Aberrant Methylation Of Promoter Region Research Articles

Hypermethylation in Ovarian Cancer of Long Non-Coding RNA Genes: <i>HOTAIR</i>, <i>GAS5</i>, <i>LINC00472</i>, <i>LINC00886</i>, <i>TUG1</i>

Recently, more and more data have been accumulating indicating the role of long non-coding RNAs (lncRNAs) in the regulation of biological processes in cells, as well as in the mechanisms of cancer development and progression. Aberrant methylation of promoter regions of both protein genes and lncRNA genes can disrupt their expression and functional activity. Using bioinformatics databases, six lncRNA genes (GAS5, HOTAIR, LINC00472, LINC00886, SNHG17 and TUG1) with CpG islands, differentially expressed and presumably hypermethylated in tumors of patients with ovarian cancer (OC) were selected. A statistically significant (p 0.05) increase in the methylation level in tumours was demonstrated in a sample of 93 OC specimens using methylation-specific real-time PCR assay. Moreover, for the genes LINC00472, LINC00886, SNHG17 and TUG1, hypermethylation in OC was detected for the first time. 5 genes (except SNHG17) showed a further increase in methylation levels at a more advanced stage, and 4 genes (except SNHG17 and LINC00886) showed a significant association with metastasis. Using real-time RT-PCR, differential changes in the expression level of the GAS5, HOTAIR, SNHG17 and TUG1 genes and a significant correlation of methylation with expression for the GAS5 gene were shown. Thus, hypermethylation associated with the progression and/or development of OC was detected for six lncRNA genes, which is important for elucidating the epigenetic processes involved in the pathogenesis of OC and can be used as new biomarkers of OC.

Abstract B013: Epigenetic clocks and breast cancer outcomes: A scoping review. [R

Abstract Introduction: Age is a strong predictor for breast cancer (BCa) risk. The median age at diagnosis varies by race/ethnicity. In the United States (U.S.), non-White women are more likely to be diagnosed at a younger age than non-Hispanic White (NHW) women. Epigenetic age, measured as changes to DNA via methylation (DNAm) using epigentic clocks, is highly accurate at estimating cellular age using several tissues and cell types may differ from chronological age. Epigenetic age is more robust than other molecular markers of aging, such as telomere length. Differences between epigenetic age and chronological age, termed epigenetic age acceleration, may be the driver of age and race/ethnicity discrepancies when estimating BCa risk. Age-related methylation between disease-free and tumor breast tissue is significantly differentially methylated. For example, aberrant methylation of promoter regions of polycomb group protein target genes and CpG islands associated with BCa risk and progression. Therefore, the purpose of this scoping review is to gather data on epigenetic clocks as a predictor for BCa incidence. Study objective: Synthesize the existing evidence on epigenetic accelerated aging measured with epigenetic clocks and breast cancer risk in the U.S. Methods: The PROSPERO Scoping Review Protocol was used for this project. We performed multiple PubMed searches utilizing search language developed by an experienced search librarian. The searches were conducted from January 2022 to April 2022. The searches yielded a total of 2,908 studies in the search process. The eligibility for inclusion applied during article review was: 1) the study must have used an epigenetic clock to assess epigenetic accelerated age in their sample, 2) the study’s primary outcome needed to be breast cancer risk, 3) the study needed to use a U.S. based study sample 3) the study needed to be published in a peer-reviewed PubMed indexed journal, 4) the study must have been published by April 30, 2022. Results: A total of seven articles from the 2,908 were determined eligible for inclusion. Data extraction from these seven articles demonstrated epigenetic accelerated age measured with epigenetic clocks does have an associative relationship with the development of breast cancer. Discussion: BCa risk is also known to drastically increase after menopause, although there are significant differences of menopausal transition in race/ethnicity. On average, African American, Asian, and Latina women begin menopause earlier than non-Hispanic White women. Emerging evidence suggests that earlier menopause in these populations may be a result of structural factors experienced by historically disadvantaged communities. The studies included in this review focused primarily on NHW women. More research is needed to better understand the role of DNAm and accelerated epigenetic age in diverse populations. Citation Format: Jennifer Daw, Devin Saunders, Adria Vasquez, Celina Valencia. Epigenetic clocks and breast cancer outcomes: A scoping review. [R] [abstract]. In: Proceedings of the AACR Special Conference: Aging and Cancer; 2022 Nov 17-20; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2022;83(2 Suppl_1):Abstract nr B013.

