Methylation Patterns Of CpG Islands Research Articles

CpG Island methylation patterns in various histotypes of endometrial cancer

Our laboratory is interested in defining molecular pathways that help distinguish various histotypes of endometrial cancer. Type 1 endometrial cancer (low grade endometrioid adenocarcinoma) is characterized by superficial myometrial invasion, low clinical stage, and excellent prognosis. Type 2 endometrial cancer (grade 3 endometrioid adenocarcinoma, uterine papillary serous carcinoma, and malignant mixed mullerian tumor) is associated with deep myometrial invasion, metastasis, advanced clinical stage, and poor prognosis. One approach that we have used to define the molecular characteristics of these endometrial tumors is to analyze CpG island methylation patterns. Gene methylation is an important mechanism of tumor suppressor gene inactivation in a wide variety of neoplasms. We have modified existing techniques for methylation-specific PCR for effective use in formalin-fixed, paraffin-embedded tissues. To date, we have analyzed a large series (greater than 100) different endometrial tumors for gene methylation patterns, using a panel of 12 different genes. A subset of the data for endometrioid tumors and uterine MMMT will be presented at the symposium. The endometrioid and MMMT groups have similar percentages of tumors with a methylator phenotype (tumor with two or more genes methylated). However, the pattern of gene methylation was distinct for several genes. Similar to previous studies, we found methylation of MLH1 and resulting microsatellite instability to be extremely uncommon in uterine MMMT compared to endometrioid adenocarcinoma. Methylation of MINT 31, a common event in endometrioid tumors, is also significantly less common in MMMT. Several important genes showed similar methylation frequencies in the endometrioid and MMMT groups. These include apoptosis-promoting genes (DAP-K and RASSF1) and cell cycle regulatory genes (p14 and p16). RASSF1, which encodes a pro-apoptotic protein downstream from RAS, is methylated in a majority of the endometrioid tumors and MMMT. These results are intriguing, especially considering that the established frequency of RAS mutation in endometrial cancer is relatively low (12–25% of cases). It has been proposed by several different groups that RAS mutation and methylation of RASSF1 may be mutually exclusive events. Our preliminary findings for endometrial cancer certainly support this assertion. Finally, analysis of COX-2 methylation has yielded exciting results. Previously, it had been proposed that induction of COX-2 leads to PGE2 synthesis, which causes induction of aromatase. Increased aromatase levels contribute to increased local production of estrogens, which contributes to tumor cell growth. Therefore, we were surprised to find that nearly half of the endometrioid tumors and MMMT demonstrated methylation of COX-2. Since gene methylation usually implies decreased gene expression, these findings did not support the “COX-2/aromatase/estrogen” pathway in endometrial cancer. Quantitative PCR and immunohistochemistry supported these findings. Collectively, these results suggest that elevated COX-2 is not an important mechanism in at least half of endometrial cancers.

Expression of PRV-1 Decreases Dependency of Cells on Growth Factors for Proliferation.

