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Results for “DNA Modification Methylases”

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PubMed2001

Induction of MAGE-3 expression in lung and esophageal cancer cells.

BACKGROUND: Although MAGE-3 has been detected in approximately 40% of lung and esophageal cancers, expression of this cancer testis antigen appears to be below the threshold for immune recognition in patients with these malignancies. The aim of this study was to determine if the demethylating agent, 5-Aza-2'-deoxycytidine (DAC) and if the histone deacetylase inhibitor Depsipeptide FR901228 (DP) could enhance MAGE-3 expression in lung and esophageal cancer cells. METHODS: Eleven lung and esophageal cancer lines and cultured normal human bronchial epithelial (NHBE) cells were exposed to normal media (NM), DAC, DP, or combination DAC/DP at varying concentrations and exposure durations. MAGE-3 expression was evaluated by quantitative RT-PCR (TaqMan) and immunohistochemistry techniques. Trypan blue exclusion techniques were used to examine the proliferation of cancer cells after drug exposure. RESULTS: Relative to untreated controls, MAGE-3 expression was enhanced 32-fold (range 3.9 to 110) by DAC alone (0.1 micromol/L x 72 h), 2.1-fold (0.4 to 4.2) by DP alone (25 ng/mL x 6h), and 57-fold (4.6 to 209) by sequential DAC/DP exposure. Increased MAGE-3 mRNA copy numbers coincided with enhanced protein levels in these cells. MAGE-3 expression persisted after drug exposure. Flow cytometry confirmed the presence of functional HLA class I expression in these cells. Sequential DAC/DP treatment mediated pronounced growth inhibition in cancer cells but not NHBE. CONCLUSIONS: Sequential DAC/DP treatment may be a novel strategy to simultaneously augment MAGE-3 expression and induce growth arrest in thoracic malignancies.

Adenocarcinoma↗
PubMed2003

Coffee and its chemopreventive components Kahweol and Cafestol increase the activity of O6-methylguanine-DNA methyltransferase in rat liver--comparison with phase II xenobiotic metabolism.

A lower rate of colon cancer was observed in consumers of coffee with a high content of the diterpenes Kahweol and Cafestol (K/C). In animal models, K/C have been found to protect against the mutagenic/carcinogenic effects of compounds such as 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP), aflatoxin B1, and 7,12-dimethylbenz[a]anthracene. Thus far, such chemoprotection by K/C has been attributed to modifications of xenobiotic metabolism, e.g. enhanced detoxification by UDP-glucuronosyltransferase (UDPGT) and/or glutathione transferase (GST). In the present study, we investigated the potential of several coffee-related treatments (K/C [1:1], Cafestol-alone, Turkish coffee) to modify the expression level of the DNA repair protein O(6)-methylguanine-DNA methyltransferase (MGMT) which is involved in the reversal of the precarcinogenic DNA damage O(6)-alkylguanine induced by alkylating agents. The results show that, in the male F344 rat, K/C and Cafestol increase hepatic MGMT in a dose-dependent manner up to a maximum of 2.6-fold at 0.122% K/C in the feed. Turkish coffee led to enhancements of up to 16%, the more moderate increase being associated with the lower estimated K/C intake through the beverage. In the livers of the rats receiving Turkish coffee, we also found 10-30% increases in several GST-related parameters (overall GST, GST-pi, glutathione, gamma-glutamylcysteine-synthetase) and a two-fold increase in UDPGT activity. Dose-response studies with K/C revealed that MGMT increased in parallel with three of the four GST-related parameters whereas the dose-response curves of UDPGT and of GST-pi activity displayed a steeper slope. Increased expression level of MGMT may extend the antimutagenic/anticarcinogenic potential of coffee components to protection against DNA alkylating agents.

Animals↗
PubMed2002

Age-dependent DNA methylation changes in the ITGAL (CD11a) promoter.

DNA methylation patterns change with age in a complex fashion, typically with an overall decrease in genomic deoxymethylcytosine (d(m)C) content, but with local increases in some promoters that contain GC-rich sequences known as CpG islands. While the consequences of age-dependent CpG island methylation have recently been studied in organs such as the colon, less is known about the functional significance of the progressive hypomethylation of promoters lacking CpG islands, and the significance of age-dependent changes in T cell DNA methylation is completely unexplored. We asked if age-dependent DNA hypomethylation might contribute to overexpression of the T cell ITGAL gene, which encodes CD11a, a subunit of LFA-1. CD11a mRNA increased with age as well as with experimentally induced DNA hypomethylation. This increase correlated with hypomethylation of sequences flanking the ITGAL promoter in vitro and in aging. 'Patch' methylation of the region suppressed promoter function. DNA methyltransferases 1 and 3a also decreased with aging. These results indicate that hypomethylation of regions flanking the ITGAL promoter may increase CD11a expression, and suggest that age-dependent hypomethylation of promoters lacking CpG islands, perhaps due to decreased DNA methyltransferase expression, may be one mechanism contributing to increased T cell gene expression with aging.

