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PubMed1999

DNA methylation analysis of the promoter region of the human telomerase reverse transcriptase (hTERT) gene.

The promoter of the hTERT gene encoding the catalytic subunit of telomerase was recently cloned and has a dense CG-rich CpG island, suggesting a role for methylation in regulation of hTERT expression. In this study, we have initiated the analysis of the regulation of hTERT expression by examining the methylation status of up to 72 CpG sites extending from 500 bases upstream of the transcriptional start site of the hTERT gene into the first exon in 37 cell lines. These cell lines represent a variety of cell and tissue types, including normal, immortalized, and cancer cell lines from lung, breast, and other tissues. Using bisulfite genomic sequencing, we did not find a generalized pattern of site-specific or region-specific methylation that correlated with expression of the hTERT gene: most of the hTERT-negative normal cells and about one-third of the hTERT-expressing cell lines had the unmethylated/hypomethylated promoter, whereas the other hTERT-expressing cell lines showed partial or total methylation of the promoter. The promoter of one hTERT-negative fibroblast cell line, SUSM-1, was methylated at all sites examined. Treatment of SUSM-1 cells with the demethylating agent 5-aza-2'-deoxycytidine and the histone deacetylase inhibitor trichostatin A induced the cells to express hTERT, suggesting a potential role for DNA methylation and/or histone deacetylation in negative regulation of hTERT. This study indicates that there are multiple levels of regulation of hTERT expression in CpG island methylation-dependent and -independent manners.

Cell Line↗

PubMed2000

Frequent methylation of estrogen receptor in prostate cancer: correlation with tumor progression.

Prior studies have shown that the estrogen receptor (ER) gene is down-regulated in prostate cancer, but the mechanism of its inactivation is not known. We hypothesize that inactivation of the ER gene in prostate cancer is through promoter methylation. To test this hypothesis, we investigated the methylation status of the ER gene in prostate cancer cell lines, prostate cancer, and benign prostatic hyperplasia (BPH) tissues samples using the bisulfite genomic sequencing method. Our results show that the ER gene promoter was methylated in 100% (six of six) of the prostate cancer cell lines tested and all were accompanied by loss of ER mRNA expression. Treatment of these cell lines with demethylating agent 5-aza-2'-deoxycytidine restored ER mRNA expression in all of the ER-negative cell lines. In addition, elevated expression of DNA methyltransferase mRNA was found in all of the prostate cancer cell lines. Of the prostate tissue samples analyzed, 60% (6 of 10) in the BPH samples, 80% (8 of 10) in the low-grade cancer samples (grades I and II), and 95% (20 of 21) in the high-grade cancer samples (grades III-V) exhibited promoter methylation of the ER gene. The overall methylation levels in the cancer samples were higher than that in the BPH samples. The differences between the high-grade cancer samples and BPH samples were significant at all CpG sites. Only at three CpG sites were the differences significant between the low-grade cancer samples and BPH samples. This study presents the first evidence that ER gene is transcriptionally inactivated by DNA methylation in prostate cancer. Our data suggest that ER may be involved in the pathogenesis of prostate cancer, as well as BPH.

DNA (Cytosine-5-)-Methyltransferase 1↗

PubMed2000

Methylation patterns in human androgen receptor gene and clonality analysis.

Tumor clonality, an important issue in tumor biology, has been analyzed using X-chromosome inactivation studies based on the differential methylation patterns of active and inactive alleles. Recently, a PCR-based androgen receptor gene (AR) analysis method was developed that takes advantage of highly polymorphic CAG repeats and nearby HpaII and HhaI sites in exon 1 of AR at the Xq13 region. However, the data from this assay, which is now widely used, are sometimes uninterpretable and irreproducible for some currently unclear reason. To determine that reason, we analyzed a panel of lung cancer cell lines, using HpaII or HhaI restriction enzymes, methylation-specific PCR, and bisulfite genomic sequencing of the polymorphic CAG repeat site of AR exon 1, including nearby CpG sites. We found direct evidence of a variable methylation pattern at the restriction sites that prevented proper enzyme cleavage in two lung cancer cell lines (NCI-H292 and NCI-H1944) obtained from female patients who had a polymorphic CAG repeat in AR exon 1. Our data suggest that methylation patterns at the CpG sites of AR exon 1 are complicated and vary among different individuals. Therefore, the reliability of the PCR-based clonality analysis may require further evaluation.

