Methylation profiling of CpG islands in human breast cancer cells

CpG island hypermethylation is known to be associated with gene silencing in cancer. This epigenetic event is generally accepted as a stochastic process in tumor cells resulting from aberrant DNA methyltransferase (DNA-MTase) activities. Specific patterns of CpG island methylation could result from clonal selection of cells having growth advantages due to silencing of associated tumor suppressor genes. Alternatively, methylation patterns may be determined by other, as yet unidentified factors. To explore further the underlying mechanisms, we developed a novel array-based method, called differential methylation hybridization (DMH), which allows a genome- wide screening of hypermethylated CpG islands in tumor cells. DMH was used to determine the methylation status of >276 CpG island loci in a group of breast cancer cell lines. Between 5 and 14% of these loci were hypermethylated extensively in these cells relative to a normal control. Pattern analysis of 30 positive loci by Southern hybridization indicated that CpG islands might differ in their susceptibility to hypermethylation. Loci exhibiting pre-existing methylation in normal controls were more susceptible to de novo methylation in these cancer cells than loci without this condition. In addition, these cell lines exhibited different intrinsic abilities to methylate CpG islands not directly associated with methyltransferase activities. Our study provides evidence that, aside from random DNA-MTase action, additional cellular factors exist that govern aberrant methylation in breast cancer cells.      

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.     

结直肠癌细胞中脾酪氨酸激酶基因甲基化状态和表达的关系

目的：探讨结直肠癌细胞中脾酪氨酸激酶基因甲基化和表达的关系。方法：应用亚硫酸盐修饰测序、甲基化特异性聚合酶链反应和蛋白印迹技术检测结直肠癌细胞脾酪氨酸激酶的甲基化状态以及表达情况；荧光素酶报告分析法研究启动子区域CpG岛的甲基化与启动子活性的关系；甲基化转移酶抑制剂处理脾酪氨酸激酶甲基化失表达的结直肠癌细胞株，观察处理前后细胞内脾酪氨酸激酶基因甲基化状态和表达情况。结果：（1）23个结直肠癌细胞中，9个细胞启动子发生甲基化而失去蛋白质表达；其余则正常表达，甲基化发生率为39.2％；（2）9个甲基化的细胞中，7个存在微卫星不稳定；而14个未发生甲基化的细胞中，仅有4个存在微卫星不稳定。二者之间的差异显著（P<0.05）；（3）脾酪氨酸激酶启动子全长和未甲基化启动子荧光素酶的活性分别是甲基化组的4.5和4.7倍；5-Aza-CdR可恢复甲基化启动子的活性；（4）5-Aza-CdR可去甲基化而使脾酪氨酸激酶基因重新表达，而且具有时间依从性。结论：结直肠癌细胞中，启动子区域的甲基化导致Syk基因丧失表达，5-Aza-CdR可以去甲基化而恢复脾酪氨酸激酶基因的表达。     

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.     

Methylation patterns of IGFBP7 in colon cancer cell lines are associated with levels of gene expression

Altered expression of insulin-like growth factor binding protein 7 (IGFBP7) has been found in colon cancer, but the exact regulatory mechanism has not been fully investigated. In order to elucidate the mechanisms underlying aberrant IGFBP7 expression in colon cancer, we used bisulphite sequencing PCR (BSP) to detect the detailed methylation profiles of the IGFBP7 5' CpG island. Exon 1 of the IGFBP7 gene was highly methylated in IGFBP7-negative cell lines but unmethylated in IGFBP7-positive lines. The methylation status of the promoter region and the intron 1 region was not so discriminating in IGFBP7-positive and -negative cell lines. Methylation-specific PCR (MSP) confirmed the hypermethylation of IGFBP7 exon 1 in IGFBP7-negative cell lines. Treatment with 5-aza-2'-deoxycytidine (5-aza-dC) induced demethylation of the CpG island in exon 1 of IGFBP7, as examined by both MSP and bisulphate genomic sequencing. Furthermore, the expression of IGFBP7 was restored, as detected by both RT-PCR and immunocytochemistry. Our study is the first to provide detailed methylation profiles of the IGFBP7 5' CpG island and shows that hypermethylation of the CpG island in exon 1 of IGFBP7 is closely related to the absence of its expression in colon cancer cells.     