Adult↗
PubMed1998

Epigenetic control of programmed cell death: inhibition by 5-azacytidine of 1,25-dihydroxyvitamin D3-induced programmed cell death in C6.9 glioma cells.

In mammalian DNA cytosine methylation occurs specifically at CpG dinucleotide. Although the full array of function of DNA methylation is yet to be elucidated, it is well established that DNA methylation is an important mechanism involved in gene expression, DNA replication and cancer. Rat glioma C6.9 cells undergo programmed cell death (PCD) after treatment with 1,25-dihydroxyvitamin D3 (1,25-D3). Hence, these cells were used to study whether DNA methylation was involved in the control of PCD. We found that 1,25-D3-mediated PCD of C6.9 cells was suppressed by exposure of the cells to the DNA demethylating agents 5-azacytidine (5-AzaC) and 5-aza-2'-deoxycytidine. This effect remains detectable several cell divisions following removal of 5-AzaC and, therefore, involves DNA methylation as an epigenetic regulatory mechanism of PCD. Accordingly, internucleosomal fragmentation, a feature of apoptosis that is detected in 1,25-D3-treated cells, is no longer observable after treatment of these cells with 5-AzaC. However, 5-AzaC does not totally suppress the responsiveness of C6.9 cells to 1,25-D3 since the induction of the c-myc gene remains unaffected. These results suggest that a change in DNA methylation pattern could suppress 1,25-D3-mediated PCD through the expression of previously hypermethylated genes such as proto-oncogenes with death-repressor activity, endogenous virus sequences or even genes inducing change in the differentiated state of these cells.

Animals↗
PubMed1996

A novel positive detection system of in vivo mutations in rpsL (strA) transgenic mice.

To positively detect the in vivo mutations accumulated in different mouse organs, we have developed a transgenic mouse system. This transgenic mouse carried an Escherichia coli (E. coli) plasmid pML4 as a shuttle vector that consisted of a replication origin (ori), the kanamycin-resistant gene (KanR) and the rpsL+ gene (strAS) derived from E. coli. These E. coli elements were expected to be inert in the transgenic mouse system; thus, neutral mutations would be accumulated on the shuttle plasmid in the transgenic mice. The shuttle plasmid vector was recovered from the mouse genomic DNA and introduced into kanamycin-sensitive (KmS) and streptomycin-resistant (SmR) E. coli cells by using electroporation. The original pML4 shuttle plasmid transformed the host E. coli to KmR and SmS, since both the KanR and rpsL genes exhibited dominant traits of KmR and SmS, respectively. On the other hand, when the retrieved pML4 shuttle plasmid carried a mutated rpsL gene, it could be positively detected as both KmR and SmR. Based on this principle, we were able to positively detect the in vivo mutations accumulated in the rpsL transgene of the shuttle vector pML4 integrated into the mouse genome. The total number of rescued shuttle plasmids were counted on the plates containing Km alone, while only mutants were detected on the plates containing both Km and Sm. We have so far established 22 independent transgenic mouse lines that carried up to approx. 750 copies of the shuttle plasmid pML4 in a haploid genome. By using high-copy-number transgenic mouse lines which carried 350 copies or more of the shuttle vector, we also developed a simple and proficient method for retrieving the shuttle plasmid from various tissues of the transgenic mice. The background mutant frequency was approx. 5 x 10(-5). In order to validate the applicability of the positive-detection transgenic system for the induced mutagenicity assay, methylnitrosourea (MNU) was administered to the transgenic mice, and an increase in the number of mutant frequencies was seen in all tested organs including spleen, liver and brain. The rpsL transgenic mouse system was therefore considered to provide a quick-and-easy risk assessment test for in vivo tissue-specific mutagenicity, using positive detection by streptomycin.

Animals↗
PubMed2001

Potential of 5-aza-2'-deoxycytidine (Decitabine) a potent inhibitor of DNA methylation for therapy of advanced non-small cell lung cancer.