Novel DNA methylation markers with potential prognostic relevance in advanced malignant melanoma identified using COBRA assays.

Aberrant methylation of promoter regions involved in silencing of tumor suppressor genes is a key feature of many human cancers including melanoma. These DNA methylation events occur early in cancer development, increase with progression, and may therefore serve as biomarkers for the detection and staging of cancer. In our study, we used an epigenomic reactivation screening approach including Combined Bisulfite Restriction Analyses (COBRA) assays to identify novel methylation markers in late-stage melanoma. Two human xenograft melanoma models have been used to identify genes methylated in cancer and reactivated upon treatment with a histone deacetylase inhibitor. Gene expression analysis and promoter scanning for DNA methylation by COBRA assays and bisulfite sequencing were used to identify candidate genes. The methylation status of the CpG island promoter region of genes related to melanoma pathophysiology in skin, lymph node, and visceral metastatic metastases in 28 patients (samples n=35) were assessed. These methylation markers have been evaluated in melanoma metastasis tissue and in control samples from normal skin. The screening in in-vitro and in-vivo systems for methylated genes in melanoma samples showed 10 candidate genes. Using COBRA assays, we detected a methylation pattern in the promoter region of 10 genes with two genes (BASP1, CDH11), together with the patient's age and the log-S100B-level at biopsy, constructing a descriptor with a trend to correlate with shorter time to death.

Relation between Different Stages of Oral Cancer and Aberrant Methylation in Taiwan: Preliminary Report

Objectives: During the past decade, oral cancer has become the fourth cause of death in Taiwan. After receiving treatment at the early stages of oral cancer (stage I and stage II), the 5-year survival rate is up to 81.9%. However, as soon as cancer cells spread to the neighboring tissues accompanied with lymph node metastasis in the neck (stage III and stage IV), the 5-year survival rate drops to 23-25%. This indicates the importance of the early detection of carcinogenesis to high survival rate. Aberrant methylation in promoter regions of certain genes has long been noticed and evidenced as strongly correlative to carcinogenesis. Methods: The tissue samples were obtained from the patients of one medical center, and the tissue was divided into 3 parts: tumor, nearby tissue, and further (normal) tissue. Then, the tissue was deposited in liquid nitrogen for DNA and RNA extractions. Patient data were collected including age, sex, risk factors (alcohol/betelnut/cigarette use), clinical and pathological stage, and operation. Results: This study investigates the aberrant methylation level on seven genes, E-cadherin, cyclinA1, cytoglobin, IGF2, MGMT, P16, and RARβ, in tissue samples of oral cancer. Conclusions: By means of the high sensitivity and accuracy technology, pyrosequencing, the methylation profiles of studied genes should be able to provide insight and evaluation of biomarkers of aberrant methylation in carcinogenesis.

HOXA11 gene is hypermethylation and aberrant expression in gastric cancer.