Alleles↗

PubMed2000

Activation of the MN/CA9 gene is associated with hypomethylation in human renal cell carcinoma cell lines.

The MN/CA9 (G250) gene expressed in the normal alimentary tract in a tissue-specific manner is often activated in renal cell carcinomas. To cast light on the activation mechanism, we examined the methylation status of this gene in seven human renal cell carcinoma cell lines (SKRC-01, -06, -10, -12, -14, -44, and -59) and three normal kidney tissue samples by using the bisulfite genomic sequencing protocol. CpG methylation was measured at seven locations in the MN/CA9 5' region. MN/CA9 transcripts were detected by reverse transcription-polymerase chain reaction in five of the renal cell carcinoma cell lines (SKRC-01, -06, -10, -44, and -59). These MN/CA9 positive cell lines showed hypomethylation, whereas the remaining two cell lines (SKRC-12, and -14), and three normal kidney tissue samples without transcripts demonstrated hypermethylation. Treatment with the demethylating agent 5-aza-2'-deoxycytidine resulted in activation of the MN/CA9 gene in the negative cell lines (SKRC-12 and -14). These data suggest that hypomethylation in the 5' region may have a major role in expression of the MN/CA9 gene in renal cell carcinoma cells.

Antigens, Neoplasm↗

PubMed2000

Hypermethylation of multiple genes in pancreatic adenocarcinoma.

Hypermethylation of CpG islands is a common mechanism by which tumor suppressor genes are inactivated. We studied 45 pancreatic carcinomas and 14 normal pancreata for aberrant DNA methylation of CpG islands of multiple genes and clones using methylation-specific PCR (MSP) and bisulfite-modified sequencing. Using MSP, we detected aberrant methylation of at least one locus in 60% of carcinomas. The genes analyzed included RARbeta (methylated in 20%), p16 (18%), CACNA1G (16%), TIMP-3 (11%), E-cad (7%), THBS1 (7%), hMLH1 (4%), DAP kinase (2%), and MGMT (0%). In addition, aberrant methylation was found in three CpG islands (MINT31, -1, and -2) in 38, 38, and 14% of carcinomas, respectively. Hypermethylation was largely confined to the carcinomas with only three loci (E-cad, DAP kinase, and MINT2) harboring methylation in some normal pancreata (36, 21, and 14%, respectively). Simultaneous methylation of at least four loci was observed in 5 of 36 (14%) pancreatic adenocarcinomas. We defined this subgroup of pancreatic adenocarcinomas as "CpG island-methylator-phenotype positive (CIMP+)." Two of four carcinomas with microsatellite instability harbored promoter hypermethylation of hMLH1, and both cases were CIMP+. Thus, we conclude that many pancreatic carcinomas hypermethylate a small percentage of genes, whereas a subset displays a CIMP+ phenotype.

Adenocarcinoma↗

PubMed2000

Aberrant methylation of the Cyclooxygenase 2 CpG island in colorectal tumors.

