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Results for “Promoter hypermethylation”

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At least 19 recordsLinked to original sources

PubMed2005

Promoter hypermethylation profile of RASSF1A, FHIT, and sFRP1 in intracranial primitive neuroectodermal tumors.

Medulloblastomas (MBs) and supratentorial primitive neuroectodermal tumors (SPNETs) are histologically alike intracranial PNETs found in different anatomical locations of the brain. Current evidence suggests that hypermethylation of promoter CpG islands is a common epigenetic event in a variety of human cancers. The aim of this study was to investigate whether promoter hypermethylation of putative tumor suppressor genes was involved in both types of intracranial PNETs. We examined the methylation status at promoter regions of RASSF1A, FHIT, and sFRP1 by methylation-specific polymerase chain reaction in a cohort of 25 primary MBs, 9 primary SPNETs, and 3 MB and 2 SPNET cell lines. Our results revealed no promoter hypermethylation of RASSF1A, FHIT, and sFRP1 in 2 normal cerebellar and 5 normal cerebral tissue specimens examined. In contrast, promoter hypermethylation of RASSF1A was detected in 100% of primary MBs, 67% (6/9) of primary SPNETs, and all PNET cell lines. The frequency of promoter hypermethylation of RASSF1A was significantly lower in SPNETs than in MBs (Fisher exact test, P = .014). Treatment of RASSF1A-deficient PNET cell lines with 5-aza-2'deoxycytidine, a demethylating agent, restored RASSF1A expression, providing evidence that promoter hypermethylation contributes to transcriptional silencing. In addition, promoter hypermethylation of FHIT and sFRP1 was detected in 22% (2/9) and 11% (1/9) of SPNETs, respectively, but not in any MBs studied. In conclusion, our study demonstrates that promoter hypermethylation of RASSF1A is a common event in intracranial PNETs, whereas FHIT and sFRP1 are epigenetically affected in a fraction of SPNETs.

Acid Anhydride Hydrolases↗

PubMed2003

Downregulation of ID4 by promoter hypermethylation in gastric adenocarcinoma.

Promoter hypermethylation has become apparent as a common mechanism of gene silencing in cancer. Based on our published microarray expression data, we noticed a prominent downregulation of ID4 in gastric adenocarcinoma. The dense 5' CpG island covering the previously mapped upstream promoter of ID4 has prompted us to relate its downregulation to promoter hypermethylation. ID proteins are distinct members in the helix-loop-helix family of transcriptional regulators, which modulate various key developmental processes. Emerging data have suggested the involvement of ID genes in tumorigenesis. In this study using bisulfite genomic sequencing, we have found hypermethylation of ID4 promoter in most gastric cancer cell lines and 30% of primary tumors. This correlated with decreased level of ID4 expression. Restoration of ID4 expression in various gastric cancer cell lines was achieved by treatment with the DNA methyltransferase inhibitor 5-aza-2'-deoxycytidine, which at times required the synergistic action of the histone deacetylase inhibitor trichostatin A, but not with trichostatin A alone. Re-expression was accompanied by the corresponding ID4 promoter demethylation. Furthermore, we have found significant association of ID4 promoter methylation with hMLH1 promoter methylation (P=0.008) and microsatellite instability (P=0.006). Overall, our results have shown that transcriptional silencing of ID4 is related to the aberrant methylation of its promoter in gastric cancer. The significant association of ID4 and hMLH1 promoter hypermethylation suggested that ID4 may also be among the genes being targeted in the CpG island methylator phenotype tumorigenic pathway.

Adenocarcinoma↗

PubMed2006

CDH1 gene promoter hypermethylation in gastric cancer: relationship to Goseki grading, microsatellite instability status, and EBV invasion.