Polycythemia rubra vera-1 (PRV-1) is a GPI-linked protein expressed on surface of neutrophils. Polycythemia vera and essential thrombocythemia were found to be associated with an increase in the level of PRV-1 mRNA; however, the function of PRV-1 remains unknown. We have studied the functional effect of expression of PRV-1, both in heterologous cell line and in neutrophils. cDNA encoding PRV-1 was subcloned from a leukocyte cDNA library and expressed in Chinese hamster ovary cells (CHO). After seeding equal numbers of CHO cells stably transfected with PRV-1 or empty plasmid, cell count was conducted at different time intervals and under different concentration of fetal bovine serum (FBS) in growth media. The numbers of PRV-1 transfected cells were higher than sham-transfected cells at 24, 48, 96 and 144 hrs after seeding, and this difference was enhanced with cells growing in a medium with a reduced concentration of serum with the maximal effect seen in serum-free medium. The percentage of apoptotic cells was similar between PRV-1 and sham-transfected cells. These results are suggestive of that CHO cells expressing PRV-1 proliferate faster than sham-transfected cells. The difference in proliferation rate was more significant at lower concentrations of serum and was the highest in serum-free medium. An immunoblot analysis with anti-phosphotyrosine antibody on the whole cell lysate demonstrated that the presence of PRV-1 alters cell signaling. A 60kDa protein band that disappeared after removal of serum in growth medium of sham-transfected cells continued to be present in the PRV-1 transfected cell lysate regardless of the presence or absence of FBS in the media. In order to understand the function of PRV-1 in neutrophils, we used the fact that in 85% of individuals PRV-1 is expressed only by a subgroup of neutrophils. We separated PRV-1 expressing neutrophils of an individual from those lacking this protein in the same donor, using anti-PRV-1 monoclonal antibody. We then compared the gene expression profile of the two groups of neutrophil using DNA microarray technique. Expression of PRV-1 was associated with several folds increase in expression of growth factors (fracture callus-1, X 14.9 times; Insulin-like 3, X 6.7; neural proliferation differentiation, and control-1 or NPDC1, X 3.4 times) and post-translational modifying enzyme of cell surface protein (N-acetylase N sulfotransferase-1, X 18.9 times). Under physiologic conditions, transcriptional regulation of the PRV-1 gene limits its expression to only a certain percentage of neutrophils. In order to investigate the role of epigenetic factors in regulation of transcription of the PRV-1 gene, we analyzed methylation of CpG dinucleotide in promoter of the PRV-1 gene in the genomic DNA obtained from neutrophils expressing PRV-1 to those that don't express this protein in the same individual. Expression of PRV-1 was associated with a decrease in methylation of the CpG dinucleotide located 2937 bp before initiation codon (45% methylated in PRV-1 negative neutrophils compared to 30% methylated in PRV positive neutrophils). We are currently studying the methylation pattern of CpG islands inside the PRV-1 gene. Studying the effect of DNA methylation on expression of PRV-1 in physiologic conditions, may shed light to the mechanism of overexpression of PRV-1 in MPD.

Methylation profiles of thirty four promoter-CpG islands and concordant methylation behaviours of sixteen genes that may contribute to carcinogenesis of astrocytoma.

BackgroundAstrocytoma is a common aggressive intracranial tumor and presents a formidable challenge in the clinic. Association of altered DNA methylation patterns of the promoter CpG islands with the expression profile of cancer-related genes, has been found in many human tumors. Therefore, DNA methylation status as such may serve as an epigenetic biomarker for both diagnosis and prognosis of human tumors, including astrocytoma.MethodsWe used the methylation specific PCR in conjunction with sequencing verification to establish the methylation profile of the promoter CpG island of thirty four genes in astrocytoma tissues from fifty three patients (The WHO grading:. I: 14, II: 15, III: 12 and IV: 12 cases, respectively). In addition, compatible tissues (normal tissues distant from lesion) from three non-astrocytoma patients were included as the control.ResultsSeventeen genes (ABL, APC, APAF1, BRCA1, CSPG2, DAPK1, hMLH1, LKB1, PTEN, p14ARF, p15INK4b, p27KIP1, p57KIP2, RASSF1C, RB1, SURVIVIN, and VHL) displayed a uniformly unmethylated pattern in all the astrocytoma and non-astrocytoma tissues examined. However, the MAGEA1 gene that was inactivated and hypermethylated in non-astrocytoma tissues, was partially demethylated in 24.5% of the astrocytoma tissues (co-existence of the hypermethylated and demethylated alleles). Of the astrocytoma associated hypermethylated genes, the methylation pattern of the CDH13, cyclin a1, DBCCR1, EPO, MYOD1, and p16INK4a genes changed in no more than 5.66% (3/53) of astrocytoma tissues compared to non-astrocytoma controls, while the RASSF1A, p73, AR, MGMT, CDH1, OCT6,, MT1A, WT1, and IRF7 genes were more frequently hypermethylated in 69.8%, 47.2%, 41.5%, 35.8%, 32%, 30.2%, 30.2%, 30.2% and 26.4% of astrocytoma tissues, respectively. Demethylation mediated inducible expression of the CDH13, MAGEA1, MGMT, p73 and RASSF1A genes was established in an astrocytoma cell line (U251), demonstrating that expression of these genes is likely regulated by DNA methylation. AR gene hypermethylation was found exclusively in female patients (22/27, 81%, 0/26, 0%, P < 0.001), while the IRF7 gene hypermethylation preferentially occurred in the male counterparts (11/26, 42.3% to 3/27, 11%, P < 0.05). Applying the mathematic method "the Discovery of Association Rules", we have identified groups consisting of up to three genes that more likely display the altered methylation patterns in concert in astrocytoma.ConclusionsOf the thirty four genes examined, sixteen genes exhibited astrocytoma associated changes in the methylation profile. In addition to the possible pathological significance, the established concordant methylation profiles of the subsets consisting of two to three target genes may provide useful clues to the development of the useful prognostic as well as diagnostic assays for astrocytoma.