BackgroundAberrant DNA methylation is an acquired epigenetic alteration that serves as an alternative to genetic defects in the inactivation of tumor suppressor genes and other genes in diverse human cancers. Gastric carcinoma is one of the tumors with a high frequency of aberrant methylation in promoter region. Hence we investigated the promoter methylation status and expression level of HOXA11 gene which may involve in GC development.MethodsThirty-two surgical excised gastric cancer specimens, twelve paired adjacent non-cancerous specimens and seven normal gastric mucosas were examined. The methylation status and expression level of HOXA11 gene were determined by bisulfite sequencing polymerase chain reaction (BSP), real-time polymerase chain reaction (RT-PCR) and immunohistochemistry (IHC) respectively. HOXA11 expression was knocked-down with siRNA to mimic HOXA11 gene hypermethylation and ability of cell proliferation and migration was determinate. In addition, we analyzed and correlated the findings with clinicopathological features.ResultsThe methylation level of HOXA11 gene in gastric cancer tissues and adjacent non-cancerous tissues were higher than those in normal gastric mucosa (P < 0.05). The methylation level was higher in TNM III and IV patients of GC than those in TNM I and II patients (P < 0.05). The expression of HOXA11 mRNA and protein decreased in normal gastric mucosa, peri-cancer tissue and GC (P < 0.05). HOXA11 expression was inversely correlated with DNA methylation (P < 0.05). Knocked-down of HOXA11 expression with siRNA in BGC-823 cells enhanced cell proliferation compared with control, but no significant different was observed in migration ability.ConclusionHypermethylation and decreased expression of HOXA11 gene may be involved in the carcinogenesis and development of GC and may provide useful information for the prediction of the malignant behaviors of GC. And the expression of HOXA11 is impaired by DNA methylation. However, repression of HOXA11 expression promoted BGC-823 cell proliferation.

Methylation status of CDH1 promoter and E-cadherin expression in colorectal cancer

The E-cadherin plays a crucial role in epithelial cell cell adhesion and in the maintenance of a tissue. In colorectal cancer (CRC), changes in E-cadherin expression have been correlated with histopathology and differentiation, but results are still inconsistent. Further studies should aid the development of novel strategies for both prevention and treatment of cancer. Aberrant methylation in promoter regions of the genes might lead to reduce gene expression and result in cancer. Thus in the present study for better understanding role of the Ecadherin in CRC, methylation status of the CDH1 promoter region was determined by methylation specific PCR following bisulphite modification, and compared with mRNA E-cadherin expression by qPCR. 43 of 70 cancers (61.4%) were aberrant methylated in the CDH1 promoter region and this correlated with reduced E-cadherin expression (p<0.05). Generally, our study demonstrated aberrant methylation of CDH1 can significantly reduce its expression among tumor tissues in CRC, and contribute to tumor staging, tumor differentiation and progression or prognosis.

DNA methylation as an early diagnostic marker of cancer (Review).

DNA methylation is one of the essential epigenetic mechanisms that are closely correlated with the mechanisms underlying cell growth, differentiation and transformation in eukaryotes. Global changes in the epigenetic landscape are considered to be a hallmark of cancer. The initiation and progression of cancer are mediated through epigenetic modifications along with genetic alterations. Aberrant methylation of promoter regions is an epigenetic abnormality of the human genome that is highly characteristic of cancer. In this review, we aimed to summarize our current understanding of the alterations in the epigenetic landscape and investigate the potential use of DNA and RNA methylation in effective molecular treatment strategies.

Evaluation of tumor suppressor gene expressions and aberrant methylation in the colon of cancer-induced rats: a pilot study

Altered expression and methylation pattern of tumor suppressor and DNA repair genes, in particular involved in mismatch repair (MMR) pathway, frequently occur in primary colorectal (CRC) tumors. However, little is known about (epi)genetic changes of these genes in precancerous and early stages of CRC. The aim of this pilot study was to analyze expression profile and promoter methylation status of important tumor suppressor and DNA repair genes in the early stages of experimentally induced colorectal carcinogenesis. Rats were treated with azoxymethane (AOM), dextran sodium sulphate (DSS) or with their combination, and sacrificed 1 or 4 months post-treatment period. The down-regulation of Apc expression in left colon, detectable in animals treated with DSS-AOM and sacrificed 1 month after the end of treatment, represents most early marker of the experimental colorectal carcinogenesis. Significantly reduced gene expressions were also found in 5 out of 7 studied MMR genes (Mlh1, Mlh3, Msh3 Pms1, Pms2), regarding the sequential administration of DSS-AOM at 4 months since the treatment. Strong down-regulation was also discovered for Apc, Apex1, Mgmt and TP53. Tumors developed in rectum-sigmoid region displayed significantly lower Apc and Pms2 expressions. The decreased expression of studied genes was not in any case associated with aberrant methylation of promoter region. Present data suggest that down-regulation of Apc and MMR genes are prerequisite for the development of CRC. In this study we addressed for the first time early functional alterations of tumor suppressor genes with underlying epigenetic mechanisms in experimentally induced CRC in rats.