Hypermethylation of the CDH1 promoter region seems to be the most common epigenetic mechanism in this gene silencing in gastric cancer. In this study, CDH1 promoter hypermethylation was observed in 54.8% (46/84) of the analyzed sporadic gastric carcinomas. We introduce a new relation: clustering of Goseki grading into 3 grade was determined by CDH1 promoter hypermethylation. The percentage of methylation in Goseki III cancers was significantly higher (83%) when compared with other grades; the lowest proportion was detected in IV (36%) and II (38%) groups, whereas grade I demonstrated typical percentage of promoter hypermethylation. A novel polymorphism R732R in exon 14 of the CDH1 gene was detected by mutational analysis. Additionally, all cases with the MSI-high phenotype revealed CDH1 promoter hypermethylation. In MSI-low and MSS gastric cancers, this percentage was lower, reaching 71% and 41%, respectively. Moreover, the methylation status was correlated with the LOH phenotype. We detected CDH1 promoter hypermethylation in all EBV-positive gastric cancers (5/5), whereas methylation in the EBV-negative group occurred in 58% of cases. We also report that "methylated" tumors were slightly larger than "nonmethylated," whereas the second group revealed a higher probability of longer patient survival, though these relationships were not statistically significant. These results suggest that downregulation of E-cadherin, caused by promoter hypermethylation, in sporadic gastric carcinomas may be associated with a worse prognosis and specific tumor phenotype.

Adult↗

PubMed2004

Frequent epigenetic inactivation of RIZ1 by promoter hypermethylation in human gastric carcinoma.

The retinoblastoma protein-interacting zinc finger gene, RIZ1 (GenBank accession number U17838), is involved in chromatin-mediated gene expression and is also a target for frameshift mutation in microsatellite-unstable cancers. Methylation of the RIZ1 promoter CpG island has been shown to be a common mechanism in inactivating the RIZ1 gene in human liver and breast cancers. We investigated levels of RIZ1 mRNA in 45 gastric carcinoma tissues by quantitative RT-PCR and in gastric carcinoma cell lines by RT-PCR. In addition, we examined CpG island methylator phenotype (CIMP) status, p53 mutation status, and the correlation between promoter methylation status and RIZ1 mRNA expression. CIMP status was investigated by examining the methylation status of MINT1, MINT2, MINT12, MINT25 and MINT31. p53 mutation status was examined by PCR-single strand conformation polymorphism and promoter methylation status was examined by methylation-specific PCR. Promoter hypermethylation of the RIZ1 gene was found in 31 (69%) of 45 gastric carcinoma tissues and in 3 (21%) of 14 corresponding non-neoplastic mucosae, the incidence being significantly different (p = 0.002). None of the 12 normal gastric tissues from young non-cancer individuals showed hypermethylation. Promoter hypermethylation was associated with reduced RIZ1 expression in gastric carcinoma tissues (p = 0.029). Promoter hypermethylation of the RIZ1 gene was significantly associated with CIMP (p = 0.002). Mutation status of the p53 gene was not associated with methylation status or RIZ1 expression in gastric carcinoma. In gastric carcinoma cell lines MKN-28 and KATO-III, the RIZ1 promoter was hypermethylated and RIZ1 transcription was inactive. Treatment of these cells with demethylating agent 5-aza-2'-deoxycytidine restored RIZ1 transcription. Our results suggest that transcriptional inactivation of the RIZ1 gene by promoter hypermethylation may participate in stomach carcinogenesis.

Cell Line, Tumor↗

PubMed2004

BRAF/K-ras mutation, microsatellite instability, and promoter hypermethylation of hMLH1/MGMT in human gastric carcinomas.

BACKGROUND: The BRAF and K-ras genes are the most frequently mutated oncogenes in various human malignancies. We examined BRAF and K-ras mutations in human gastric cancer, and investigated their relationship with microsatellite instability (MSI) and the hypermethylation of promoter regions in hMLH1 and O6-methylguanine DNA methyltransferase (MGMT). METHODS: Sixteen gastric cancer cell lines and 62 gastric cancer tissue samples were screened for BRAF and K-ras mutations by direct sequencing. We also performed a microsatellite assay and investigated methylation status in the promoter regions of hMLH1 and MGMT. RESULTS: mutation was not found in any of the cancer cell lines examined. One (1.6%) cancer tissue sample showed a point mutation in the BRAF gene (GTG --> GAG; V599E). K-ras mutation (GGT --> GAT, G12D) was detected in five (31%) gastric cancer cell lines and in 1 (1.6%) gastric cancer tissue sample. In the gastric cancer tissue samples examined, MSI was detected in 23 (37%) samples. Hypermethylated promoter regions in hMLH1 and MGMT, respectively, were detected in 6 (10%) and 13 (21%) gastric cancer tissue samples. Microsatellite stable (MSS) tumors showed frequent lymphatic invasion (P = 0.050). CONCLUSION: Although BRAF mutation has been reported in a variety of other human cancers, it is a rare event in the carcinogenesis and progression/development of gastric cancer.