Cyclooxygenases (COXs) are key enzymes that convert arachidonic acid to prostaglandins. Overexpression of one of the COX isozymes, COX2, has been shown to play an important role in colorectal cancer progression. Recently, however, low expression of COX2 has been reported in a subset of colorectal and gastric cancers. Aberrant CpG island methylation and associated transcriptional silencing are common in colorectal cancer, and we therefore investigated the potential role of methylation in the transcriptional silencing of COX2. We examined the methylation status of the COX2 5' CpG island in a series of tumor cell lines. Among the 33 cell lines examined, dense methylation (>70%) of COX2 was detected in 5 cell lines, and partial methylation was detected in 10 cell lines. Detailed methylation mapping using bisulfite genomic sequencing revealed that loss of expression of COX2 mRNA was closely correlated with methylation of a region upstream of exon 1, and expression could be restored by demethylation using the DNA methyltransferase inhibitor 5-aza-deoxycytidine. Aberrant methylation of COX2 was also detected in 12 of 92 (13%) unselected sporadic primary colorectal cancers and 7 of 50 (14%) colorectal adenomas. COX2 methylation was strongly associated with the presence of the CpG island methylator phenotype (P<0.01), inversely related to p53 gene mutation (P<0.01), and unrelated to microsatellite instability status. We propose that COX2 expression in colorectal tumors is modulated by functional factors that favor high expression and by the CpG island methylator phenotype that favors silencing in a subset of cases. These results raise the possibility that tumors with COX2 methylation may be less sensitive to treatment using specific COX2 inhibitors.

Colorectal Neoplasms↗

PubMed2001

Methylation of the E-cadherin gene promoter correlates with progression of prostate cancer.

PURPOSE: We studied the methylation status of E-cadherin gene promoter in prostate cancer and its relationship with E-cadherin inactivation in prostate cancer. MATERIALS AND METHODS: Seven human prostate cell lines and 35 microdissected prostate cancer specimens were analyzed for E-cadherin promoter methylation using the bisulfite genome sequencing technique. E-cadherin messenger (m)RNA expression and protein expression were also studied in prostate cell lines by reverse transcriptase-polymerase chain reaction and in prostate cancer specimens by immunostaining, respectively. RESULTS: The overall methylation of E-cadherin promoter was evident in 14 of 20 grades III to V (70%) and in 5 of 15 grades I to II (33%) prostate cancer samples. It correlated with absent or reduced E-cadherin immunostaining. Methylation in low grade tumors was present mainly in the exon region, whereas in high grade tumors methylation was also present in the promoter region. Methylation was noted in 2 of 6 prostate cancer cell lines (33%) and correlated well with decreased E-cadherin mRNA in these cell lines. Treatment with the demethylating agent 5-aza-2'-deoxycytidine restored E-cadherin mRNA levels in the E-cadherin negative prostate cancer cell lines TSUPr1 and DuPro. CONCLUSIONS: Methylation of the E-cadherin gene is common in prostate cancer and the severity of E-cadherin methylation correlates with tumor progression. This study implies that the invasion and metastasis suppressor function of E-cadherin may often be compromised in human prostate cancer by epigenetic rather than by mutational events.

Cadherins↗

PubMed2001

Frequent epigenetic inactivation of RASSF1A in human bladder carcinoma.

Allelic deletion or transcriptional silencing of RASSF1, a putative tumor suppressor at 3p21.3, has been found in a considerable proportion of lung, breast, and ovarian cancers. In this study, we analyzed the expression and mutation status of three RASSF1 isoforms (-A, -B, and -C) in 55 primary bladder carcinomas and 10 bladder and prostate cancer cell lines. The RASSF1A transcript was not found in 80% (4 of 5) and 100% (4 of 4) of bladder and prostate cell lines, respectively. Compared with normal bladder tissues, loss or significant reduction of RASSF1A was identified in 62% (34 of 55) of primary bladder carcinomas and 10 (83%) of 12 matched sets showed tumor-specific alteration of RASSF1A expression. Moreover, loss or abnormal down-regulation of RASSF1A correlated with advanced tumor stage. RASSF1B was undetectable in 60% (3 of 5) of bladder cell lines and in 31% (17 of 55) of primary tumors, but none of these tumors showed altered expression exclusively in RASSF1B. RASSF1C transcript was detected in all cell lines and primary tumors we examined. Expression of RASSF1A and RASSF1B was reactivated in all nonexpressor cell lines by treatment with the demethylating agent 5-aza-2'-deoxycytidine. Bisulfite DNA sequencing analysis revealed that aberrant hypermethylation at the CpG island in the RASSF1A promoter is strongly associated with the loss of RASSF1A expression in cell lines and uncultured primary tumors. Methylation-specific PCR and BstUI digestion analyses also demonstrated that 97% (33 of 34) of RASSF1A-nonexpressing primary tumors are methylated. Although somatic mutations were not identified in RASSF1 transcripts expressed in unmethylated tumors, 24% (9 of 37) of methylated cell lines and primary tumors showed detectable reductions in genomic levels of RASSF1, suggesting that RASSF1A inactivation might be caused by both epigenetic and genetic mechanisms in a subset of bladder tumors. Together, our data suggest that RASSF1A inactivation may play a critical role in the malignant progression of human bladder carcinomas.