Methylation profile of the promoter CpG islands of 14 “drug-resistance” genes in hepatocellular carcinoma

To establish the DNA methylation patterns of the promoter CpG islands of 14 "drug-resistance" genes in hepatocellular carcinoma (HCC). The methylation specific polymerase chain reaction in conjunction with sequencing verification was used to establish the methylation patterns of the 14 genes in the liver tissues of four healthy liver donors, as well as tumor and the paired non-cancerous tissues of 30 HCC patients. While 11 genes (ATP-binding cassette, sub-family G (WHITE), member 2(ABCG2), activating transcription factor (ATF2), beta-2-microglobulin (B2M), deoxycytidine kinase (DCK), occludin (OCLN), v-raf-1 murine leukemia viral oncogene homolog (RAF1), ralA binding protein 1 (RALBP1), splicing factor (45 kD) (SPF45), S-phase kinase-associated protein 2 (p45) (SKP2), tumor protein p53 (Li-Fraumeni syndrome) (TP53) and topoisomerase (DNA) II beta (TOP2B)) maintained the unmethylated patterns, three genes displayed to various extents the hypermethylation state in tumor tissues in comparison with the normal counterparts. The catalase (CAT) was hypermethylated in tumor and the neighboring non-cancerous tissue of one case (3.3%). Both glutathione S-transferase pi (GSTpi) (80%, 24/30 in tumor and 56.7%, 17/30 in the paired non-cancerous tissues) and cystic fibrosis transmembrane conductance regulator, ATP-binding cassette (sub-family C, member 7) (CFTR) (77%, 23/30 in tumor and 50%, 15/30 in the paired non-cancerous tissues) genes were prevalently hypermethylated in HCC as well as their neighboring non-cancerous tissues. No significant difference in the hypermethylation occurrence was observed between the HCC and its neighboring non-cancerous tissues. Hypermethylation of promoter CpG islands of both CFTR and GSTpi genes occurs prevalently in HCC, which may correlate with the low expression of these two genes at the mRNA level and has the profound etiological and clinical implications. It is likely to be specific to the early phase of HCC carcinogenesis.

Endothelin B receptor gene hypermethylation in prostate adenocarcinoma

Background: Alterations in the methylation patterns of promoter CpG islands have been associated with the transcriptional inhibition of genes in many human cancers. These epigenetic alterations could be used as...

Epigenetic marks by DNA methylation specific to stem, germ and somatic cells in mice.

DNA methylation is involved in many gene functions such as gene-silencing, X-inactivation, imprinting and stability of the gene. We recently found that some CpG islands had a tissue-dependent and differentially methylated region (T-DMR) in normal tissues, raising the possibility that there may be more CpG islands capable of differential methylation. We investigated the genome-wide DNA methylation pattern of CpG islands by restriction landmark genomic scanning (RLGS) in mouse stem cells (ES, EG and trophoblast stem) before and after differentiation, and sperm as well as somatic tissues. A total of 247 spots out of 1500 (16%) showed differences in the appearance of their RLGS profiles, indicating that CpG islands having T-DMR were numerous and widespread. The methylation pattern was specific, and varied in a precise manner according to cell lineage, tissue type and during cell differentiation. Genomic loci with altered methylation status seem to be more common than has hitherto been realized. The formation of DNA methylation patterns at CpG islands is one of the epigenetic events which underlies the production of various cell types in the body. These findings should have implications for the use of embryonic stem cells and cells derived from them therapeutically, and also for the cloning of animals by the transfer of somatic cell nuclei.