Adaptor Proteins, Signal Transducing↗

PubMed2004

Promoter hypermethylation downregulates RUNX3 gene expression in colorectal cancer cell lines.

It was recently reported that RUNX3 gene expression is significantly downregulated in human gastric cancer cells due to hypermethylation of its promoter region or hemizygous deletion (Cell, 109, 2002). To verify the genetic alterations and methylation status of the RUNX3 gene in colorectal carcinogenesis, we analysed for mutations, loss of heterozygosity (LOH), and RUNX3 gene promoter hypermethylation, in 32 colorectal cancer cell lines. RT-PCR analysis showed undetectable or low RUNX3 expression in 16 cell lines, and no mutations were found in the RUNX3 gene by PCR-SSCP analysis. Of these 16 cell lines, hypermethylation of the RUNX3 promoter was confirmed in 12. The following observations were made: (i) RUNX3 was re-expressed after 5-aza-2'-deoxycytidine treatment, (ii) the RUNX3 promoter was found to be methylated by MS-PCR, and (iii) hypermethylation of the RUNX3 promoter was confirmed by direct sequencing analysis after sodium bisulfite modification in the above 12 cell lines. RUNX3 was neither methylated nor expressed in four cell lines. Of these four, microsatellite instability (MSI) at the RUNX3 locus was found in three, SNU-61 (D1S246), SNU-769A, and SNU-769B (D1S199). This study suggests that transcriptional repression of RUNX3 is caused by promoter hypermethylation of the RUNX3 CpG island in colorectal cancer cell lines, and the results of these experiments may contribute to an understanding of the role of RUNX3 inactivation in the pathogenesis of colorectal cancers.

Base Sequence↗

PubMed2006

Mutually exclusive promoter hypermethylation patterns of hMLH1 and O6-methylguanine DNA methyltransferase in colorectal cancer.

Hypermethylation of CpG islands in gene promoter regions is an important mechanism of gene inactivation in cancer. Many cellular pathways, including DNA repair, are inactivated by this type of epigenetic lesion, resulting in proposed mutator phenotypes. Promoter hypermethylation of hMLH1 has been implicated in a subset of colorectal cancers that show microsatellite instability (MSI). Transcriptional silencing of O6-methylguanine DNA methyltransferase (MGMT) has also been described in a variety of neoplasms and has been associated with a consequent mutational spectrum. We investigated the relationship between hMLH1 promoter hypermethylation and MGMT promoter hypermethylation in 110 colorectal cancers using methylation-specific polymerase chain reaction. Expression of hMLH1 and MGMT was assessed by immunohistochemistry. MSI testing was performed using the National Cancer Institute consensus panel of five microsatellite markers. Promoter hypermethylation of hMLH1 was detected in 12% of tumors. This was significantly associated with the MSI-high phenotype (P < 0.01) and loss of hMLH1 expression (P < 0.01). Methylation of the MGMT promoter was detected in 43% of tumors, which were mostly microsatellite stable or MSI-low (P = 0.041) and showed loss of MGMT expression (P < 0.01). We demonstrated an inverse relationship between hMLH1 promoter hypermethylation and MGMT promoter hypermethylation (P = 0.041), suggesting that a number of distinct hypermethylation-associated pathways may exist in colorectal cancer.

Adaptor Proteins, Signal Transducing↗

PubMed2004

Frequent loss of RUNX3 expression by promoter hypermethylation in gastric carcinoma.

