Epigenetic silencing of PEG3 gene expression in human glioma cell lines

Genomic imprinting, the phenomenon in which alleles of genes are expressed differentially depending on their parental origins, has important consequences for mammalian development, and disturbance of normal imprinting leads to abnormal embryogenesis and some inherited diseases and is also associated with various cancers. In the context of screening for novel imprinted genes on human chromosome 19q13.4 with mouse A9 hybrids, we identified a maternal allele-specific methylated CpG island in exon 1 of paternally expressed imprinted gene 3 (PEG3), a gene that exhibits paternal allele-specific expression. Because PEG3 expression is downregulated in some gliomas and glioma cell lines, despite high-level expression in normal brain tissues, we investigated whether the loss of PEG3 expression is related to epigenetic modifications involving DNA methylation. We found monoallelic expression of PEG3 in all normal brain tissues examined and five of nine glioma cell lines that had both unmethylated and methylated alleles; the remaining four glioma cell lines exhibited gain of imprinting with hypermethylated alleles. In addition, treatment of glioma cell lines with the DNA demethylating agent 5-aza-2'-deoxycytidine reversed the silencing of PEG3 biallelically. In this article, we report that the epigenetic silencing of PEG3 expression in glioma cell lines depends on aberrant DNA methylation of an exonic CpG island, suggesting that PEG3 contributes to glioma carcinogenesis in certain cases.     

Less frequent promoter hypermethylation of DLC-1 gene in primary breast cancers

Absence or low expression of DLC-1, a tumor suppressor gene, in breast cancers has been shown recently. LOH of 8p12-p22, on which DLC-1 is located, is frequent in breast cancers, but the correlation between low expression of DLC-1 and LOH has not been confirmed. To determine the implication of aberrant methylation, one of the most frequent mechanisms of silencing the tumor suppressor or cancer-related genes, we examined the methylation status of DLC-1 promoter region in breast cancer cell lines and primary breast tumors. The hypermethylation status was examined by MSP and 25% of cell lines harbored a methylated allele. The gene silencing by methylation was also confirmed by the re-expression of DLC-1 by the 5-aza-2'-deoxycytidine treatment in DLC-1 hypermethylated cell line. But the methylation of DLC-1 gene was less frequently shown in primary breast cancers (10%). These data suggest that hypermethylation is responsible for silencing of DLC-1 gene in a limited portion of breast cancers.     

Genome-wide promoter analysis uncovers portions of the cancer methylome

DNA methylation has a role in mediating epigenetic silencing of CpG island genes in cancer and other diseases. Identification of all gene promoters methylated in cancer cells "the cancer methylome" would greatly advance our understanding of gene regulatory networks in tumorigenesis. We previously described a new method of identifying methylated tumor suppressor genes based on pharmacologic unmasking of the promoter region and detection of re-expression on microarray analysis. In this study, we modified and greatly improved the selection of candidates based on new promoter structure algorithm and microarray data generated from 20 cancer cell lines of 5 major cancer types. We identified a set of 200 candidate genes that cluster throughout the genome of which 25 were previously reported as harboring cancer-specific promoter methylation. The remaining 175 genes were tested for promoter methylation by bisulfite sequencing or methylation-specific PCR (MSP). Eighty-two of 175 (47%) genes were found to be methylated in cell lines, and 53 of these 82 genes (65%) were methylated in primary tumor tissues. From these 53 genes, cancer-specific methylation was identified in 28 genes (28 of 53; 53%). Furthermore, we tested 8 of the 28 newly identified cancer-specific methylated genes with quantitative MSP in a panel of 300 primary tumors representing 13 types of cancer. We found cancer-specific methylation of at least one gene with high frequency in all cancer types. Identification of a large number of genes with cancer-specific methylation provides new targets for diagnostic and therapeutic intervention, and opens fertile avenues for basic research in tumor biology.     

Aberrant CpG island methylation in cancer cell lines arises in the primary cancers from which they were derived

A higher prevalence of epigenetic inactivation of tumor suppressor genes has been reported in cancer cell line populations compared to primary cancer populations. Cancer-related genes are commonly methylated in cancer cell lines but it is not known the extent to which tumor suppressor genes may be artificially methylated in vitro. We therefore examined 10 pancreatic cancer cell lines and corresponding primary tumors for aberrant DNA methylation of promoter CpG islands of eight genes and seven CpG islands. Using methylation-specific PCR (MSP), methylation was not detected at any of the 15 CpG islands in 15 normal pancreata or in an immortalized normal pancreatic duct epithelial (HPDE) cell line. Of 150 loci examined, 49 loci were methylated in both primary carcinomas and their corresponding cell lines, 95 loci were not methylated in either cell lines or their corresponding primary carcinomas. There were four loci methylated only in cell lines while another two loci were methylated only in primary carcinomas. Overall, the methylation status of primary carcinomas and their cell lines were concordant in 96% of cases (144 of 150) (J statistic; J=0.92, P<0.0001). We conclude that most of the DNA methylation of tumor suppressor genes observed in cancer cell lines is present in the primary carcinomas from which they were derived.     