Adult↗

PubMed2001

Frequent epigenetic inactivation of RASSF1A by aberrant promoter hypermethylation in human gastric adenocarcinoma.

Methylation associated inactivation of RASSF1, a putative tumor suppressor identified at 3p21.3, has been frequently observed in several human malignancies, including lung and breast cancers. To explore the penetrance of RASSF1 in gastric carcinogenesis, we performed expression and mutation analyses of 3 isotypes of RASSF1 (A, B, and C) in 150 gastric specimens, including 15 carcinoma cell lines. RASSF1A and RASSF1B transcripts were not expressed in 60% (9 of 15) and 33% (5 of 15) of gastric carcinoma cell lines, respectively, whereas RASSF1C was detectable in all cell lines. Bisulfite DNA sequencing analysis revealed that the CpG island in the RASSF1A promoter is hypermethylated in all RASSF1A-nonexpressing cell lines. In addition, both RASSF1A and RASSF1B were re-expressed by treatment with the demethylating agent 5-aza-2'-deoxycytidine. Among 90 primary gastric adenocarcinomas examined, 41 (46%) and 19 (21%) expressed no or abnormally low levels of RASSF1A and RASSF1B, respectively, and 12 (13%) tumors showed no expression of both isoforms. Loss or abnormal down-regulation of RASSF1A correlated with tumor stage and grade but not with histological types of tumors. Methylation-specific PCR analysis demonstrated that 95% (39 of 41) of RASSF1A-nonexpressing primary tumors are methylated at the CpG sites in the promoter, whereas none of the adjacent noncancerous or normal tissues are methylated. No somatic mutations were detected in RASSF1 transcripts expressed in unmethylated tumors. However, 10 methylated tumors, including 4 cell lines, showed low genomic levels of RASSF1 and expressed no RASSF1A transcripts, suggesting that RASSF1A inactivation might be caused by both epigenetic and genetic mechanisms in a subset of gastric adenocarcinomas. In conclusion, our data indicate that epigenetic transcriptional silencing of RASSF1, especially RASSF1A isoform, is a frequent event in gastric tumorigenesis and might play an important role in the malignant progression of gastric adenocarcinomas.

Adenocarcinoma↗

PubMed2002

[Study on abnormal expression of the p73 gene in childhood acute lymphoblastic leukemia].