Hypermethylation of tumor-related genes in genitourinary cancer cell lines.

Hypermethylation of CpG island is a common mechanism for the inactivation of tumor-related genes. In the present study, we analyzed 13 genitourinary cancer cell lines for aberrant DNA methylation of 5 tumor-related genes using methylation- specific polymerase chain reaction (MSP). GSTP1 was methylated in 5 (38.5%), E-cadherin in 1 (8%), VHL in 1 (8%), and MGMT and hMLH1 in none (0%). Six out of thirteen genitourinary cancer cell lines had methylation of at least one of five genes; 5 had one gene methylated, and, 1 had two genes methylated. Methylation of these 5 genes was not detected in any of the bladder cancer cell lines. GSTP1 was methylated in all of the 3 prostate cancer cell lines. We conclude that aberrant hypermethylation may be an important mechanism for the inactivation of cancer-related genes in kidney and prostate cancer cell lines.     

Hypermethylation of CpG islands is more prevalent than hypomethylation across the entire genome in breast carcinogenesis

This study aimed to establish a high-throughput, genome-wide and non-gene-specific approach to assess the methylation status of multiple CpG islands in parallel and employ it to detect the CpG island methylation profiling alterations in breast carcinogenesis. We used methylation-sensitive restriction fingerprint (MSRF) to screen the permutations of primers that could detect varied and specific methylation profiling in genomic DNA isolated from four different cell lines. Five permutations of nine arbitrary primers were determined for the following experiments based on the above test. We then examined the methylation profiling alterations of CpG islands in 31 breast cancer tissue samples relative to their adjacent non-neoplastic tissues with modified MSRF that replaced silver staining with denatured high-performance liquid chromatography for size fraction. We found that two pairs of primers could reveal specific alterations of CpG methylation in the examined tissues, and 83.9% (26/31) of breast cancer tissues exhibited specific CpG island methylation profiling relative to their adjacent non-neoplastic tissues. Size fraction analysis revealed that hypermethylation of CpG islands was responsible for the aberrant methylation profiling in breast cancer tissues. Our work not only established a relative high-throughput, genome-wide and economic method to detect methylation alterations of CpG island profiling, but also revealed that hypermethylation of CpG islands was more prevalent than hypomethylation across the entire genome in our examined cancer tissues. The methylation profiling alterations revealed by two primer pairs used in the present study might be a novel marker for breast cancer.     

Abnormal CpG island methylation occurs during in vitro differentiation of human embryonic stem cells

Directed differentiation of human embryonic stem cells (hESCs) into specific somatic cells holds great promise for cell replacement therapies. However, it is unclear if in vitro hESC differentiation causes any epigenetic abnormality such as hypermethylation of CpG islands. Using a differential methylation hybridization method, we identified 65 CpG islands (out of 4608 CpG islands or 1.4%) that exhibited increased DNA methylation during the conversion of hESCs into neural progenitor/stem cells (NPCs). These methylated CpG islands belong to genes in cell metabolism, signal transduction and cell differentiation, which are distinctively different from oncogenic CpG island hypermethylation observed in cancer-related genes during tumorigenesis. We further determined that methylation in these CpG islands, which is probably triggered by de novo DNA methyltransferase Dnmt3a, is abnormally higher in hESC-NPCs than in primary NPCs and astrocytes. Correlating with hypermethylation in promoter CpG islands of metabolic enzyme gene CPT1A and axoneme apparatus gene SPAG6, levels of CPT1A and SPAG6 mRNAs are significantly reduced in hESC-NPCs when compared with hESCs or primary neural cells. Because CPT1A is involved in lipid metabolism and CPT1A deficiency in human is associated with the hypoketotic hypoglycemia disorder, the reduced CPT1A expression in hESC-NPCs raises a potential concern for the suitability of these cells in cell transplantation. Collectively, our data show that abnormal CpG island methylation takes place in a subset of genes during the differentiation/expansion of hESC derivatives under current culture conditions, which may need to be monitored and corrected in future cell transplantation studies.     