Methyl-CpG Binding Domain Protein 2 Represses Transcription from Hypermethylated π-Class Glutathione S-Transferase Gene Promoters in Hepatocellular Carcinoma Cells

During the pathogenesis of human hepatocellular carcinoma (HCC), the CpG island encompassing the pi-class glutathione S-transferase gene (GSTP1) becomes hypermethylated. Repression of transcription accompanying CpG island hypermethylation has been proposed to be mediated by methyl-CpG binding domain (MBD) proteins. We report here that inhibition of transcription from hypermethylated GSTP1 promoters in Hep3B HCC cells, which fail to express GSTP1 mRNA or GSTP1 polypeptides, appears to be mediated by MBD2. Treatment of Hep3B cells with 5-azadeoxycytidine (5-aza-dC), a methyltransferase inhibitor, activated GSTP1 expression, whereas treatment with trichostatin A, a histone deacetylase inhibitor, had little effect. To more precisely assess the contribution of the pattern of GSTP1 CpG island methylation on GSTP1 mRNA expression, Hep3B cells were treated for 72 h with 5-aza-dC and then subjected to limiting dilution cloning. Bisulfite sequencing was used to map the methylation patterns of the GSTP1 promoter region in GSTP1-expressing and -non-expressing clones. In the clone that expressed GSTP1 mRNA determined by Northern blot analysis and quantitative reverse transcriptase (RT)-PCR, widespread demethylation of at least one GSTP1 allele was evident. Chromatin immunoprecipitation experiments revealed the presence of MBD2, but not Sp1, at the GSTP1 promoter in Hep3B cells. In contrast, Sp1 was detected at the GSTP1 promoter in a GSTP1-expressing Hep3B 5-aza-dC subclone. To test whether MBD2 might be responsible for the inhibition of GSTP1 transcription from hypermethylated GSTP1 promoters, siRNAs were used to reduce MBD2 polypeptide levels in Hep3B cells. SssI-catalyzed methylation of GSTP1 promoter sequences resulted in diminished luciferase reporter activity after transfection into Hep3B cells. However, when hypermethylated GSTP1 promoter sequences were transfected into Hep3B cells that had been treated with siRNA-targeting MBD2 mRNA, no repression of luciferase reporter expression was evident. These findings implicate MBD2 in the repression of GSTP1 expression associated with GSTP1 CpG island hypermethylation in HCC cells.

Bisulfite genomic sequencing of microdissected cells.

Mapping of methylation patterns in CpG islands has become an important tool for understanding tissue-specific gene expression in both normal and pathological situations. However, the inherent cellular heterogeneity of any given tissues can affect the outcome and interpretation of molecular studies. In order to analyse genomic DNA methylation on a pure cell population from tissue sample, we have developed a simple technique of single-cell microdissection from cryostat sections which can be combined with bisulfite-mediated sequencing of 5-methylcytosine. We report here our results on the methylation status of the androgen receptor gene studied by bisulfite genomic sequencing on purified cells isolated from human testis.