The RUNX3 gene is a member of the Runt domain family of transcription factors that are master regulators of gene expression in major developmental pathways. Recently, lack of RUNX3 function was found to be associated with genesis and progression of gastric carcinoma. We studied methylation of CpG islands in the RUNX3 gene by methylation-specific PCR in 80 gastric carcinoma specimens, 45 corresponding non-neoplastic mucosae, and 7 gastric carcinoma cell lines. We also measured levels of RUNX3 mRNA in 50 of the gastric carcinoma cases by quantitative RT-PCR and in the gastric carcinoma cell lines by RT-PCR. Hypermethylation of the RUNX3 promoter was found in 57 (71%) of 80 gastric carcinomas, and promoter hypermethylation of RUNX3 occurred more frequently in intestinal and diffuse-adherent type tumors than in diffuse-scattered type tumors (p = 0.046). Reduced RUNX3 expression was associated with promoter hypermethylation (p = 0. 036), however, there was no correlation between RUNX3 mRNA expression levels and T grade, N grade, tumor stage, or histological type. In corresponding non-neoplastic mucosae, hypermethylation of the RUNX3 promoter was found in 38 (84%) of 45 specimens. Among seven gastric carcinoma cell lines, three cell lines (MKN-28, MKN-74, TMK-1) with diminished expression of RUNX3 had promoter methylation and three cell lines (MKN-1, MKN-7, MKN-45) with RUNX3 expression showed no promoter methylation. Our results overall suggest that transcriptional inactivation of RUNX3 by promoter hypermethylation may participate in the stomach carcinogenesis.

Adenocarcinoma↗

PubMedDate not supplied

Aberrant promoter hypermethylation of multiple genes in head and neck squamous cell carcinoma.

PURPOSE: Epigenetic alteration, via promoter hypermethylation, inactivates genes important for the development of head and neck squamous cell carcinoma (SCCHN). The aim of this study is to characterize and correlate, with clinical parameters, the promoter methylation profile of DNA repair genes, hMLH1 and O6-methylguanine-DNA methyltransferase (MGMT), and tumor-suppressor gene p16. MATERIALS AND METHODS: Fifty-one cases of SCCHN, collected from the paraffin block archives (1997-1999) in the Department of Pathology at the University of Arkansas for medical sciences, provided DNA for methylation-specific PCR using primers specific for hMLH1, MGMT, and p16. RESULTS: Sixty-two percent displayed promoter hypermethylation in at least one gene, with 23% seen for hMLH1, 30% for MGMT, and 36% for p16. Promoter hypermethylation of these genes separately or in combination was not associated with history of smoking and alcohol use, tumor size, nodal status, clinical stage, and overall survival. Promoter hypermethylation of more than 1 gene was significantly associated with increased 2-year disease-free survival. The probability of surviving 2 years without tumor recurrence was 100% with promoter hypermethylation in 2 or 3 genes and 46% with promoter hypermethylation in none or just 1 gene (P=.013). Promoter hypermethylation of 2 or 3 genes was independently related to increased 2-year cumulative disease-free survival (P=.028). CONCLUSIONS: Promoter hypermethylation of hMLH1, MGMT, and p16 genes was commonly detected in 47 SCCHN cases with up to 65% showing aberrant promoter hypermethylation in at least 1 gene. Promoter hypermethylation of 2 or 3 genes was significantly associated with increased 2-year disease-free survival, suggesting that promoter hypermethylation of multiple genes might improve survival. Significant correlation was also noted between a positive alcohol use history and promoter hypermethylation of the MGMT gene with no promoter hypermethylation of the p16 gene.

Adaptor Proteins, Signal Transducing↗

PubMed2001

Promoter hypermethylation of multiple genes in carcinoma of the uterine cervix.

