TMS1, a novel proapoptotic caspase recruitment domain protein, is a target of methylation-induced gene silencing in human breast cancers

Gene silencing associated with aberrant methylation of promoter region CpG islands is an acquired epigenetic alteration that serves as an alternative to genetic defects in the inactivation of tumor suppressor and other genes in human cancers. The hypothesis that aberrant methylation plays a direct causal role in carcinogenesis hinges on the question of whether aberrant methylation is sufficient to drive gene silencing. To identify downstream targets of methylation-induced gene silencing, we used a human cell model in which aberrant CpG island methylation is induced by ectopic expression of DNA methyltransferase. Here we report the isolation and characterization of TMS1 (target of methylation-induced silencing), a novel CpG island-associated gene that becomes hypermethylated and silenced in cells overexpressing DNA cytosine-5-methyltransferase-1. We also show that TMS1 is aberrantly methylated and silenced in human breast cancer cells. Forty percent (11 of 27) of primary breast tumors exhibited aberrant methylation of TMS1. TMS1 is localized to chromosome 16p11.2-12.1 and encodes a 22-kDa predicted protein containing a COOH-terminal caspase recruitment domain, a recently described protein interaction motif found in apoptotic signaling molecules. Ectopic expression of TMS1 induced apoptosis in 293 cells and inhibited the survival of human breast cancer cells. The data suggest that methylation-mediated silencing of TMS1 confers a survival advantage by allowing cells to escape from apoptosis, supporting a new role for aberrant methylation in breast tumorigenesis.     

Aberrant methylation and silencing of ARHI,an imprinted tumor suppressor gene in which the function is lost in breast cancers

ARHI is a maternally imprinted tumor suppressor gene that maps to a site on chromosome 1p31 where loss of heterozygosity has been observed in 40% of human breast and ovarian cancers. ARHI is expressed in normal ovarian and breast epithelial cells, but ARHI expression is lost in a majority of ovarian and breast cancers. Expression of ARHI from the paternal allele can be down-regulated by multiple mechanisms in addition to loss of heterozygosity. This article explores the role of DNA methylation in silencing ARHI expression. There are three CpG islands in the ARHI gene. CpG islands I and II are located in the promoter region, whereas CpG island III is located in the coding region. Consistent with imprinting, we have found that all three CpG islands were partially methylated in normal human breast epithelial cells. Additional confirmation of imprinting has been obtained by studying DNA methylation and ARHI expression in murine A9 cells that carry either the maternal or the paternal copy of human chromosome 1. All three CpG islands were methylated, and ARHI was not expressed in A9 cells that contained the maternal allele. Conversely, CpG islands were not methylated and ARHI was expressed in A9 cells that contained the paternal allele of human chromosome 1. Aberrant methylation was found in several breast cancer cell lines that exhibited decreased ARHI expression. Hypermethylation was detected in 67% (6 of 9) of breast cancer cell lines at CpG island I, 33% (3 of 9) at CpG island II, and 56% (5 of 9) at CpG island III. Hypomethylation was observed in 44% (4 of 9) of breast cancer cell lines at CpG island II. When methylation of CpG islands was studied in 20 surgical specimens, hypermethylation was not observed in CpG island I, but 3 of 20 cases exhibited hypermethylation in CpG island II (15%), and 4 of 20 cases had hypermethylation in CpG island III (20%). Treatment with 5-aza-2'-deoxycytidine, a methyltransferase inhibitor, could reverse aberrant hypermethylation of CpG island I, II and III and partially restore ARHI expression in some, but not all of the cell lines. Treatment with 5-aza-2'-deoxycytidine partially reactivated ARHI expression in cell lines with hypermethylation of CpG islands I and II but not in cell lines with partial methylation or hypomethylation of these CpG islands. To test the impact of CpG island methylation on ARHI promoter activity more directly, constructs were prepared with the ARHI promoter linked to a luciferase reporter and transfected into SKBr3 and human embryo kidney 293 cells. Methylation of the entire construct destroyed promoter activity. Selective methylation of CpG island II alone or in combination with CpG island I also abolished ARHI promoter activity. Methylation of CpG I alone partially inhibited promoter activity of ARHI. Thus, hypermethylation of CpG island II in the promoter region of ARHI is associated with the complete loss of ARHI expression in breast cancer cells. Other epigenetic modifications such as hypermethylation in CpG island III may also contribute to the loss of ARHI expression.     

Silencing of the UCHL1 gene in human colorectal and ovarian cancers

Aberrant DNA methylation is associated with many types of human cancers. To identify genes silenced in human colorectal cancers, we performed a microarray analysis for genes whose expression was induced by treatment of HCT116 human colon cancer cells with a demethylating agent, 5-aza-2'-deoxycitidine (5-aza-dC). Seven known genes were identified as being upregulated (> or =8-fold) and expressed at more than twice as high as the average level. Among these was the UCHL1 gene (also known as PGP9.5), which is involved in regulation of cellular ubiquitin levels. A dense CpG island in its promoter region was completely methylated in HCT116 cells, and no mRNA was detected. 5-Aza-dC treatment of HCT116 cells induced dose-dependent demethylation of the CpG island, and restored UCHL1 mRNA and protein expression. UCHL1 silencing was observed in 11 of 12 human colorectal cancer cell lines, and its methylation was detected in 8 of 17 primary colorectal cancers. Further, UCHL1 silencing was observed in 6 of 13 ovarian cancer cell lines, and its methylation was detected in 1 of 17 primary ovarian cancers. These results showed that UCHL1 is inactivated in human colorectal and ovarian cancers by its promoter methylation, and suggest that disturbance of cellular ubiquitin levels is present.     