Aberrant methylation of T-cadherin gene in human esophageal carcinoma cells

To explore the methylation status of T-cadherin promoter region in human esophageal carcinoma cell lines EC1 and EC109 and elucidate the effects of 5-azacytidine-2'-deoxycytidine (5-Aza-CdR) on their abilities of proliferation and invasion, the methylation status and the expression of T-cadherin. The expression level of T-cadherin was measured by Western blot. And the methylation status of T-cadherin promoter region was analyzed by methylation-specific PCR (polymerase chain reaction), separately before and after a treatment of demethylating agent 5-Aza-CdR. The T-cadherin gene was hypermethylated in EC109 cells but semi-methylated in EC1 cells. After a treatment of 5-Aza-CdR, T-cadherin gene methylation was reversed. And the gene expression was strongly up-regulated in both cells. 5-Aza-CdR could decrease the proliferation and effectively inhibit the ability of cell invasion (P < 0.01). The aberrant methylation in promoter region is an important mechanism of T-cadherin gene inactivation in human esophageal carcinoma cells. 5-Aza-CdR can increase the expression of T-cadherin by a reversal of hypermethylation and effectively inhibit the proliferation and invasion of tumor cells.

Polymorphisms in genes involved in folate metabolism and plasma DNA methylation in colorectal cancer patients

Aberrant methylation of promoter regions associated with gene silencing is one of the major mechanisms underlying the inactivation of tumor suppressor genes in carcinogenesis. Previous studies have proposed that methylated DNA from tumor cells is released into the circulation and might be widely used as a marker for non-invasive tumor detection. Catalytic activities of folate metabolism-related enzymes and adequate synthesis of methyl donors are important elements for the cellular methylation reaction. In the present study, we sought to determine the following: i) genotype frequencies of MTHFR and MTR involved in folate metabolism in cases and cancer-free controls; and ii) the methylation status of three candidate genes (p16INK4A, p73 and hMLH1) in plasma related to the folate and homocysteine levels. From genotype frequency analysis, individuals homozygous for the MTHFR 677TT genotype had a significantly reduced risk of developing colorectal cancer compared with those harboring the MTHFR 677CC genotype (OR, 0.206; 95% CI, 0.070-0.604; P=0.005), and had a lower plasma folate concentration than those with the MTHFR 677CC+CT genotype (P<0.05). Using methylation-specific PCR, p73 was shown to be more frequently methylated in the high folate group [50% (8 of 16)] than in the medium [16.7% (3 of 18)] or low folate subgroups [11.1% (2 of 18)]. In conclusion, subjects with the variant MTHFR 677TT genotype appeared to have a significantly lower risk for colorectal cancer than those with the MTHFR 677CC genotype, and the methylation status of circulating p73 genomic DNA was substantially altered by the plasma folate level.

The value of epigenetic markers in esophageal cancer

Developing esophageal cancer is a multi-step process that begins with the accumulation of genetic and epigenetic alterations, and leads to the activation of oncogenes and the inactivation or loss of tumor suppressor genes (TSG). In addition to genetic alteration, epigenetic modifications, and in particular DNA methylation, are recognized as a common molecular alteration in human tumors. In esophageal cancer, aberrant methylation of promoter regions occurs not only in advanced cancer, but also in premalignant lesions. DNA methylation is related to survival time and sensitivity of chemoradiotherapy. This review is mainly focused on epigenetic changes in esophageal cancer and the value of early detection for patient prognosis, treatment choices, and potential targeting therapy.