OBJECTIVE: To investigate the relationship between p73 gene and the development and progression of childhood acute lymphoblastic leukemia (ALL). METHODS: The levels of p73 transcripts in 61 ALL cell lines and 53 childhood ALL patients were assayed using reverse transcriptase-polymerase chain reaction (RT-PCR), and their relationship with the clinicopathological characteristics was analyzed. Besides, the methylation status of p73 exon 1 was analysed by restriction-enzyme related PCR, methylation-specific PCR and bisulfite genomic sequencing in the 61 ALL cell lines. RESULTS: Of the 61 ALL cell lines, 42 showed expression of p73 mRNA, with a negative rate of 31.1%, and of the 53 primary childhood ALL samples, 39 showed expression of p73 mRNA, with a negative rate of 26.4%. Loss of p73 expression was significantly associated with the reduced disease free survival and overall survival of the patients. 39.3% (24/61) of the ALL cell lines showed hypermethylation of p73 exon 1, while normal lymphocytes and most cell lines expressed p73 mRNA were not hypermethylated. CONCLUSION: There was a higher negative expression rate of p73 mRNA in childhood ALL. The main mechanism of the loss expression would be the hypermethylation of p73 gene. p73 gene inactivation might play an important role in the pathogenesis of ALL. Examination of p73 mRNA might have clinical significance in predicting prognosis of childhood ALL.

Adolescent↗

PubMed2003

Hypomethylation of the synuclein gamma gene CpG island promotes its aberrant expression in breast carcinoma and ovarian carcinoma.

Recent studies indicate that synuclein gamma (SNCG) gene, located in chromosome 10, participates in the pathogenesis of the breast and ovary. SNCG, also known as breast cancer-specific gene 1 (BCSG1), is not expressed in normal mammary or ovarian surface epithelial cells but is highly expressed in the vast majority of advanced staged breast and ovarian carcinomas. When overexpressed, SNCG significantly stimulates breast cancer proliferation and metastasis. To fully understand the molecular mechanisms underlying the abnormal expression of SNCG in neoplastic diseases, in this study, we extensively examined the methylation status of a CpG island located in exon 1 of SNCG gene in a panel of breast and ovarian tumor-derived cell lines to determine whether DNA methylation plays a crucial role in SNCG expression. In vivo bisulfite DNA sequencing of genomic DNA isolated from breast cancer cell lines showed that the 15 CpG sites within the CpG island were completely unmethylated in all SNCG-positive cell lines (5 of 5), but were densely and heterogeneously methylated in the majority of SNCG-negative cell lines (3 of 4). The methylation occurred primarily at the CpG sites 2, 5, 7, and 10-15. Similarly, we observed a strong correlation of hypomethylation of the CpG island and SNCG expression in ovarian cancer cell lines (5 of 5). Intriguingly, the methylation pattern in ovarian cancer cells is different from that in breast cancer cells. In SNCG-nonexpressing ovarian cancer cells, all 15 of the CpG sites were completely methylated instead of selective methylation at certain sites shown in breast cancer cells, thereby suggesting a tissue-specific methylation pattern. A correlation between hypomethylation of the exon 1 and expression of SNCG mRNA was also observed in primary breast tumor tissues. The importance of DNA methylation in the control of SNCG expression in cancer cells is further strengthened by demonstration of re-expression of SNCG mRNA in SNCG-negative ovarian and breast cancer cells with a demethylating agent 5-aza-2'-deoxycytidine. In addition, we demonstrate that inhibition of cell growth leads to a decreased mRNA expression and an increased DNA methylation of SNCG gene. Taken together, these new findings strongly suggest that DNA hypomethylation is a common mechanism underlying the abnormal expression of this candidate oncogene in breast and ovarian carcinomas.

Azacitidine↗

PubMed2003

Epigenetic inactivation of laminin-5-encoding genes in lung cancers.