DNA Methylation and Ovarian Cancer: I. Analysis of CpG Island Hypermethylation in Human Ovarian Cancer Using Differential Methylation Hybridization

Objective. The aim of this study was to examine CpG island methylation patterns in ovarian cancer and determine whether epigenetic information can be related to clinical data of patients. CpG island (CpGI) hypermethylation is commonly associated with cancer progression, but little is currently known about the role of methylation in ovarian cancer.Methods. Differential methylation hybridization (DMH) analysis at 742 loci was performed to determine methylation signatures for 20 primary epithelial ovarian carcinomas (Stages II, III, and IV adenocarcinomas, serous papillary), 6 ovarian cancer cell lines, and normal ovarian surface epithelial cells.Results. Between 23 and 108 methylated CpGIs were seen in the ovarian carcinomas. Fewer (P < 0.05) methylated CpGIs were observed in the ovarian cancer cell lines; however, a number of CpGIs were commonly hypermethylated in both the cell lines and the tumor samples. A methylation signature, consisting of frequently (P < 0.05) methylated CpGIs, was determined for the samples. The observed pattern of methylation in ovarian cancers included several (11) CpGI tags that were previously reported to be hypermethylated in human breast cancer.Conclusions. Epigenetic signatures in ovarian cancer were determined using DMH. This proof-of-concept study lays the foundation for genome-wide screening of methylation to examine epigenotype–phenotype relationships in ovarian cancer.

Aberrant CpG-island methylation has non-random and tumour-type-specific patterns.

CpG islands frequently contain gene promoters or exons and are usually unmethylated in normal cells. Methylation of CpG islands is associated with delayed replication, condensed chromatin and inhibition of transcription initiation. The investigation of aberrant CpG-island methylation in human cancer has primarily taken a candidate gene approach, and has focused on less than 15 of the estimated 45,000 CpG islands in the genome. Here we report a global analysis of the methylation status of 1,184 unselected CpG islands in each of 98 primary human tumours using restriction landmark genomic scanning (RLGS). We estimate that an average of 600 CpG islands (range of 0 to 4,500) of the 45,000 in the genome were aberrantly methylated in the tumours, including early stage tumours. We identified patterns of CpG-island methylation that were shared within each tumour type, together with patterns and targets that displayed distinct tumour-type specificity. The expression of many of these genes was reactivated by experimental demethylation in cultured tumour cells. Thus, the methylation of particular subsets of CpG islands may have consequences for specific tumour types.

CpG island methylator phenotypes in aging and cancer

CpG islands are short stretches of CpG rich regions that are frequently associated with the promoter region of genes. Aberrant methylation of CpG islands is one mechanism of inactivating tumor suppressor genes (TSGs) in neoplasia, and there is growing evidence that altered cytosine methylabtion play important roles in cancer development. However, the differences in global CpG island methylation patterns between normal and cancer cells remain poorly understood. By examining a large number of loci in a series of cancers, global methylation profiles can be constructed. Such studies revealed that in colorectal cancer, there appears to be two types of methylation that are associated with cancer progression: type A (for age-related) methylation, and type C (for cancer-specific) methylation. Initially, type A methylation arises as a function of age in normal colorectal epithelial cells. By affecting genes that regulate the growth and/or differentiation of these cells, such methylation may result in a predisposition state that precedes tumor formation in the colon. Type C methylation, by contrast, was found exclusively in a subset of cancers, which display a CpG island methylator phenotype (CIMP). CIMP is a novel molecular instability pathway that appears to be responsible for most cases of aberrant TSG methylation in colorectal cancer, and which has important interactions with genetic pathways as well. In fact, CIMP+ tumors account for the majority of sporadic colorectal cancers with microsatellite instability, through methylation of the mismatch repair gene hMLH1. This model whereby age-related methylation increases cell-susceptibility to transformation and cancer-specific methylabtion results in neoplastic progression in a subset of cases may be applicable to many human neoplasms.

CpG island methylator phenotype in colorectal cancer.

