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.     

Hypermethylation of the human telomerase catalytic subunit (hTERT) gene correlates with telomerase activity

DNA methylation is an epigenetic process involved in embryonic development, differentiation and aging. It is 1 of the mechanisms resulting in gene silencing in carcinogenesis, especially in tumor suppressor genes (e.g., p16, Rb). Telomerase, the DNA polymerase adding TTAGGG repeats to the chromosome end, is involved in the regulation of the replicative life span by maintaining telomere length. This enzyme is activated in germ and stem cells, repressed in normal somatic cells and reactivated in a large majority of tumor cells. The promoter region of the hTERT gene, encoding for the catalytic subunit of human telomerase, has been located in a CpG island and may therefore be regulated at least in part by DNA methylation. We analyzed the methylation status of 27 CpG sites within the hTERT promoter core region by methylation-sensitive single-strand conformation analysis (MS-SSCA) and direct sequencing using bisulfite-modified DNA in 56 human tumor cell lines, as well as tumor and normal tissues from different organs. A positive correlation was observed among hypermethylation of the hTERT promoter, hTERT mRNA expression and telomerase activity (p < 0.00001). Furthermore, this correlation was confirmed in normal tissues where hypermethylation of the hTERT promoter was found exclusively in hTERT-expressing telomerase-positive samples and was absent in telomerase-negative samples (p < 0.00002). Since tumor tissues contain also nonneoplastic stromal elements, we performed microdissection to allow confirmation that the hTERT promoter methylation truly occurred in tumor cells. Our results suggest that methylation may be involved in the regulation of hTERT gene expression. To our knowledge, this is the first gene in which methylation of its promoter sequence has been found to be positively correlated with gene expression.     

Methylation of the human telomerase gene CpG island

The acquisition of expression of hTERT, the catalytic subunit of the telomerase enzyme, seems to be an essential step in the development of a majority of human tumors. However, little is known about the mechanisms preventing telomerase gene expression in normal and transformed cells that do not express hTERT. Using a methylation-specific PCR-based assay, we have found that the CpG island associated with the hTERT gene is unmethylated in telomerase-negative primary tissues and nonimmortalized cultured cells, indicating that mechanisms independent of DNA methylation are sufficient to prevent hTERT expression. The hTERT CpG island is methylated in many telomerase-negative and telomerase-positive cultured cells and tumors, but the extent of methylation did not correlate with expression of hTERT. Demethylation of DNA with 5-azacytidine in two cell lines induced expression of hTERT, suggesting that DNA methylation can contribute to hTERT repression in some cells. Together, these data show that the hTERT CpG island can undergo cytosine methylation in cultured cells and tumors and that DNA methylation may contribute to the regulation of the hTERT gene, but that CpG island methylation is not responsible for repressing hTERT expression in most telomerase-negative cells.     

hTERT methylation is necessary but not sufficient for telomerase activity in colorectal cells

Colorectal cancers exhibit a high telomerase activity, usually correlated with the hypermethylation of the promoter of its hTERT catalytic subunit. Although telomerase is not expressed in normal tissue, certain proliferative somatic cells such as intestinal crypt cells have demonstrated telomerase activity. The aim of this study was to determine whether a correlation exists between telomerase activity, levels of hTERT methylation and telomere length in tumoral and normal colorectal tissues. Tumor, transitional and normal tissues were obtained from 11 patients with a colorectal cancer. After bisulfite modification of genomic DNA, hTERT promoter methylation was analyzed by methylation-sensitive single-strand conformation analysis (MS-SSCA). Telomerase activity and telomere length were measured by a fluorescent-telomeric repeat amplification protocol assay and by Southern blotting, respectively. A significant increase of hTERT methylation and telomerase activity, and a reduction of the mean telomere length were observed in the tumor tissues compared to the transitional and normal mucosa. In the transitional and normal mucosa, telomerase activity was significantly lower than that in tumor tissues, even with high levels of hTERT methylation. Nevertheless, hTERT promoter methylation was not linearly correlated to telomerase activity. These data indicate that hTERT promoter methylation is a necessary event for hTERT expression, as is telomerase activity. However, methylation is not sufficient for hTERT activation, particularly in normal colorectal cells.     