PURPOSE: We investigated the loss of expression of three laminin-5 (LN5)-encoding genes in lung cancer cell lines and elucidated the mechanism of inactivation of the genes in lung cancer cell lines and tumors. EXPERIMENTAL DESIGN: We examined the expression of LN5-encoding genes by reverse transcription-PCR in 49 lung cancer cell lines. To elucidate the mechanism of gene silencing, we treated expression-negative cell lines (two for each gene) with a demethylating agent and examined the restoration of expression by reverse transcription-PCR. We dissected out the methylation patterns of CpG sites unique to the promoter regions of LN5-encoding genes by bisulfite genomic sequencing of expression-negative cell lines. We designed methylation-specific primers and validated the methylation status of the promoter regions in lung cancer cell lines using methylation-specific PCR. We further studied the methylation patterns of primary non-small cell lung cancer [NSCLC (n = 36)], small cell lung cancer [SCLC (n = 26)], and carcinoids (n = 24) tumors. RESULTS: We observed frequent losses of expression in NSCLC (20-60%) and SCLC (65-86%) cell lines. Expression of one or more genes was lost in 90% of SCLC cell lines and 65% of NSCLC cell lines. Treatment of expression-negative cell lines with demethylating agent restored expression in all of the cases. Methylation of LN5-encoding genes was present more frequently in SCLC cell lines (60-80%) than in NSCLC cell lines (15-60%), and at least one gene was methylated in 95% of SCLC and 60% of NSCLC cell lines. The concordances between loss of expression and methylation in 40 lung cancer cell lines for the three genes (90-95%) were statistically significant. Methylation was more frequent in SCLC tumors (58-77%) than in NSCLC tumors (22-42%) and carcinoids (13-33%), and at least one gene was methylated in 92% of SCLC tumors, 47% of NSCLC tumors, and 33% of carcinoids. CONCLUSIONS: Our results demonstrate frequent epigenetic inactivation of LN5-encoding genes in lung cancers, and these findings are of biological interest and are potentially of clinical importance.

Azacitidine↗

PubMed2003

Epigenetic down-regulation of death-associated protein kinase in lung cancers.

PURPOSE: Death-associated protein kinase (DAPK) is a pro-apoptotic serine/threonine kinase involved in apoptosis. Aberrant methylation of DAPK was reported in lung cancers by methylation-specific PCR. However, we were unable to relate methylation with gene silencing with the same methodology. Our goals were to develop a methodology that related methylation with gene silencing and use it to study the state of the gene in lung cancers. EXPERIMENTAL DESIGN AND RESULTS: Using a semiquantitative real-time reverse transcription-PCR, DAPK expression was lower in lung cancers than in corresponding nonmalignant bronchial epithelial cells in five of six primary short-term cultures. In continuous cell lines, mRNA expression was down-regulated, as well as compared with nonmalignant bronchial epithelial cells, and its protein was not detected by Western blotting in 17 of 23 (74%) cell lines. We investigated methylation status of 5' flanking region of DAPK by combined bisulfite restriction analysis and bisulfited DNA sequencing. Aberrant methylation was detected in 21 of 48 (44%) cell lines, 2 of 6 primary cultured tumors, and 14 of 38 (37%) primary lung cancers, although varying degrees of methylation were noticed. Furthermore, bisufite sequence data suggested that aberrant methylation might occur selectively at some CpG dinucleotides in cell lines which had absent expression. Treatment with 5-aza-2'-deoxycytidine restored DAPK expression in heavily methylated cell lines tested, and histone deacetylase inhibitor trichostatin A alone restored DAPK expression in some methylated cell lines as well. CONCLUSIONS: Our major findings are: (a) DAPK expression is frequently down-regulated in lung cancers; (b) aberrant methylation of DAPK is frequent in lung cancers, although considerable heterogeneity of methylation is present, and some specific CpG dinucleotides are often methylated in expression negative lung cancers; and (c) besides methylation and histone deacetylation, there may be other mechanisms for down-regulation of DAPK expression.

Apoptosis↗

PubMed2003

A tumor-suppressive role for trypsin in human cancer progression.

Trypsin is a serine protease family member with a potential role in cancer invasion. We investigated trypsinogen expression at the RNA level in 49 esophageal squamous cell carcinomas (ESCCs) and 72 gastric adenocarcinomas. Almost all primary ESCC tissues (95%) showed reduced expression, and 9 of 13 ESCC cell lines were silenced for trypsinogen expression. Absent expression correlated with promoter hypermethylation of trypsinogen-4 by bisulfite DNA sequence. Moreover, we detected promoter hypermethylation in 50% of primary ESCCs by methylation-specific PCR. A subset of gastric adenocarcinomas (71%) also showed reduced trypsinogen accompanied by reduction in PAR2, a G protein activated by trypsin, and a propensity to penetrate beyond the gastric wall (P = 0.001). Our results support the notion that trypsin plays a tumor-suppressive role in human carcinoma.

