Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:2.1.1.37 (DNA methyltransferase)
4,983 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Dietary folate/methyl deficiency provides a unique model of endogenous hepatocarcinogenesis in which to study progressive alterations in DNA methylation patterns during tumor progression in vivo. Weanling male F344 rats were given a semi-purified diet deficient in the methyl donors choline, methionine and folic acid for a period of 9 weeks. Using a genomic sequencing procedure based on the PCR amplification of bisulfite-modified DNA, the methylation status of individual CpG sites within exons 6 and 7 of the p53 gene in liver samples from control and deficient rats was determined. Treatment of denatured nuclear DNA with sodium bisulfite quantitatively converts all cytosine residues to uracil which are then amplified as thymine in the PCR reaction. In contrast, 5-methylcytosine is resistant to bisulfite deamination under the reaction conditions and is amplified as cytosine. Automated sequencing of bisulfite-modified DNA will then elucidate the methylation status of each cytosine residue within a defined gene sequence. In addition to evaluation of the methylation status of the p53 gene, the relative activity of the DNA methyltransferase was also quantified in nuclear extracts from control and folate/methyl deficient rats. The results indicate that specific 5-methyl cytosines within the hepatic p53 gene from methyl deficient rats are resistant to demethylation despite the diet-induced decrease in S-adenosylmethionine and the increase in cell proliferation associated with this dietary intervention. Progressive demethylation was observed at other methylated cytosine residues in folate/methyl deficient rats after 9 weeks despite a paradoxical increase in DNA methyltransferase activity. The application of this sequence-specific technology will allow the definition of the methylation status of every CpG site within a coding sequence or promoter region and should provide new insights into mechanisms and consequences of methylation dysregulation during progressive multistage carcinogenesis.
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PMID:Differential sensitivity to loss of cytosine methyl groups within the hepatic p53 gene of folate/methyl deficient rats. 758 11

Male weanling Fischer 344 rats were fed either a semipurified diet deficient in the methyl donors methionine, choline, and folic acid or a supplemented control diet for a period of 9 weeks. At intervals of 2, 5, and 7 days, 3 weeks, and 9 weeks after initiation of the respective diets, the relative level of DNA strand breaks and the degree of cytosine methylation were quantified in high molecular weight DNA and also within the p53 gene in liver samples from these rats. Genome-wide strand break accumulation was associated with progressive genomic hypomethylation and increased DNA methyltransferase activity. With the use of quantitative PCR as a gene-specific DNA strand break assay, unique DNA strand breaks were detected in exon 5 but not in exons 6-8 of the p53 gene, and were accompanied by significant p53 gene hypomethylation. DNA hypomethylation has been shown to alter the conformation and stability of the chromatin structure, rendering affected regions more accessible to DNA-damaging agents. To determine whether methylation status alters the sensitivity of DNA to strand breakage, DNA in isolated nuclei was methylated in vitro and exposed to endogenous calcium/magnesium-dependent endonuclease activated under defined conditions. The incidence of enzyme-induced DNA strand breaks was decreased significantly with increased DNA methylation. In nuclei isolated from livers of methyl-deficient rats, the hypomethylated DNA was found to be more sensitive to enzyme- and oxidant-induced DNA strand break induction. Taken together, these results provide evidence that DNA strand breaks are induced in high molecular weight DNA and also within the p53 gene in liver tissue from methyl-deficient rats. The increased incidence of these strand breaks in DNA from methyl-deficient rats may be related to alterations in chromatin accessibility associated with DNA hypomethylation.
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PMID:Breaks in genomic DNA and within the p53 gene are associated with hypomethylation in livers of folate/methyl-deficient rats. 779 83

Changes in the pattern of DNA methylation have been a consistent finding in cancer cells. The mostly descriptive nature of these studies and the fact that both hypo- and hypermethylation have been observed at various loci have made it difficult to assess whether these changes are causally involved in the transformation process or whether they reflect the altered physiology of rapidly dividing cancer cells. It is clear, however, that DNA methylation plays an important role in the generation of mutations in human tumors. The high incidence of C-to-T transitions found in the p53 tumor-suppressor gene is attributed to the spontaneous deamination of 5-methylcytosine residues. The multiple observations linking DNA methylation to cancer can be resolved in a model proposing that the high rate of mutation at CpG dinucleotides is due in part to methyltransferase-facilitated deamination. Support for a role of DNA methyltransferase as a mutator enzyme is provided by work with a prokaryotic DNA methyltransferase under S-adenosyl-methionine methyl-donor limiting conditions. Methyl-donor limiting conditions might arise in early stages of tumor development, leading to high rates of methyltransferase-mediated CpG mutagenesis, as seen in human tumors. Such a mechanism is consistent with the frequently reported methionine auxotrophy of cancer cells and with the tumorigenic effects of methyl-deficient diets. Methyl deficiency in tumor cells is also consistent with the commonly observed global hypomethylation of tumor cell DNA, despite normal or even high levels of DNA methyltransferase expression.
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PMID:DNA methylation and cancer. 784 43