Although new agents and drug combinations have increased the response rate in advanced non-small cell lung cancer (NSCLC), long-term survivors are rare. There is an urgent need to develop new chemotherapeutic approaches for disease. In a previous pilot phase I-II study on 5-aza-2'-deoxycytidine (5-AZA-CdR) in patients with stage IV NSCLC, we observed several interesting responses, including one patient that was still alive (68 months) at the time of publication of our results. In the present report, we want to point out the long-term follow up of this patient, who survived 81 months, and discuss the interesting mechanism of action of 5-AZA-CdR that may have been responsible for this interesting response. 5-AZA-CdR is a potent inhibitor of DNA methylation. Recent progress in this field has shown that aberrant methylation of the promoter region of tumor suppressor genes inhibits their expression. This epigenetic event can contribute to tumorigenesis. Since 5-AZA-CdR can reactivate these genes by blocking DNA methylation, it has the potential to reverse tumorigenesis. This novel mode of action makes it an interesting agent to investigate for the chemotherapy of malignant disease, including lung cancer.

Antimetabolites, Antineoplastic↗
PubMed2001

E-cadherin expression is commonly downregulated by CpG island hypermethylation in esophageal carcinoma cells.

E-cadherin, a cell adhesion molecule, is regarded as an invasion-suppressor molecule and a prognostic marker in many types of human cancers. Downregulation of E-cadherin is common in esophageal carcinoma and is associated with an increase in invasive and metastatic potential. To study the mechanisms responsible for inactivation of this gene in esophageal squamous cell carcinoma (ESCC), we investigated the methylation status around the 5' promoter region of E-cadherin gene of six ESCC cell lines by methylation-specific polymerase chain reaction, and compared it with E-cadherin protein and mRNA expression. We also studied the methylation status of 20 ESCC clinical specimens. Methylation was noted in four of the six cell lines (one fully methylated and three partially methylated). The completely methylated cell line lacked E-cadherin protein expression and mRNA transcription. E-cadherin expression and transcription were reduced in a partially methylated cell line but preserved in the other partially methylated cell lines. Treatment of E-cadherin-negative carcinoma cells with the demethylating agent, 5-aza-2'-deoxycytidine, induced re-expression of the gene. A high frequency of methylation (16/20, 80%) was also noted in the 20 ESCC clinical samples. Our results indicate that 5' CpG island methylation is common in esophageal carcinoma and may play an important role in downregulation of E-cadherin.

Azacitidine↗
PubMed2003

Clinicopathological significance and molecular regulation of maspin expression in ductal adenocarcinoma of the pancreas.

We evaluated the biological relevance of maspin expression in pancreatic ductal adenocarcinoma and studied regulatory mechanisms of maspin gene activation in pancreatic carcinoma cell lines. Maspin expression was immunohistochemically detected in a series of 57 pancreatic ductal adenocarcinomas, 51 (90%) of which were classified as high-expressers. In lymph node metastases, maspin expression was somewhat decreasingly found in 39/49 (80%). Maspin high-expressers showed predominantly a low histological grade (p=0.013). Moreover, maspin expression was found in two mixed ductal-endocrine carcinomas, but not in 10 endocrine tumors and the surrounding normal pancreatic tissues. Using a luciferase reporter system, maspin promoter activity was induced in the maspin-positive pancreatic cancer cell lines as well as maspin-negative PANC-1 cells. Additionally, treatment with the DNA methyltransferase inhibitor, 5-aza-2' deoxycytidine, and histone deacetylase inhibitor, trichostatin A, led to re-expression of maspin mRNA in PANC-1 cells. Our results indicate that maspin expression is up-regulated in most if not all pancreatic ductal adenocarcinomas and may be related to the development and differentiation, and that DNA methylation and histone deacetylation may suppress maspin gene activation in pancreatic cancer cells.

Acetylation↗
PubMed1997

Alterations in hepatic p53 gene methylation patterns during tumor progression with folate/methyl deficiency in the rat.

Chronic dietary methyl deficiency in F344 rats was used as an in vivo mammalian model in which to evaluate the gene-specific alterations in DNA methylation patterns during multistage hepatocarcinogenesis. Using bisulfite mapping, the site-specific methylation profile within exons 6-7 of the 53 gene was determined in control liver, preneoplastic nodules (after 36 weeks of folate/methyl deficiency) and in hepatocellular carcinoma (after 54 weeks of deficiency). A progressive loss of methyl groups was observed at most CpG sites on both coding and non-coding strands during the first 36 weeks of folate/methyl deficiency, with the greatest loss occurring on the coding strand. When the same sequence was evaluated in tumor DNA after 54 weeks of deficiency, the majority of cytosines were unexpectedly found to have become remethylated. CpG sites that had previously lost methyl groups on both strands during preneoplasia as well as CpG sites that had been constitutively non-methylated, had undergone de novo methylation in tumor DNA. Maintenance methyltransferase and de novo methyltransferase activity in nuclear extracts were assessed using hemimethylated and non-methylated DNA substrates, respectively. In tumor, de novo methyltransferase capacity was increased approximately 4-fold relative to control or preneoplastic liver and associated with a relative increase in both p53 and genome-wide methylation density. In the preneoplastic nodules, the level p53 mRNA was increased and associated with hypomethylation in the coding region of the gene, whereas in tumor tissue, p53 mRNA was decreased and associated with relative hypermethylation. Taken together, these results provide additional insights into the dysregulation and instability in DNA methylation that accompanies the transition to tumor.