Aberrant methylation of promoter region CpG islands is associated with transcriptional inactivation of tumor-suppressor genes in neoplasia. To understand global patterns of CpG island methylation in colorectal cancer, we have used a recently developed technique called methylated CpG island amplification to examine 30 newly cloned differentially methylated DNA sequences. Of these 30 clones, 19 (63%) were progressively methylated in an age-dependent manner in normal colon, 7 (23%) were methylated in a cancer-specific manner, and 4 (13%) were methylated only in cell lines. Thus, a majority of CpG islands methylated in colon cancer are also methylated in a subset of normal colonic cells during the process of aging. In contrast, methylation of the cancer-specific clones was found exclusively in a subset of colorectal cancers, which appear to display a CpG island methylator phenotype (CIMP). CIMP+ tumors also have a high incidence of p16 and THBS1 methylation, and they include the majority of sporadic colorectal cancers with microsatellite instability related to hMLH1 methylation. We thus define a pathway in colorectal cancer that appears to be responsible for the majority of sporadic tumors with mismatch repair deficiency.

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.

Mapping Patterns of CpG Island Methylation in Normal and Neoplastic Cells Implicates Both Upstream and Downstream Regions inde Novo Methylation

Promoter region CpG island methylation is associated with tumor suppressor gene silencing in neoplasia. GenBank sequence analyses revealed that a number of CpG islands are juxtaposed to multiple Alu repeats, which have been proposed as "de novo methylation centers." These islands also contain multiple Sp1 elements located upstream and downstream of transcription start, which have been shown to protect CpG islands from methylation. We mapped the methylation patterns of the E-cadherin (E-cad) and von Hippel-Lindau (VHL) tumor suppressor gene CpG island regions in normal and neoplastic cells. Although unmethylated in normal tissue, these islands were embedded between densely methylated flanking regions containing multiple Alu repeats. These methylated flanks were segregated from the unmethylated, island CpG sites by Sp1-rich boundary regions. Finally, in human fibroblasts overexpressing DNA methyltransferase, de novo methylation of the E-cad CpG island initially involved sequences at both ends of the island and the adjacent, flanking regions and progressed with time to encompass the entire CpG island region. Together, these data suggest that boundaries exist at both ends of a CpG island to maintain the unmethylated state in normal tissue and that these boundaries may be progressively overridden, eliciting the de novo methylation associated with tumor suppressor gene silencing in neoplasia.

Methylation-specific PCR: a novel PCR assay for methylation status of CpG islands.

Precise mapping of DNA methylation patterns in CpG islands has become essential for understanding diverse biological processes such as the regulation of imprinted genes, X chromosome inactivation, and tumor suppressor gene silencing in human cancer. We describe a new method, MSP (methylation-specific PCR), which can rapidly assess the methylation status of virtually any group of CpG sites within a CpG island, independent of the use of methylation-sensitive restriction enzymes. This assay entails initial modification of DNA by sodium bisulfite, converting all unmethylated, but not methylated, cytosines to uracil, and subsequent amplification with primers specific for methylated versus unmethylated DNA. MSP requires only small quantities of DNA, is sensitive to 0.1% methylated alleles of a given CpG island locus, and can be performed on DNA extracted from paraffin-embedded samples. MSP eliminates the false positive results inherent to previous PCR-based approaches which relied on differential restriction enzyme cleavage to distinguish methylated from unmethylated DNA. In this study, we demonstrate the use of MSP to identify promoter region hypermethylation changes associated with transcriptional inactivation in four important tumor suppressor genes (p16, p15, E-cadherin, and von Hippel-Lindau) in human cancer.

Reproducible alterations of DNA methylation at a specific population of CpG islands during blast formation of peripheral blood lymphocytes.

We investigated the changes in the methylation patterns of CpG islands associated with blast formation of human peripheral blood lymphocytes activated by anti-CD3 and interleukin-2 (IL-2), using restriction landmark genomic scanning with a methylation-sensitive restriction enzyme (RLGS-M) system. Of about 2,100 NotI spot/loci which were analyzed, only 10 showed changes, whereas drastic changes have been observed in cases of malignant and SV40 transformation. These changes were highly reproducible for samples from both the same and different individuals. Even the timing of the changes after cultivation was the same. Thus, we concluded that at least the genomic DNA methylation state in vivo was essentially retained in T blast cells activated in vitro by induction with IL-2 and anti-CD3, which are commonly used in biological experiments as well as clinical diagnosis and therapy.