Decytabine enhances cytotoxicity induced by oxaliplatin and 5-fluorouracil in the colorectal cancer cell line Colo-205

BackgroundDNA methylation is an epigenetic phenomenon known to play an important role in the development of cancers, including colorectal cancer (CRC). Aberrant methylation of promoter regions of genes is potentially reversible, and if methylation is important for cancer survival, demethylation should do the opposite. To test this we have addressed the hypothesis that DNA methyltransferase inhibitors (DNMTi), decytabine and zebularine, potentiate inhibitory effects of classical anti-CRC cytostatics, oxaliplatin and 5-fluorouracil (5-FU), on survival of CRC cells in vitro.ResultsIsobole and median effect analysis revealed that decytabine shows potent synergistic interaction with oxaliplatin and 5-FU and that this is probably not the class effect of DNMTi as zebularine shows strong antagonistic interaction with oxaliplatin. The synergistic combination treatment was also applied to the cultures to investigate their mechanisms of action. We have shown that combinations of decytabine with cytostatics produced dose-dependent growth inhibition and treatment-induced apoptosis.ConclusionThe observed synergism between decytabine and cytostatics is most probably related to the augmented apoptotic signal and allowed for significant (both biologically and statistically) reduction of the cytotoxic doses of cytostatics used.

DNA Hypermethylation Profiles in Squamous Cell Carcinoma of the Vulva

Gene silencing through promoter hypermethylation is a growing concept in the development of human cancers. In this study, we examined the contribution of aberrant methylation of promoter regions in methylation-prone tumor suppressors to the pathogenesis of vulvar cancer. Thirteen cell lines from 12 patients with squamous cell carcinoma of the vulva were evaluated for aberrant methylation status and gene copy number alterations, concomitantly, using the methylation-specific multiplex ligation-dependent probe amplification assay. Of the 22 tumor suppressor genes examined, aberrant methylation was observed for 9 genes: tumor protein p73 (TP73), fragile histidine triad (FHIT), von Hippel-Lindau (VHL), adenomatosis polyposis coli (APC), estrogen receptor 1 (ESR1), cyclin-dependent kinase inhibitor 2B (CDKN2B), death-associated protein kinase 1 (DAPK1), glutathione S-transferase pi (GSTP1), and immunoglobin superfamily, member 4 (IGSF4). The most frequently methylated genes included TP73 in 9 of 13 cell lines, and IGSF4, DAPK1, and FHIT in 3 of 13 cell lines. Methylation-specific polymerase chain reaction was performed for TP73 and FHIT to confirm aberrant methylation by methylation-specific multiplex ligation-dependent probe amplification. In the context of gene copy number and methylation status, both copies of the TP73 gene were hypermethylated. Loss or decreased mRNA expression of TP73 and IGSF4 by reverse transcription polymerase chain reaction confirmed aberrant methylation. Frequent genetic alterations of loss and gain of gene copy number included gain of GSTP1 and multiple endocrine neoplasia type 1 (MEN1), and loss of malignant fibrous histiocytoma amplified sequence 1 (MFHAS1) and IGSF4 in over 50% of the squamous cell carcinoma of the vulva cell lines. These findings underscore the contribution of both genetic and epigenetic events to the underlying pathogenesis of squamous cell carcinoma of the vulva.

High frequency of p16 promoter methylation in non-small cell lung carcinomas from Chile.

The inactivation of tumour suppressor genes by aberrant methylation of promoter regions has been described as a frequent event in neoplasia development, including lung cancer. The p16 gene is a tumour suppressor gene involved in the regulation of cell cycle progression that has been reported to be inactivated by promoter methylation in lung carcinomas at variable frequencies around the world in a smoking habit dependent manner. The purpose of this study was to investigate the methylation status of the promoter region of the p16 gene in 74 non-small cell lung carcinomas from Chile. The frequency of p16 gene inactivation by promoter methylation was determined as 79.7% (59/74). When we considered histological type, we observed that p16 promoter methylation was significantly higher in squamous cell carcinomas (30/33, 91%) compared with adenocarcinomas (21/30, 70%) (p=0.029). In addition, no association between p16 promoter methylation and gender, age or smoking habit was found (p=0.202, 0.202 and 0.147 respectively). Our results suggest that p16 promoter hypermethylation is a very frequent event in non-small cell lung carcinomas from Chile and could be smoking habit-independent.