Promoter hypermethylation is an important pathway for the repression of gene transcription in cancer. We investigated promoter hypermethylation of six genes, p16, APC, HIC-1, death-associated protein kinase (DAPK), O(6)-methylguanine-DNA-methyltransferase (MGMT), and E-cadherin, in uterine cervical carcinoma from 53 patients including 31 cases of squamous cell carcinoma (SCC) and 22 cases of adenocarcinoma (AC). Aberrant methylation of at least one of these genes was detected in 79% (42 of 53) of cases including 71% (22 of 31) of SCC and 91% (20 of 22) of AC cases. No aberrant methylation was detected in normal cervical tissue from 24 control hysterectomy specimens. There was no correlation between promoter hypermethylation at any gene and the presence of human papillomavirus-16 or -18 E7 DNA. In AC cases, promoter hypermethylation of the APC and HIC-1 genes was detected at a statistically significant higher frequency than in the SCC cases (APC, 60% versus 13%, P < 0.001; HIC-1, 63% versus 32%, P < 0.03). Conversely, promoter hypermethylation of p16 and DAPK was more common in SCC cases than in AC cases. Our results suggest that promoter hypermethylation is a frequent epigenetic event in cervical carcinoma. The pattern of gene promoter hypermethylation is distinctly different between AC and SCC. The absence of these epigenetic alterations in normal cervical tissue suggests that they may also be valuable as cancer markers.

Adenocarcinoma↗

PubMed2004

Prognostic analysis of E-cadherin gene promoter hypermethylation in patients with surgically resected, node-positive, diffuse gastric cancer.

PURPOSE: Recent investigations have demonstrated that hypermethylation is a frequent mechanism for silencing tumor suppressor genes. This is a potentially reversible epigenetic change, and it is the target of a novel class of anticancer compounds with demethylating activity. Better understanding of the clinical implications of hypermethylation will allow the optimal planning of future trials with demethylating drugs. In this perspective, we investigated whether hypermethylation in the CDH1 promoter region is correlated with poor prognosis of patients with surgically resected, node-positive, diffuse gastric cancer. EXPERIMENTAL DESIGN: Consecutive cases of diffuse gastric cancer were considered eligible for study entry. Additional inclusion criteria were radical surgery with a minimum of D1 lymphadenectomy, complete follow-up information, and availability of tumor specimens for methylation-specific PCR and immunohistochemistry analyses. RESULTS: CDH1 promoter hypermethylation was found in 40 of 73 cases (54%), and it was significantly associated with worse prognosis. In patients with and without hypermethylation, the 5-year event-free survival rate was 30% and 62%, respectively, and the 5-year overall survival rate was 35% and 67%, respectively. CDH1 promoter hypermethylation retained its prognostic role for disease-free survival (P < 0.001) and overall survival (P < 0.001) in multivariate analysis. Immunohistochemistry showed a significant association between CDH1 methylation and E-cadherin expression (P < 0.001). CONCLUSIONS: This study shows adverse prognostic effect of CDH1 promoter hypermethylation in patients with diffuse gastric cancer. This form of cancer, and other types with frequent hypermethylation and silencing of critical tumor suppressor genes, would make appropriate targets for the testing of novel compounds with demethylating activity.

Adult↗

PubMed2005

Promoter hypermethylation and inactivation of O(6)-methylguanine-DNA methyltransferase in esophageal squamous cell carcinomas and its reactivation in cell lines.

The purpose of this study was to characterize the role of DNA hypermethylation in the loss of expression of O(6)-methylguanine-DNA methyltransferase (MGMT) during the development of esophageal squamous cell carcinoma (ESCC) and to investigate its reactivation in cell lines. Samples were collected from Linzhou City of the Henan province in northern China, a high-risk area of this disease. The hypermethylation of promoter CpG islands of the MGMT gene was examined by methylation-specific PCR in ESCC and neighboring non-tumorous tissues, as well as in laser capture microdissected samples with normal epithelium, basal cell hyperplasia (BCH), and dysplasia (DYS). The MGMT mRNA and protein expression were determined with RT-PCR and immunohistochemistry, respectively. Five of 17 (29%) normal, 10 of 20 (50%) BCH, 8 of 12 (67%) DYS, and 13 of 18 (72%) ESCC samples had DNA hypermethylation in the MGMT promoter region, showing a gradual increase with the progression of carcinogenesis. The frequency of the loss of MGMT mRNA and protein expression progressively decreased from normal to BCH, DYS, and ESCC, and it was highly correlated with MGMT promoter hypermethylation according to Fisher's exact tests. When each individual sample was considered, good concordance between DNA hypermethylation and the loss of expression of MGMT was also observed. In samples from the same patient, if hypermethylation was detected in earlier lesions, it was usually observed in more severe lesions. In the ESCC cell line KYSE 510, treatment with a DNA methyltransferase inhibitor, 2'-deoxy-5-azacytidine partially reversed the CpG hypermethylation status and restored mRNA expression of the MGMT gene. Similar reactivation of MGMT gene by dietary polyphenols, (-)-epigallocatechin-3-gallate and genistein, has also been observed. The results suggest that the DNA hypermethylation of MGMT is an important mechanism for MGMT gene silencing in the development of ESCC, and this epigenetic event may be prevented or reversed by these polyphenols for the prevention of carcinogenesis.