Adenocarcinoma↗

PubMed2003

Hypermethylation of XIAP-associated factor 1, a putative tumor suppressor gene from the 17p13.2 locus, in human gastric adenocarcinomas.

X-linked inhibitor of apoptosis (XIAP) is the most potent member of the IAP family that exerts antiapoptotic effects by interfering with the activities of caspases. Recently, XIAP-associated factor 1 (XAF1) and two mitochondrial proteins, Smac/DIABLO and HtrA2, have been identified to negatively regulate the caspase-inhibiting activity of XIAP. To explore the candidacy of XAF1, Smac/DIABLO, and HtrA2 as a tumor suppressor in gastric tumorigenesis, we investigated the expression and mutation status of the genes in 123 gastric tissues and 15 cancer cell lines. Whereas Smac/DIABLO and HtrA2 transcripts were normally expressed in all cancer specimens we examined, XAF1 transcript was not expressed or present at extremely low levels in 40% (6 of 15) of cancer cell lines and in 23% (20 of 87) of primary carcinomas. Abnormal reduction of XAF1 expression showed a strong correlation with stage and grade of tumors, and a tumor-specific down-regulation of XAF1 was observed in 45% (9 of 20) of matched sets. Unlike XAF1, XIAP expression exhibited no detectable alteration in cancers. Whereas loss of heterozygosity within the XAF1 region or somatic mutations of the gene was not detected, expression of XAF1 transcript was reactivated in all nonexpressor cell lines after 5-aza-2-deoxycytidine treatment. The 5' upstream region of the XAF1 gene encompasses no gastric cell-rich region that rigorously satisfies the formal criteria for CpG islands. However, bisulfite DNA sequencing analysis for 34 CpG sites in the promoter region revealed a strong association between hypermethylation and gene silencing. Moreover, transcriptional silencing of XAF1 was tightly associated with hypermethylation of seven CpGs located in the 5' proximal region (nucleotides -23 to -234). Additionally, loss or abnormal reduction of XAF1 expression was found to inversely correlate with p53 mutations, suggesting that epigenetic inactivation of XAF1 and mutational alteration of p53 might be mutually exclusive events in gastric tumorigenesis. Collectively, our study suggests that epigenetic silencing of XAF1 by aberrant promoter methylation may contribute to the malignant progression of human gastric tumors.

Adaptor Proteins, Signal Transducing↗

PubMed2004

CpG methylation of the ERalpha and ERbeta genes in breast cancer.

Aberrant methylation of promoter CpG islands is known to be responsible for the alteration and silencing of genes in cancer. The data presented here describes that the CpG methylations of ERalpha and ERbeta are found in breast cancer tissues, and methylation exerts a considerable effect on gene silencing, investigated by bisulfite genomic sequencing and reverse transcriptase polymerase chain reaction. Consequently, hypermethylation of the ERalpha and ERbeta genes was found in 66.0% and 50.0% of 50 breast cancers, respectively. Eleven of 50 samples (22.0%) did not show any methylation of either ERalpha or ERbeta, whereas 19 samples (38.0%) showed methylation of both ERalpha and ERbeta. The tumors that showed aberrant methylation of ERalpha and ERbeta did not express mRNA, compared with unmethylated cases (p < 0.01). The methylated case was negatively correlated with the expression of the ERalpha protein (p < 0.01). In addition, ERbeta methylation demonstrated significant associations with the lower level of Ki67 (9.36 +/- 2.43 versus 19.68 +/- 3.42, p = 0.02). Although the number of samples was relatively small, our results suggest that DNA methylation in ERalpha and ERbeta are present in breast cancer tissue, and that the methylation causes a significant effect on transcriptional silencing. Furthermore, the CpG methylation of the ERbeta gene seems to play a role in cell proliferation of breast cancer tissue.