4株乳腺癌细胞中内皮素受体B基因的甲基化状态及对MCF-7细胞增殖的影响

目的 探讨内皮素受体B（EDNRB）基因在4株乳腺癌细胞中的表达、甲基化状态以及恢复EDNRB表达对MCF-7细胞增殖的影响。 方法 采用甲基化特异性PCR（MS-PCR）和亚硫酸盐测序法（BSP）分析4株乳腺癌细胞中EDNRB的甲基化状态;反转录聚合酶链反应（RT-PCR）检测EDNRB mRNA的表达水平;采用四甲基偶氮唑蓝（MTT）法和集落形成实验检测EDNRB表达恢复对MCF-7细胞增殖的影响。 结果 EDNRB在乳腺癌细胞MCF-7和ZR-75-1中表达缺失,并呈高甲基化状态;而在EDNRB表达最高的MDA-MB-231细胞中其启动子呈低甲基化,表明乳腺癌细胞中EDNRB基因启动子甲基化状态与其表达成负相关。5-氮杂胞苷（5-Aza-CR）能够反转EDNRB基因的表达,EDNRB表达恢复后MCF-7细胞的增殖受到抑制。 结论 EDNRB基因启动子区CpG岛频繁甲基化可能在乳腺癌发生发展中发挥重要作用,EDNRB有望成为乳腺癌早期诊断的新的分子标志物。     

Identification of Novel Methylation Markers in Hepatocellular Carcinoma using a Methylation Array

Promoter CpG island hypermethylation has become recognized as an important mechanism for inactivating tumor suppressor genes or tumor-related genes in human cancers of various tissues. Gene inactivation in association with promoter CpG island hypermethylation has been reported to be four times more frequent than genetic changes in human colorectal cancers. Hepatocellular carcinoma is also one of the human cancer types in which aberrant promoter CpG island hypermethylation is frequently found. However, the number of genes identified to date as hypermethylated for hepatocellular carcinoma (HCC) is fewer than that for colorectal cancer or gastric cancer, which can be attributed to fewer attempts to perform genome-wide methylation profiling for HCC. In the present study, we used bead-array technology and coupled methylation-specific PCR to identify new genes showing cancer-specific methylation in HCC. Twenty-four new genes have been identified as hypermethylated at their promoter CpG island loci in a cancer-specific manner. Of these, TNFRSF10C, HOXA9, NPY, and IRF5 were frequently hypermethylated in hepatocellular carcinoma tissue samples and their methylation was found to be closely associated with inactivation of gene expression. Further study will be required to elucidate the clinicopathological implications of these newly found DNA methylation markers in hepatocellular carcinoma.     

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.     

Loss of TET2 in hematopoietic cells leads to DNA hypermethylation of active enhancers and induction of leukemogenesis

DNA methylation is tightly regulated throughout mammalian development, and altered DNA methylation patterns are a general hallmark of cancer. The methylcytosine dioxygenase TET2 is frequently mutated in hematological disorders, including acute myeloid leukemia (AML), and has been suggested to protect CG dinucleotide (CpG) islands and promoters from aberrant DNA methylation. In this study, we present a novel Tet2-dependent leukemia mouse model that closely recapitulates gene expression profiles and hallmarks of human AML1-ETO-induced AML. Using this model, we show that the primary effect of Tet2 loss in preleukemic hematopoietic cells is progressive and widespread DNA hypermethylation affecting up to 25% of active enhancer elements. In contrast, CpG island and promoter methylation does not change in a Tet2-dependent manner but increases relative to population doublings. We confirmed this specific enhancer hypermethylation phenotype in human AML patients with TET2 mutations. Analysis of immediate gene expression changes reveals rapid deregulation of a large number of genes implicated in tumorigenesis, including many down-regulated tumor suppressor genes. Hence, we propose that TET2 prevents leukemic transformation by protecting enhancers from aberrant DNA methylation and that it is the combined silencing of several tumor suppressor genes in TET2 mutated hematopoietic cells that contributes to increased stem cell proliferation and leukemogenesis.     