Carcinoma, Squamous Cell↗

PubMed2000

Gene promoter hypermethylation in tumors and serum of head and neck cancer patients.

Promoter hypermethylation is an important pathway for repression of gene transcription in cancer cells. We analyzed aberrant DNA methylation at four genes in primary tumors from 95 head and neck cancer patients and then used the presence of this methylation as a marker for cancer cell detection in serum DNA. These four genes were tested by methylation-specific PCR and included: p16 (CDKN2A), O6-methylguanine-DNA-methyltransferase, glutathione S-transferase P1, and death-associated protein kinase (DAP-kinase). Fifty-five % (52 of 95) of the primary tumors displayed promoter hypermethylation in at least one of the genes studied: 27% (26/95) at p16, 33% (31 of 95) at O6-methylguanine-DNA-methyltransferase; and 18% (17 of 92) at DAP-kinase. No promoter hypermethylation was observed at the glutathione S-transferase P1 gene promoter. We detected a statistically significant correlation between the presence of DAP-kinase gene promoter hypermethylation and lymph node involvement (P = 0.014) and advanced disease stage (P = 0.016). In 50 patients with paired serum available for epigenetic analysis, the same methylation pattern was detected in the corresponding serum DNA of 21 (42%) cases. Among the patients with methylated serum DNA, 5 developed distant metastasis compared with the occurrence of metastasis in only 1 patient negative for serum promoter hypermethylation (P = 0.056). Promoter hypermethylation of key genes in critical pathways is common in head and neck cancer and represents a promising serum marker for monitoring affected patients.

Apoptosis Regulatory Proteins↗

PubMed2002

Distinct promoter hypermethylation of p16INK4a, CDH1, and RAR-beta in intestinal, diffuse-adherent, and diffuse-scattered type gastric carcinomas.

Hypermethylation of CpG islands in gene promoters is associated with silencing of various tumour suppressor genes. Recent studies of colorectal and gastric carcinomas have defined a CpG island methylator phenotype (CIMP), which involves the targeting of multiple genes by promoter hypermethylation. In this study, methylation-specific polymerase chain reaction (PCR) was performed to study methylation of CpG islands in the promoters of the p16(INK4a), cadherin 1 (CDH1), and retinoic acid receptor-beta (RAR-beta) genes in 45 gastric carcinomas and to investigate whether CDH1 and RAR-beta promoter hypermethylation is associated with CIMP-positive gastric carcinoma. CpG island hypermethylation of the p16(INK4a), CDH1, and RAR-beta promoters was detected in 12 (27%), 26 (58%), and 24 (53%) of the 45 gastric carcinomas, respectively. Hypermethylation of the p16(INK4a) promoter was more common in intestinal type than in diffuse type gastric carcinomas (p = 0.0023; Fisher's exact test) and was inversely associated with p53 mutations (p = 0.0225; Fisher's exact test). However, CDH1 and RAR-beta promoter hypermethylation was observed more frequently in diffuse-scattered type gastric carcinoma than in other types (intestinal and diffuse-adherent types) (p = 0.0175 and p = 0.0335, respectively; Fisher's exact test) and was not associated with p53 mutation status. Moreover, hypermethylation of the CDH1 and RAR-beta promoters occurred concordantly (p < 0.0001; Fisher's exact test). These results suggest that at least two types of promoter methylation status are involved in the development of the intestinal (p16(INK4a) promoter hypermethylation) and diffuse-scattered types (CDH1 and RAR-beta promoter hypermethylation) of gastric carcinoma.

