qPCR和Western blot实验结果表明ENDOD1在雄激素非依赖性前列腺癌细胞系PC3和DU145中存在着特异性低表达(P0.05)。同时,MTT实验显示,在DU145细胞中,过表达ENDOD1肿瘤细胞活力显著下降(P0.05);而流式细胞术检测结果表明过表达ENDOD1能够使DU145细胞周期停滞在G_0/G_1期,但细胞凋亡率无明显差异。此外,在Transwell实验中,过表达ENDOD1的DU145细胞迁移和侵袭能力明显下降(P0.05)。结论:ENDOD1在Gleason评分越高的前列腺癌中表达越低,同时在雄激素非依赖性前列腺癌细胞系存在着特异性低表达;而过表达ENDOD1能明显抑制雄激素非依赖性前列腺癌细胞的生长、迁移和侵袭能力。     

Correlation between histone acetylation and expression of the MYO18B gene in human lung cancer cells

Recently, we isolated a candidate tumor-suppressor gene, MYO18B, which was inactivated in approximately 50% of human lung cancers by deletion, mutation, and promoter methylation. However, more frequent reduction or loss of MYO18B expression and restoration of MYO18B expression by trichostatin A (TSA) treatment suggested the contribution of other mechanisms, especially histone deacetylation, for epigenetic inactivation of the MYO18B gene. In this study, we examined histone modification of the promoter region of the MYO18B gene in 8 human lung cancer cell lines by a chromatin immunoprecipitation assay. In 6 of 7 cell lines with reduced or silenced MYO18B expression, the levels of histones H3 and H4 acetylation surrounding the MYO18B promoter region were lower than those in a cell line with MYO18B expression. By treatment with TSA, the levels of histone H3 and H4 acetylation were increased in all 6 cell lines whose MYO18B expression was restored by TSA, whereas neither H3 nor H4 acetylation was increased in cells whose MYO18B expression was not restored by TSA. Significant correlations were observed between the levels of histone H3/H4 acetylation and MYO18B expression. These results suggest that acetylation of both histones H3 and H4 contributes to regulation of MYO18B expression in lung cancer cells and that histone deacetylation surrounding the promoter region plays an important role in MYO18B silencing and is involved in lung carcinogenesis.     

Glutathione S-transferase pi (GSTP1) hypermethylation in prostate cancer: review 2007

Prostatic carcinoma is characterised by the silencing of the pi-class glutathione S-transferase gene (GSTP1), which encodes a detoxifying enzyme. The silencing of GSTP1 results from aberrant methylation at the CpG island in the promoter-5' and occurs in the vast majority of cases of high-grade prostatic intraepithelial neoplasia (PIN) and prostate cancers. We review the potential novel role of GSTP1 and its related expression in prostate cancer. The loss of expression (silencing) of the GSTP1 gene is the most common (>90%) genetic alteration reported to date in prostate cancer. Quantitative methylation-specific PCR assays allow detection of GSTP1 methylation in prostate biopsies and may improve the sensitivity of cancer detection. Advances in the epigenetic characterisation of prostate cancer have enabled the development of DNA methylation assays that may soon be used in diagnostic testing of serum and tissue for prostate cancer. Inhibition of aberrant promoter methylation could theoretically prevent carcinogenesis.     

Down-regulation of the progesterone receptor by the methylation of progesterone receptor gene in endometrial cancer cells

Progesterone plays an important role in the regulation of normal endometrium function by binding to progesterone receptor (PR). In endometrial cancer, however, PR is always down-regulated. Previous reports showed that methylation in the promoter region of the PR gene may be responsible for PRB isoform repression. However, the CpG islands in the exon region of the PR gene are much richer and longer than in the promoter region. We hypothesize that methylation in the exon region may also take part in the down-regulation of the PR gene. The methylation status of the first exon of the PR gene in endometrial cell cultures was investigated. Aberrant methylation patterns were observed in the first exon of PR gene, and the methylation density is correlated with the differentiation of different types of endometrial cancer cells. DNA methyltransferase (DNMT) and histone deacetylase inhibitor 5-aza-2'-deoxycytidine (ADC), as well as trichostatin A (TSA), which reverses PR gene expression, were also studied. A combination of ADC and TSA resulted in synergistic effects in inducing PR expression, down-regulation of DNMT1 and DNMT3A, and could also have antigrowth effect on endometrial cancer cells by inducing apoptosis.     

DNA methylation-dependent silencing of CST6 in human breast cancer cell lines

Cystatin M (CST6) is a candidate breast cancer tumor suppressor that is expressed in normal and premalignant breast epithelium, but not in metastatic breast cancer cell lines. CST6 is subject to epigenetic silencing in MCF-7 breast cancer cells related to methylation of the CpG island that encompasses the CST6 proximal promoter region and exon 1. In the current study, CST6 CpG island methylation and expression status was examined in a panel of breast cancer cell lines. Seven of 12 (58%) cell lines lack detectable expression of CST6 and treatment of these cells with 5-aza-2'-deoxycytidine resulted in a significant increase in CST6 expression, suggesting that the loss of expression may be related to methylation-dependent epigenetic silencing. Bisulfite sequencing of CST6 in a subset of breast cancer cell lines revealed CpG island hypermethylation in CST6-negative cells, and an absence of CpG island methylation in cells that express CST6. The extent of regional methylation was strongly associated with the lack of expression of CST6 among these cell lines. In particular, hypermethylation of the proximal promoter was significantly associated with CST6 gene silencing, and methylation of a number of individual CpGs was found to be statistically correlated with extinction of gene expression. These results establish a strong link between CST6 promoter hypermethylation and loss of CST6 expression in breast cancer cell lines, and suggest that methylation-dependent epigenetic silencing of CST6 may represent an important mechanism for loss of CST6 during breast carcinogenesis in vivo.     

DNA promoter hypermethylation contributes to down-regulation of galactocerebrosidase gene in lung and head and neck cancers

Galactocerebrosidase (GALC) is a lysosomal enzyme responsible for glycosphingolipids degradation byproducts of which are important for synthesis of apoptosis mediator ceramide. Reduced expression of GALC has been identified in human malignancies; however, molecular mechanisms underlying down-regulation of GALC expression in cancer remain unknown. We performed methylation and expression analysis on GALC gene in a panel of head and neck cancer (HNC) and lung cancer cell lines, attempting to understand the regulation of GALC in human cancer. QRT-PCR and western blot analysis were performed to detect the expression of GALC in HNC. Bisulfite DNA sequencing and real-time qMSP were used to detect the methylation of GALC in HNC and lung cancer cell lines. 5aza-dC treatment assay was used to analysis the functional effect of GALC methylation on GALC expression in HNC. Reduction or complete absence of GALC expression was observed in more than a half of the tested HNC cell lines (8/14). 7 out of 8 cell lines with down-regulated expression harbored heavy CpG island methylation, while all cell lines with abundant expression of the gene contained no methylation. Hypermethylation was also found in primary HNC tumor tissues and lung cancer cell lines whereas absent in normal oral mucosa tissues. Demethylating treatment demonstrated that 5aza-dC significantly restored GALC expression in cell lines with methylated promoter while showed no effect on cell lines without promoter hypermethylation. Our findings for the first time demonstrated that promoter hypermethylation contributed to down-regulation of GALC Gene, implicating epigenetic inactivation of GALC may play a role in tumorigenesis of cancer.