Aberrant methylation in promoter regions of cyclin‐dependent kinase inhibitor genes in adenoid cystic carcinoma of the salivary gland

Adenoid cystic carcinoma (ACC) is a common malignant neoplasm of the salivary gland. The mechanism underlying ACC carcinogenesis is not fully elucidated, although data on associated genetic alterations are accumulating. Cyclin-dependent kinase inhibitors (CKIs) act as tumor suppressors in various cancers, and aberrant methylation in the CKI gene promoter region has been linked to gene silencing and downregulation of expression. The present study investigated methylation of CKI genes, p15, p18, p19, p21, and p27, in 34 cases of ACC. We found frequent and plural methylations of these genes in most cases (68.8% in p15, 90.3% in p18, 78.8% in p19, 92.3% in p21, and 26.5% in p27). Cell cycle disruption induced by these epigenetic aberrations might be important in the tumorigenesis of ACC.

Role of the IL-6 Pathway in Multiple Myeloma Cell Growth and Its Implications in Target Gene Hypermethylation.

Aberrant methylation of promoter regions of tumor suppressor genes (TSG) has recently become recognized as an important epigenetic event in cancer and tumor progression including multiple myeloma. Interleukin-6 (IL-6), which is known to play a significant role in the pathophysiology of myeloma, regulates DNA methylation by inducing expression of FLI-1, a transcription factor of DNA methyltransferase-1 (DNMT-1), resulting in over-expression of DNMT-1 and thus hypermethylation of TSG. Despite this understanding, attempts to bring IL-6 blockade to the clinic have had limited success. We hypothesize that IL-6 regulation of epigenetic events (hypermethylation) may be an important pathway that could eventually allow rational chemotherapeutic/anit-IL-6 combinations. We have studied the correlation of IL-6 expression and dependence in myeloma cell lines and correlated that to the methylation profile of TSG, DcR1 and CDH1. Using three well-established multiple myeloma cell lines: U266B1 (U266), RPMI 8226 (RPMI), and KAS6/1 (KAS), we have confirmed that KAS is IL-6-dependent (exogenous IL-6 is needed) whereas the U266 and RPMI are IL-6 independent. To determine if blockade of the IL-6 pathway would inhibit the growth of the myeloma cell lines, these cells were grown in the presence of an anti-IL-6 antibody (B-E8; Cell Sciences, Canton, MA) that is known to block IL-6 signaling in the cells. We found that blocking IL-6 (by B-E8, 200 ng/ml) inhibited the growth of U266 (36% inhibition; n≥3, p<0.01) and KAS (68% inhibition; n≥3, p<0.001) cells, but not RPMI cells. We examined IL-6 expression in these three cell lines by RT-PCR and found that U266 expresses IL-6 mRNA, but RPMI and KAS cells do not. This IL-6 mRNA expression pattern correlates well with the anti-IL-6 cellular proliferation findings. Since RPMI is not dependent on the addition of exogenous IL-6 and blocking the IL-6 pathway had no effect on cell growth; therefore, we would not expect the cells to express IL-6. The U266 cells are not dependent on exogenous IL-6 but cellular proliferation can be blocked with an anti-IL-6 antibody; therefore, we expected that the cells would endogenously express IL-6. Furthermore, KAS cells are dependent on exogenous IL-6 and cell growth can be inhibited by anti-IL-6 antibody; therefore, we would not expect the cells to express IL-6. To determine if IL-6 sensitivity correlated with hypermethylation of TSG, we investigated the methylation status of the DcR1(tumor necrosis factor-related apoptosis inducing ligand decoy receptor 1) and CDH1 (Cadherin 1) loci. We have shown that CDH1 is methylated in U266 cells and un-methylated in RPMI cells. Experiments are underway to determine the methylation status in KAS cells and gene expression in all cell-lines for CDH1. Finally, we found that the RPMI and KAS cells are un-methylated and U266 cells are methylated at the DcR1 locus. We have found that in the RPMI and U266 cell lines that methylation of target TSG correlates with anti-IL-6 sensitivity. These data support our hypothesis that an IL-6-dependent pathway may regulate hypermethylation of important TSG in multiple myeloma. Newer chemotherapeutic agents that may affect methylation pathways are being studied in combination with IL-6 blockade.