Aged↗

PubMed2005

[Frequent epigenetic inactivation of XAF1 by promotor hypermethylation in human colon cancers].

BACKGROUND/AIMS: X-linked inhibitor of apoptosis (XIAP) is the most potent member of the IAP family that exerts antiapoptotic effects. Recently, XIAP-associated factor 1 (XAF1) and two mitochondrial proteins, Smac/ DIABLO and HtrA2, have been identified to negatively regulate the caspase-inhibiting activity of XIAP. We explored the candidacy of XAF1, Smac/DIABLO and HtrA2 as a tumor suppressor in colonic carcinogenesis. METHODS: Expression and mutation status of the genes in 10 colorectal carcinoma cell lines and 40 primary tumors were examined by quantitative PCR analysis. RESULTS: XAF1 transcript was not expressed or present at extremely low levels in 60% (6/10) of cancer cell lines whereas Smac/DIABLO and HtrA2 are normally expressed in all cell lines examined. Tumor-specific loss or reduction of XAF1 was also found in 35% (14/40) of matched tissue sets obtained from the same patients. XAF1 transcript was reactivated in all the low expressor cell lines by treatment with the demethylating agent 5-aza-2'-deoxycytidine. Moreover, bisulfite DNA sequencing analysis for 34 CpG sites in the promoter region revealed a strong association between hypermethylation and gene silencing. Restoration of XAF1 expression resulted in enhanced apoptotic response to etoposide and 5-flurouracil, whereas knockdown of XAF1 expression by siRNA transfection significantly inhibited chemotherapeutic drug-induced apoptosis. CONCLUSIONS: XAF1 undergoes epigenetic gene silencing in a considerable proportion of human colon cancers by aberrant promoter hypermethylation, suggesting that XAF1 inactivation might be implicated in colonic tumorigenesis.

Adaptor Proteins, Signal Transducing↗

PubMed2006

Epigenetic silencing of the sulfotransferase 1A1 gene by hypermethylation in breast tissue.

Sulfotransferase 1A1 (SULT1A1) is reported to be involved in the conjugation with sulfate, resulting in the inactivation of estrogens. Aberrant methylation of promoter CpG islands is known to be responsible for the alteration and silencing of the gene in cancers. This study was intended to evaluate the methylation status and transcriptional activity of SULT1A1 in breast cancer tissue (n=56), benign breast tissue (n=20) and morphologically normal breast tissue (n=20), examined by bisulfite genomic sequencing and reverse transcription (RT)-PCR. As a result, the methylation of the proximal promoter (P1) was identified in 64.3% of breast carcinomas, 15% of normal and 20% of benign breast tissues. In terms of the distal promoter (P0), 32 of 56 cancer tissues (57.1%) were methylated, while 4 normal (20%) and 6 benign tissues (30%) were methylated. Breast cancer tissue showed a higher methylation rate of SULT1A1 than normal and benign tissue at both P1 (p=0.001) and P0 (p=0.006) promoters with statistical significance. Furthermore, cancer tissue showed a higher methylation density rate than normal and benign breast tissue at both P1 and P0 promoters (P1, p=0.001; P0, p=0.001). The tissues that showed aberrant methylation of SULT1A1 did not express mRNA significantly, compared with the unmethylated cases (P1, p=0.003; P0, p=0.023). Although the number of samples was relatively small, our results suggest that DNA methylation in the SULT1A1 gene appears to be present in breast tissue including cancer and methylation significantly impacts transcriptional silencing of the gene. In addition, it can be suggested that progressive SULT1A1 methylation within the promoter area of the gene occurs during breast carcinogenesis.

Arylsulfotransferase↗