启动子CpGs位点甲基化所致BRCA1基因转录抑制作用的位点特异性

目的 研究BRCA1基因CpG岛内各个CpGs位点甲基化与转录水平的关系，探讨甲基化所致转录抑制作用的特异性CpG位点，以阐明DNA甲基化的作用方式。方法 以Rice等研究数据为材料，用方差分析和多因素线性回归模型等方法进行统计再分析。结果 BRCA1基因CpG岛 8～ 44、-189～-19、-379～-258、-37～-19和-80～-19区段内CpGs的甲基化与基因转录抑制有关，这些区段内CpGs位点的甲基化数目越多，基因转录水平越低。多因素线性回归模型分析表明，-80～-19区段的甲基化与转录抑制关系密切。并以-29CpG位点甲基化引起的转录抑制作用最强。结论 CpG岛甲基化引起的转录抑制与CpGs位点有关，具有位点的特异性。而甲基化的程度与转录抑制程度呈正相关。     

Detailed methylation analysis of the glutathione S-transferase pi (GSTP1) gene in prostate cancer

Glutathione-S-Transferases (GSTs) comprise a family of isoenzymes that provide protection to mammalian cells against electrophilic metabolites of carcinogens and reactive oxygen species. Previous studies have shown that the CpG-rich promoter region of the pi-class gene GSTP1 is methylated at single restriction sites in the majority of prostate cancers. In order to understand the nature of abnormal methylation of the GSTP1 gene in prostate cancer we undertook a detailed analysis of methylation at 131 CpG sites spanning the promoter and body of the gene. Our results show that DNA methylation is not confined to specific CpG sites in the promoter region of the GSTP1 gene but is extensive throughout the CpG island in prostate cancer cells. Furthermore we found that both alleles are abnormally methylated in this region. In normal prostate tissue, the entire CpG island was unmethylated, but extensive methylation was found outside the island in the body of the gene. Loss of GSTP1 expression correlated with DNA methylation of the CpG island in both prostate cancer cell lines and cancer tissues whereas methylation outside the CpG island in normal prostate tissue appeared to have no effect on gene expression.     

涎腺腺样囊性癌中Runx3基因启动子区5'-CpG岛的甲基化演进规律

目的 研究涎腺腺样囊性癌(SGACC)中Runx3基因启动子区5'-CpG岛甲基化的关键位点和演进规律.方法 以定量甲基化特异性PCR(qMSP)检测41例SGACC及配对的癌旁正常涎腺组织中Runx3基因启动子区5'-CpG岛(3478 bp)10个位点的甲基化状态,分析Runx3基因甲基化与SGACC临床病理特征的关系,用Logistic模型分析Runx3基因及各位点甲基化在SGACC中发生的危险度.用Western blot法检测19例SGACC及其相应癌旁正常涎腺组织中Runx3蛋白的表达,分析Runx3基因甲基化对Runx3蛋白表达的影响.结果 SGACC及其相应癌旁正常涎腺组织中,Runx3基因启动子区CpG岛(3478 bp)第1～10个位点的甲基化阳性率分别为42.69%～85.69%和22.17%～59.43%,正常涎腺组织中Runx3基因各位点的甲基化率均显著低于SGACC(P＜0.01).其中第1、2位点甲基化程度最高,向转录起始点方向甲基化程度逐渐减弱,在转录起始点位置(第7、8位点)甲基化程度最低,向3'端方向甲基化程度又略微升高.Logistic模型分析结果显示,Runx3甲基化是SGACC发生的一个重要因素,Runx3甲基化从5'端向转录起始点方向演进,转录起始点区可能是Runx3甲基化的关键位点.Western blot检测结果显示,SGACC组织中Runx3蛋白的表达量显著低于相应的癌旁正常涎腺组织(P＜0.01).结论 SGACC组织中Runx3基因甲基化从5'端向转录起始点方向演进,转录起始点部位可能是Runx3基因甲基化的关键位点.Runx3基因甲基化是引起Runx3蛋白表达下调的原因之一,与SGACC的发生有关.     

Epigenetic silencing of maspin gene expression in human breast cancers

Maspin is a tumor suppressor whose expression is lost in many advanced breast cancers. Maspin has been shown to inhibit cell motility, invasion and metastasis; however, its precise role in normal mammary epithelium remains to be elucidated. Although expression of maspin mRNA is low or absent in most human breast cancer cells, the maspin gene is rarely re-arranged or deleted. We hypothesized that aberrant cytosine methylation and chromatin condensation of the maspin promoter participates in the silencing of maspin expression during neoplastic progression. To test this hypothesis, we compared cultured normal human mammary epithelial cells (HMECs) to 9 cultured human breast cancer cell lines. HMECs expressed maspin mRNA and displayed a completely non-methylated maspin gene promoter with an open chromatin structure. In contrast, 7 of 9 breast cancer cell lines had no detectable maspin expression and 6 of these 7 maspin-negative breast cancer cell lines also displayed an aberrant pattern of cytosine methylation of the maspin promoter. Interestingly, the maspin promoter was completely methylated in maspin-negative normal peripheral blood lymphocytes. This indicates that the maspin promoter is not a functional CpG island and that cytosine methylation of this region may contribute to normal tissue-restricted gene expression. Chromatin accessibility studies with MCF-7 cells, which lack maspin expression and have a methylated maspin promoter, showed a closed chromatin structure compared with HMECs. Moreover, maspin gene expression could be re-activated in MCF-7 cells by treatment with 5-aza-2;-deoxycytidine, a DNA demethylating agent. Thus, aberrant cytosine methylation and heterochromatinization of the maspin promoter may silence maspin gene expression, thereby contributing to the progression of human mammary cancer.