Frequent inactivation of SPARC by promoter hypermethylation in colon cancers

Epigenetic modification of gene expression plays an important role in the development of human cancers. The inactivation of SPARC through CpG island methylation was studied in colon cancers using oligonucleotide microarray analysis and methylation specific PCR (MSP). Gene expression of 7 colon cancer cell lines was evaluated before and after treatment with the demethylating agent 5-aza-2'-deoxycytidine (5Aza-dC) by oligonucleotide microarray analysis. Expression of SPARC was further examined in colon cancer cell lines and primary colorectal cancers, and the methylation status of the SPARC promoter was determined by MSP. SPARC expression was undetectable in 5 of 7 (71%) colorectal cancer cell lines. Induction of SPARC was demonstrated after treatment with the demethylating agent 5Aza-dC in 5 of the 7 cell lines. We examined the methylation status of the CpG island of SPARC in 7 colon cancer cell lines and in 20 test set of colon cancer tissues. MSP demonstrated hypermethylation of the CpG island of SPARC in 6 of 7 cell lines and in all 20 primary colon cancers, when compared with only 3 of 20 normal colon mucosa. Immunohistochemical analysis showed that SPARC expression was downregulated or absent in 17 of 20 colon cancers. A survival analysis of 292 validation set of colorectal carcinoma patients revealed a poorer prognosis for patients lacking SPARC expression than for patients with normal SPARC expression (56.79% vs. 75.83% 5-year survival rate, p = 0.0014). The results indicate that epigenetic gene silencing of SPARC is frequent in colon cancers, and that inactivation of SPARC is related to rapid progression of colon cancers.     

抗凋亡基因bcl-2启动子CpG岛的高甲基化状态见于28.3%(13/46例)的膀胱癌患者尿沉淀细胞DNA

目的:检测上海地区膀胱癌患者尿沉淀细胞中3个肿瘤相关基因启动子CpG岛的甲基化谱式异常频率,继而评估其应用前景。方法:采用甲基化特异性PCR方法分析尿沉淀细胞DNA中DAPK1、bcl2和hTERT3个基因的启动子CpG岛甲基化状态,并通过RTPCR方法评估DAPK1基因在膀胱癌细胞系中的表达状态。结果:对膀胱癌细胞系中DAPK1基因的启动子CpG岛甲基化状态及其表达(mRNA水平上)所做的分析,确立了高甲基化状态与表达静息化之间的相关性。对46例临床确诊的膀胱癌患者和84例非膀胱癌对照(包括前46例术后的36例)的尿沉淀细胞中DNA甲基化的分析发现,仅bcl2基因的高甲基化见之于28.3%(13/46例)的膀胱癌患者,而84例的对照中均为去甲基化状态。结论:在美国膀胱癌患者尿沉淀细胞中频发DNA高甲基化的靶点在上海地区膀胱癌患者人群中频率很低,因此寻找在后者中频发DNA高甲基化的新靶点实属必要。     

CpG island promoter hypermethylation of a novel Ras-effector gene RASSF2A is an early event in colon carcinogenesis and correlates inversely with K-ras mutations

We report in silico identification and characterisation of a novel member of the ras association domain family 1 (RASSF1)/NORE1 family, namely, RASSF2, located at chromosomal region 20p13. It has three isoforms, all contain a ras association domain in the C-terminus. The longest isoform RASSF2A contains a 5' CpG island. RASSF2A was cloned from a brain cDNA library and directly sequenced, confirming the genomic gene structure. In previous reports, we and others have demonstrated that RASSF1A is epigenetically inactivated in a variety of cancers, including sporadic colorectal cancer (CRC). In the present report, we analysed the methylation status of RASSF2A promoter region CpG island in sporadic CRC and compared it to K-ras mutation status. RASSF2A promoter region CpG island was hypermethylated in a majority of colorectal tumour cell lines (89%) and in primary colorectal tumours (70%), while DNA from matched normal mucosa was found to be unmethylated (tumour-specific methylation). RASSF2A expression was reactivated in methylated tumour cell lines after treatment with 5-aza 2-deoxycytidine. RASSF2A methylation is an early event, detectable in 7/8 colon adenomas. Furthermore, 75% of colorectal tumours with RASSF2A methylation had no K-ras mutations (codons, 12 and 13) (P=0.048), Fisher's exact test). Our data demonstrate that RASSF2A is frequently inactivated in CRCs by CpG island promoter hypermethylation, and that epigenetic (RASSF2A) and genetic (K-ras) changes are mutually exclusive and provide alternative pathways for affecting Ras signalling.