Antigens, CD↗

PubMed2007

Promoter hypermethylation profile of cell cycle regulator genes in pituitary adenomas.

Aberrant hypermethylation of CpG islands in the promoter region plays a causal role in the inactivation of various key genes involved in the cell cycle regulatory cascade, which could result in a loss of cell cycle control. The aim of the present study was to examine in more detail the prevalence and role of the promoter methylation of genes with a proven involvement in the cell cycle regulation of pituitary adenomas, since their tumorigenesis has not yet been clearly defined. We profiled the CpG island methylation status of a series of well-characterized cell cycle regulation genes: the RB1, p14(ARF), p15(INK4b), p16(INK4a), p21(Waf1/Cip1), p27(Kip1), and p73 genes, in 34 pituitary adenomas as determined by a methylation-specific polymerase chain reaction assay. Promoter hypermethylation of the RB1, p14(ARF), p15(INK4b), p16(INK4a), p21(Waf1/Cip1), p27(Kip1), and p73 genes was detected in 12 (35%), 2 (6%), 11 (32%), 20 (59%), 1 (3%), 0 (0%), and 4 (12%) of the adenomas, respectively. In total, 88% (30 of 34) of the adenomas displayed methylation of at least one of such cell cycle regulatory genes, especially methylation of the member genes of the RB1 pathway (29 of 34; 85%). Promoter hypermethylation of p15(INK4b) coincided with RB1 and/or p16(INK4a) methylation, whereas RB1 and p16(INK4a) methylations tended to be mutually exclusive (p = 0.0048). Furthermore, promoter hypermethylations of p14(ARF), p21(Waf1/Cip1), and p73 (not belonging to the member genes of the RB1 pathway) were also coincident with RB1 and/or p16(INK4a) methylation except in one p73 methylated case. In contrast, none of the clinicopathological features, including the cell proliferation index, was significantly correlated with any particular methylation status. Our results suggested that aberrant hypermethylation of the key cell cycle regulatory genes occurs at a relatively high frequency in pituitary adenomas, especially in RB1 pathway genes with promoter hypermethylation of the p16(INK4a) gene being the most common deregulation. We further obtained evidence to indicate that RB1 and p16(INK4a) methylations tended to be mutually exclusive, but did occasionally coincide with other cell cycle regulation gene methylations.

Adenoma↗

PubMed2000

Promoter hypermethylation and BRCA1 inactivation in sporadic breast and ovarian tumors.

BACKGROUND: Inherited mutations in the BRCA1 gene may be responsible for almost half of inherited breast carcinomas. However, somatic (acquired) mutations in BRCA1 have not been reported, despite frequent loss of heterozygosity (LOH or loss of one copy of the gene) at the BRCA1 locus and loss of BRCA1 protein in tumors. To address whether BRCA1 may be inactivated by pathways other than mutations in sporadic tumors, we analyzed the role of hypermethylation of the gene's promoter region. METHODS: Methylation patterns in the BRCA1 promoter were assessed in breast cancer cell lines, xenografts, and 215 primary breast and ovarian carcinomas by methylation-specific polymerase chain reaction (PCR). BRCA1 RNA expression was determined in cell lines and seven xenografts by reverse transcription-PCR. P values are two-sided. RESULTS: The BRCA1 promoter was found to be unmethylated in all normal tissues and cancer cell lines tested. However, BRCA1 promoter hypermethylation was present in two breast cancer xenografts, both of which had loss of the BRCA1 transcript. BRCA1 promoter hypermethylation was present in 11 (13%) of 84 unselected primary breast carcinomas. BRCA1 methylation was strikingly associated with the medullary (67% methylated; P =.0002 versus ductal) and mucinous (55% methylated; P =.0033 versus ductal) subtypes, which are overrepresented in BRCA1 families. In a second series of 66 ductal breast tumors informative for LOH, nine (20%) of 45 tumors with LOH had BRCA1 hypermethylation, while one (5%) of 21 without LOH was methylated (P =.15). In ovarian neoplasms, BRCA1 methylation was found only in tumors with LOH, four (31%) of 13 versus none of 18 without LOH (P =.02). The BRCA1 promoter was unmethylated in other tumor types. CONCLUSION: Silencing of the BRCA1 gene by promoter hypermethylation occurs in primary breast and ovarian carcinomas, especially in the presence of LOH and in specific histopathologic subgroups. These findings support a role for this tumor suppressor gene in sporadic breast and ovarian tumorigenesis.