High frequency of p 16 promoter methylation in non-small cell lung carcinomas from Chile

The inactivation of tumour suppressor genes by aberrant methylation of promoter regions has been described as a frequent event in neoplasia development, including lung cancer. The p16 gene is a tumour suppressor gene involved in the regulation of cell cycle progression that has been reported to be inactivated by promoter methylation in lung carcinomas at variable frequencies around the world in a smoking habit dependent manner. The purpose of this study was to investigate the methylation status of the promoter region of the p16 gene in 74 non-small cell lung carcinomas from Chile. The frequency of p16 gene inactivation by promoter methylation was determined as 79.7% (59/74). When we considered histological type, we observed that p16 promoter methylation was significantly higher in squamous cell carcinomas (30/33, 91%) compared with adenocarcinomas (21/30, 70%) (p=0.029). In addition, no association between p16 promoter methylation and gender, age or smoking habit was found (p=0.202, 0.202 and 0.147 respectively). Our results suggest that p16 promoter hypermethylation is a very frequent event in non-small cell lung carcinomas from Chile and could be smoking habit-independent.

Promoter hypermethylation of MGMT, CDH1, RAR-beta and SYK tumour suppressor genes in granulosa cell tumours (GCTs) of ovarian origin.

Ovarian carcinoma (OC) is a leading cause of death among women throughout the world. A number of cancer-associated genes have been shown to be inactivated by hypermethylation of CpG islands during tumorigenesis. We tested the hypothesis that methylation status of MGMT, CDH1, RAR-β and SYK could be important in the ovarian tumorigenic process and can lead to the gene(s) inactivation. Therefore, we assessed the promoter hypermethylation of MGMT, CDH1, RAR-β and SYK in 43 ovarian granulosa cell tumours (GCTs) (adult type) using methylation-specific PCR. These tumours are relatively rare, accounting for approximately 3% of all ovarian cancers. Hypermethylation of MGMT (in 14 tumours), CDH1 (in nine tumours), RAR-β (in eight tumours) and SYK (in seven tumours) have been found. Selective loss of RAR-β and RAR-β2 mRNA has been found in seven patients, while that of MGMT and SYK in three patients who also show aberrant methylation in promoter region of RAR-β in addition to MGMT, SYK and CDH1 genes. Promoter CpG hypermethylation may be an alternative to mutation(s) to inactivate tumour suppressor genes such as MGMT, CDH1, RAR-β and SYK, and this can also be an early event in the pathogenesis of OCs. Moreover, hypermethylation of the MGMT and CDH1, MGMT and RAR-β and CDH1 and RAR-β promoters occurred concordantly (P< 0.001, 0.0421 and 0.0005 respectively; Fischer's exact test). In addition to this, monosomy 22 and trisomy 14 have also been found in 10 tumours. It is clear from the results that hypermethylation of the promoter region of these tumour suppressor genes, monosomy 22 and trisomy 14, may be critical steps in the tumorigenesis, which consequently play a permissive role for tumour aggressiveness. All these events might play an important role in the early clinical diagnosis of the disease. Our results, therefore, suggest a potential role for epigenetic modification of these critical tumour suppressor genes in pathways relevant to the transformation and differentiation of rare type of ovarian cancer (GCTs).