CpG island arrays: an application toward deciphering epigenetic signatures of breast cancer.

CpG island hypermethylation is a frequent epigenetic event in cancer. We have recently developed an array-based method, called differential methylation hybridization (DMH), allowing for a genome-wide screening of CpG island hypermethylation in breast cancer cell lines (T. H-M. Huang et al., Hum. Mol. Genet., 8: 459-470, 1999). In the present study, DMH was applied to screen 28 paired primary breast tumor and normal samples and to determine whether patterns of specific epigenetic alterations correlate with pathological parameters in the patients analyzed. Amplicons, representing a pool of methylated CpG DNA derived from these samples, were used as hybridization probes in an array panel containing 1104 CpG island tags. Close to 9% of these tags exhibited extensive hypermethylation in the majority of breast tumors relative to their normal controls, whereas others had little or no detectable changes. Pattern analysis in a subset of CpG island tags revealed that CpG island hypermethylation is associated with histological grades of breast tumors. Poorly differentiated tumors appeared to exhibit more hypermethylated CpG islands than their moderately or well-differentiated counterparts (P = 0.041). This early finding lays the groundwork for a population-based DMH study and demonstrates the need to develop a database for examining large-scale methylation data and for associating specific epigenetic signatures with clinical parameters in breast cancer.     

Identification of genes uniquely involved in frequent microsatellite instability colon carcinogenesis by expression profiling combined with epigenetic scanning

Gene silencing through CpG island hypermethylation has been associated with genesis or progression of frequent microsatellite instability (MSI-H) cancers. To identify novel methylation sites unique to MSI-H colon cancers in an unbiased fashion, we conducted a global expression profiling-based methylation target search. We identified 81 genes selectively down-regulated in MSI-H cancers using cDNA microarray analysis of 41 primary colon cancers. Forty six of these 81 genes contained CpG islands overlapping their 5'untranslated regions. Initial screening of six genes in 57 primary colon cancers detected the following gene with MSI-H cancer-specific hypermethylation: RAB32, a ras family member and A-kinase-anchoring protein, was methylated in 14 of 25 (56%) MSI-H cancers but in none of 32 non-MSI-H cancers or 23 normal colonic specimens. RAB32 hypermethylation correlated with RAB32 mRNA down-regulation and with hMLH1 hypermethylation. In addition, the protein-tyrosine phosphatase receptor type O gene, PTPRO, was frequently methylated in right-sided tumors. This methylation screening strategy should identify additional genes inactivated by epigenetic silencing in colorectal and other cancers.     

CpG island hypermethylation and tumor suppressor genes: a booming present,a brighter future

We have come a long way since the first reports of the existence of aberrant DNA methylation in human cancer. Hypermethylation of CpG islands located in the promoter regions of tumor suppressor genes is now firmly established as an important mechanism for gene inactivation. CpG island hypermethylation has been described in almost every tumor type. Many cellular pathways are inactivated by this type of epigenetic lesion: DNA repair (hMLH1, MGMT), cell cycle (p16(INK4a), p15(INK4b), p14(ARF)), apoptosis (DAPK), cell adherence (CDH1, CDH13), detoxification (GSTP1), etc em leader However, we still know little of the mechanisms of aberrant methylation and why certain genes are selected over others. Hypermethylation is not an isolated layer of epigenetic control, but is linked to the other pieces of the puzzle such as methyl-binding proteins, DNA methyltransferases and histone deacetylase, but our understanding of the degree of specificity of these epigenetic layers in the silencing of specific tumor suppressor genes remains incomplete. The explosion of user-friendly technologies has given rise to a rapidly increasing list of hypermethylated genes. Careful functional and genetic studies are necessary to determine which hypermethylation events are truly relevant for human tumorigenesis. The development of CpG island hypermethylation profiles for every form of human tumors has yielded valuable pilot clinical data in monitoring and treating cancer patients based in our knowledge of DNA methylation. Basic and translational will both be needed in the near future to fully understand the mechanisms, roles and uses of CpG island hypermethylation in human cancer. The expectations are high.     

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.