Mutation of the APC gene may be a common denominator of all human colon cancer--polypoid and non-polypoid familial cancer as well as sporadic occurrences. Fearon and Vogelstein (1990) have described a series of molecular changes during the progression of human colon cancer, beginning with mutations in APC. Min is a strain of the laboratory mouse carrying a nonsense mutation in Apc, the mouse homologue of APC. The Min strain has been used to test the effect of germline alterations in certain genes identified in the progression pathway of Fearon and Vogelstein. A deficiency in DNA cytosine methylase leads to a reduction in the tumour multiplicity of Min mice contrary to the a priori expectation based on the global hypomethylation of the DNA of early colonic neoplasms. Alterations in Kras had no perceptible effect on the tumour multiplicity of Min mice but may not have been successfully directed to the proliferative cell population. Constitutional mutation of p53 did not influence the multiplicity or histopathology of early Min induced intestinal tumours. The cause and effect analysis of the genetics of colon cancer is clearly in an early phase. An unlinked genetic factor interacting with Min in controlling intestinal tumour multiplicity is Mom1. A central goal for the near future is to identify the Mom1 gene product and to identify other loci that can interact with the Min mutation and affect tumour multiplicity or progression. Mouse chimaeras will permit an analysis of the clonality and cell autonomy of Min induced neoplasms and also of the action of Mom1. The results of these analyses will inform investigators as to what modes of prevention and therapy might be designed for particular tumour types. The Min strain thereby presents an opportunity to discover protective factors against human colon cancer.
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PMID:Emergent issues in the genetics of intestinal neoplasia. 871 26

Hydrazine sulfate is a genotoxic hepatocarcinogen for the hamster. A study was conducted to follow changes in DNA maintenance methylation in selected genes in liver DNA during the 21-month induction of liver adenomas and hepatocellular carcinomas by demonstrating changes in restriction fragment length polymorphism. Male Syrian golden hamsters were exposed to hydrazine sulfate in the drinking water at three concentrations (170, 340 and 510 mg/l) shown previously to result in a dose-dependent induction of liver tumors. Liver DNA from animals exposed to the high concentration for 6, 12, 16, 20 and 21 months and animals exposed to the low or mid concentration for 21 months was digested with EcoRI, MspI, HindIII or BamHI, or a combination of one of these endonucleases and a methyl-sensitive restriction enzyme, HpaII or HhaI. The DNA digests were subjected to Southern analysis using a c-DNA probe for one of the following genes: DNA methyltransferase (DMT), c-Ha-ras, c-jun, c-fos, and c-myc proto-oncogenes, p53 tumor suppressor gene or gamma-glutamyltranspeptidase. Alteration in DNA restriction by methyl-sensitive endonucleases was detected in four (DMT, c-Ha-ras, p53 and c-jun) of the seven genes examined and as early as 6 months in animals exposed to the highest concentration of hydrazine sulfate; alteration of recognition sites in c-Ha-ras was also detected in DNA from animals exposed for 21 months to the intermediate concentration of hydrazine sulfate. Early changes in recognition sites, presumed to indicate altered methylation status of DNA cytosine and/or guanine mutations, were seen using c-DNA probes for DMT, c-Ha-ras and c-jun; in the p53 tumor suppressor gene alteration of such sites was a late event relevant to appearance of liver adenomas and hepatocellular carcinomas. Evidence for hypomethylation in the p53 and c-jun genes and hypermethylation of the c-Ha-ras and DMT genes is provided. This study supports the induction of site-specific hypomethylation and hypermethylation during the course of hydrazine carcinogenesis.
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PMID:Changes in methyl-sensitive restriction sites of liver DNA from hamsters chronically exposed to hydrazine sulfate. 900 10