DNA methylation patterns in hereditary human cancers mimic sporadic tumorigenesis.

Cancer cells have aberrant patterns of DNA methylation including hypermethylation of gene promoter CpG islands and global demethylation of the genome. Genes that cause familial cancer, as well as other genes, can be silenced by promoter hypermethylation in sporadic tumors, but the methylation of these genes in tumors from kindreds with inherited cancer syndromes has not been well characterized. Here, we examine CpG island methylation of 10 genes (hMLH1, BRCA1, APC, LKB1, CDH1, p16(INK4a), p14(ARF), MGMT, GSTP1 and RARbeta2) and 5-methylcytosine DNA content, in inherited (n = 342) and non-inherited (n = 215) breast and colorectal cancers. Our results show that singly retained alleles of germline mutated genes are never hypermethylated in inherited tumors. However, this epigenetic change is a frequent second "hit", associated with the wild-type copy of these genes in inherited tumors where both alleles are retained. Global hypomethylation was similar between sporadic and hereditary cases, but distinct differences existed in patterns of methylation at non-familial genes. This study demonstrates that hereditary cancers "mimic" the DNA methylation patterns present in the sporadic tumors.     

启动子区5′CpG岛去甲基化对人结肠癌细胞生物学表型的影响

目的：探讨DNA启动子区5′CpG岛甲基化状态与人结肠癌RKO细胞增殖凋亡等生物学特征的关系。方法： 应用特异性DNA甲基转移酶（DNMTs）抑制剂-5-氮-2′-脱氧胞苷（5-Aza-2′-deoxycytidine，5-Aza-CdR）处理肠癌RKO细胞72 h，甲基化特异性PCR（methylation-specific PCR,MSP）及DNA测序法分析p16/CDKN2基因CpG岛甲基化状态；MTT、FCM、荧光染色及透射电镜检测启动子区去甲基化后细胞生长、形态和细胞周期凋亡的影响。 结果： DNMTs抑制剂能较好地逆转启动子区胞嘧啶甲基化状态；CpG岛去甲基化后能明显地抑制肠癌细胞的生长，增加细胞群体倍增时间（P<0.01），诱导肠癌细胞凋亡，影响肠癌细胞周期分布，并具有良好的量效依赖关系。 结论： 通过逆转CpG岛高甲基化能有效地抑制肠癌细胞增殖，为临床治疗大肠癌提供新的作用靶点。     

Reactivation of the silenced and imprinted alleles of ARHI is associated with increased histone H3 acetylation and decreased histone H3 lysine 9 methylation

ARHI has been identified as a maternally imprinted tumor suppressor gene that maps to chromosome 1p31 and whose expression is markedly down-regulated in breast cancer. To explore possible mechanisms that could silence ARHI expression, we have tested the importance of DNA methylation, histone acetylation and histone methylation in regulating ARHI expression. We found that treatment with CpG demethylating agents and/or histone deacetylase inhibitors could reactivate both the silenced and the imprinted alleles of this tumor suppressor gene. Reactivation of ARHI expression by these reagents is related to the methylation status of the CpG islands in the ARHI promoter, especially CpG island II. Chromatin immunoprecipitation assays revealed that histone H3 lysine 9/18 acetylation levels associated with ARHI in normal cells were significantly higher than those in breast cancer cell lines that lacked ARHI expression. Treatment with a CpG demethylating agent and/or histone deacetylase inhibitor could increase ARHI expression in breast cancer cells, with a corresponding increase in histone H3 lysine 9/18 acetylation and decrease in histone H3 lysine 9 methylation.