Breast Neoplasms↗

PubMed2001

Increased sensitivity of transforming growth factor (TGF) beta 1 null cells to alkylating agents reveals a novel link between TGFbeta signaling and O(6)-methylguanine methyltransferase promoter hypermethylation.

Inactivation of the transforming growth factor beta (TGFbeta)-signaling pathway and gene silencing through hypermethylation of promoter CpG islands are two frequent alterations in human and experimental cancers. Here we report that nonneoplastic TGFbeta1-/- keratinocyte cell lines exhibit increased sensitivity to cell killing by alkylating agents, and this is due to lack of expression of the DNA repair enzyme O(6)-methylguanine DNA methyltransferase (MGMT). In TGFbeta1-/- but not TGFbeta1+/- cell lines, the CpG dinucleotides in the MGMT promoter are hypermethylated, as measured by restriction enzyme analysis and methylation specific polymerase chain reaction. In one unstable TGFbeta1+/- cell line, loss of the wild type TGFbeta1 allele correlates with the appearance of methylation in the MGMT promoter. Bisulfite sequencing shows that in the KO3 TGFbeta1-/- cell line nearly all of the 28 CpG sites in the MGMT promoter 475 base pairs upstream of the start site of transcription are methylated, whereas most are unmethylated in the H1 TGFbeta1+/- line. Treatment of the TGFbeta1-/- cell lines with 5-azacytidine causes reexpression of MGMT mRNA and demethylation of CpG islands in the promoter. Analysis of the time course of methylation using methylation-specific polymerase chain reaction shows a lack of methylation in primary TGFbeta1-/- keratinocytes and increasing methylation with passage number of immortalized clones. Subcloning of early passage clones reveals a remarkable heterogeneity and instability of the methylation state in the TGFbeta1-/- keratinocytes. Thus, the TGFbeta1-/- genotype does not directly regulate MGMT methylation but predisposes cells to immortalization-associated MGMT hypermethylation.

Alkylating Agents↗

PubMed2006

Wnt signaling promoter hypermethylation distinguishes lung primary adenocarcinomas from colorectal metastasis to the lung.

Promoter hypermethylation is responsible for gene inactivation during carcinogenesis. It has been proposed that there is some degree of specificity in the set of genes that become altered by this mechanism in distinct tumor types. To understand whether promoter hypermethylation may differentiate the site of origin, 49 lung adenocarcinomas from 31 lung primaries and 18 metastases from colorectal primaries, respectively, were tested for the presence of this alteration in the APC, CDH1, DAPK, GSTP1, MLH1, MGMT, P14, P16, RARbeta2, RASSF1, sFRP1 and WIF-1 genes. A distinct profile was apparent for the 2 groups of lung tumors and the frequencies of promoter hypermethylation at sFRP1 and WIF-1, 2 genes involved in Wnt signaling, and at CDH1 were significantly higher in colorectal metastases than in lung primaries, whereas methylation of the APC promoter was significantly more common in lung primary adenocarcinomas. Some tumors showed concomitant APC, sFRP1 and WIF-1 gene inactivation, indicating that multiple DNA methylation events must have occurred to definitively down-regulate the signaling through Wnt. However, promoter hypermethylation at the APC and CDH1 genes tended to be mutually exclusive (Fisher's exact test, p = 0.006), suggesting a similar role in carcinogenesis. In conclusion, we propose that inactivation by promoter hypermethylation at the APC, CDH1, sFRP1 and WIF-1 genes may contribute to the discrimination of lung primary adenocarcinomas from colorectal metastasis to the lung, and report the simultaneous presence of methylation at the promoters of multiple genes involved in the Wnt signaling. This may have biological consequences for carcinogenesis.

Adenocarcinoma↗