Animals↗
PubMed1998

Methylation-mediated regulation of the glutathione S-transferase P1 gene in human breast cancer cells.

Understanding the mechanisms that regulate the human pi class GST (GSTP1) gene expression in breast cancer cells is of particular importance to the study of breast cancer biology. In cultured human breast cancer cell lines, GSTP1 is exclusively expressed in estrogen receptor-negative (ER-) cells but is undetectable in receptor-positive (ER+) cells. Previously, we examined transiently transfected GSTP1 promoter activities, in vitro GSTP1 promoter-DNA interactions, and GSTP1 mRNA stability. These studies indicated that transiently transfected GSTP1 promoter elements and GSTP1 mRNA stability could only partially explain cell line-specific expression of endogenous GSTP1. In the present study, we examined whether the methylation status of the GSTP1 CpG island plays an important role in GSTP1 regulation. Southern blot analysis revealed that the GSTP1 CpG island is hypermethlyated in ER+, GSTP1 non-expressing cell lines but is undermethylated in ER-, GSTP1 expressing cell lines. Moreover, partial demethylation of the GSTP1 CpG island by treatment with 5-aza-2'-deoxycytidine resulted in de novo gene expression in ER+ cell lines, as detected by RT-PCR, Northern blot and Western blot analyses. Our data strongly indicate that methylation status of the promoter contributes significantly to the levels of GSTP1 expressed in ER- and ER+ breast cancer cell lines.

Azacitidine↗
PubMed2003

Functional interaction of estrogen receptor alpha and caveolin isoforms in neuronal SK-N-MC cells.

Estrogen receptors (ERs) are expressed in neuronal cells and exhibit a wide variety of activities in the central nervous system. The actions of ERs are regulated in a hormone-dependent manner as well as by a number of co-activators and -repressors. A recently identified co-activator of ERalpha is caveolin-1 which has been shown to mediate the ligand-independent activation of this steroid receptor. In the present study we have demonstrated that neuronal SK-N-MC cells lacking functional ERalpha show high levels of caveolin-1/-2 specific transcripts and proteins. Ectopic expression of ERalpha in SK-N-MC cells leads to the transcriptional suppression of caveolin-1 and -2 genes. This silencing event is accompanied by changes in the methylation pattern of the caveolin-1 promoter. Certain CpG dinucleotides were methylated in the caveolin-1 promoter region of the SK-ERalpha cells whereas the same sites were non-methylated in control SK-N-MC cells, implicating a gene silencing mechanism including hypermethylation of DNA. In addition, inhibitors of methyltransferases or histone deacetylases, enzymes involved in the establishment and maintenance of silenced chromatin status, partially restored caveolin transcription in SK-ERalpha cells. In conclusion, our observations provide a possible mechanism of negative feedback regulation of ERalpha co-activator caveolin by the steroid receptor itself in this cellular model.

Brain↗
PubMed2000

Methylation moves into medicine.

Two human genetic diseases have recently been shown to be due to mutations in genes encoding proteins involved in DNA methylation. The phenotypes of these two diseases are surprisingly distinct from each other and provide insights into the functions of DNA methylation in mammals.

DNA Methylation↗
PubMed1995

Cancer. Exploring the bowels of DNA methylation.

Recent studies of mice lacking methyltransferase, and of genes that modify cancer susceptibility, have shed light on the long-standing problem of how DNA methylation affects carcinogenesis.

Animals↗
PubMed1996

Base eversion and shuffling by DNA methyltransferases.

The structures of two DNA cytosine methyltransferases reveal two novel methods of gaining access to the substrate cytosine residue, both of which involve eversion of the cytosine in a process that may require DNA bending. In one instance there is also widespread base shuffling and distortion of the DNA.

DNA Modification Methylases↗
PubMed2000

Diversity of homology-dependent gene silencing strategies in fungi.

The experimental introduction of DNA in fungi has revealed a variety of gene inactivation strategies triggered in response to the presence of homologous nucleic acid sequences. These homology effects reveal distinct classes of gene inactivation resulting from hypermutation or relying on regulatory mechanisms that operate at either the transcriptional or the post-transcriptional level.

Ascomycota↗