The highly metastatic amelanotic C8161 human melanoma line was found to exhibit complete dominance of its undifferentiated and metastatic phenotype in multiple somatic cell hybridization studies designed to bypass the presence of potential tumor suppressor genes. In a three armed approach involving somatic cell fusions of C8161 with recipient lines of greater differentiation, different lineage, and different tumorigenicity status, the metastatic and undifferentiated phenotype of C8161 was promiscuously dominant. In somatic cell hybrids produced between the C8161 and a group of non-metastatic human melanoma lines which exhibited melanocyte differentiation markers including S100, HMB-45, NKI/C3, and melanin, the fusions were uniformly metastatic and undifferentiated. In somatic cell hybrids of C8161 and MCF-7 the fusions exhibited an estrogen independent and unresponsive, estrogen receptor (ER) negative, and highly metastatic phenotype. In fusions between C8161 and HMS-1, an immortalized 'benign' human myoepithelial line which produced an abundant extracellular matrix (ECM) and high levels of protease and angiogenic inhibitors including maspin, tissue inhibitor of metalloproteinase-1 (TIMP-1), alpha1-antitrypsin (alpha1-AT), protease nexin II (PN-II), thrombospondin-1 and soluble basic fibroblast growth factor (bFGF) receptors, the hybrids showed complete absence of matrix, absent maspin expression, markedly decreased protease inhibitor and angiogenic inhibitor production, high levels of proteases and angiogenic factors, and a highly metastatic phenotype. In our somatic cell fusions, the human-human hybrids represented true and complete fusions and not hybrid clones selected for by loss of dominant-acting growth suppressor genes. This finding was supported by detailed comparative genomic hybridization (CGH) studies, Q-banding karyotype analysis, and autofusions of representative clones. The purposeful creation of inherently unstable human-murine fusions between C8161 and B16-F1 where loss of putative suppressor loci would be expected, resulted in fusions exhibiting decreased growth and non-metastatic behavior with progressive chromosomal loss. Neither p53, nm23, DNA methyltransferase, activated ras, fibroblast growth factor-4 (FGF-4), or epidermal growth factor receptor (EGFR) mediated the acquisition of the metastatic or undifferentiated phenotype within the C8161-human fusions. These studies are the first studies ever to successfully transfer the complete metastatic phenotype by somatic cell fusion and support the presence of a new high level regulatory pathway(s) involving dominant trans-acting factors which act pleiotropically to regulate an undifferentiated and highly metastatic phenotype.
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PMID:Evidence of a dominant transcriptional pathway which regulates an undifferentiated and complete metastatic phenotype. 936 25

Using a yeast based p53 functional assay we previously demonstrated that the UVC-induced p53 mutation spectrum appears to be indistinguishable from the one observed in Non Melanoma Skin Cancer (NMSC). However, position 742 (codon 248, CpG site) represented the major hot spot in NMSC but was not found mutated in the yeast system. In order to determine whether UVC-induced mutagenic events may be facilitated at methylated cytosine (5mC), a yeast expression vector harbouring a human wild-type p53 cDNA (pLS76) was methylated in vitro by HpaII methylase. Methylation induced 98% protection to HpaII endonuclease. Unmethylated and methylated pLS76 vectors were then UVC irradiated (lambda(max): 254 nm) and transfected into a yeast strain containing the ADE2 gene regulated by a p53-responsive promoter. The results revealed that: (i) 5mC at HpaII sites did not cause any difference in the UVC-induced survival and/or mutagenicity; (ii) none of the 20 mutants derived from methylated pLS76 showed p53 mutations targeted at HpaII sites; (iii) the UVC-induced p53 mutation spectra derived from methylated and unmethylated pLS76 were indistinguishable not only when classes of mutations and hot spots were concerned, but also when compared through a rigorous statistical test to estimate their relatedness (P = 0.85); (iv) the presence of 5mC did not increase the formation of photo-lesions at codon 248, as determined by using a stop polymerase assay. Although based on a limited number of mutants, these results suggest that the mere presence of 5mC at position 742 does not cause a dramatic increase of its mutability after UVC irradiation. We propose that position 742 is a hot spot in NMSC either because of mutagenic events at 5mC caused by other UV components of solarlight and/or because not all the NMSC are directly correlated with UV mutagenesis but may have a "spontaneous" origin.
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PMID:5-methylcytosine at HpaII sites in p53 is not hypermutable after UVC irradiation. 1065 89

Altered patterns of the 5-cytosine methylation of genomic DNA are associated with the development of a wide range of human cancers. We have studied the mechanisms and genetic pathways by which a targeted heterozygous deficiency in the murine 5-cytosine DNA methyltransferase gene (Dnmt1(N/+)) diminishes intestinal tumorigenesis in C57BL/6-multiple intestinal neoplasia (Min)/+ mice. We found that Dnmt1(N/+) retards the net growth rate of intestinal adenomas and reduces tumor multiplicity by approximately 50%. This tumor resistance affects the entire intestinal tract and is independent of the status of modifier of Min 1 and p53, two loci that have been found to confer strong resistance to Min-induced neoplasia Interestingly, Dnmt/(N/+) and modifier of Min 1 resistance interact synergistically, together virtually eliminating tumor incidence. This finding may provide an insight into potential combinatorial therapeutic approaches for treating human colon cancer.
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PMID:Dnmt1N/+ reduces the net growth rate and multiplicity of intestinal adenomas in C57BL/6-multiple intestinal neoplasia (Min)/+ mice independently of p53 but demonstrates strong synergy with the modifier of Min 1(AKR) resistance allele. 1091 75

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.
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PMID:Aberrant methylation of the Cyclooxygenase 2 CpG island in colorectal tumors. 1094 6

The gene expression pattern of mesothelial cells in vitro was determined after 4 or 12 h exposure to the rat mesothelial, kidney, and thyroid carcinogen and oxidative stressor potassium bromate (KBrO(3)). Gene expression changes observed using cDNA arrays indicated oxidative stress, mitotic arrest, and apoptosis in treated immortalized rat peritoneal mesothelial cells. Increases occurred in oxidative stress responsive genes HO-1, QR, HSP70, GADD45, GADD153, p21(WAF1/CIP16), GST's, GAPDH, TPX, and GPX-1(0); transcriptional regulators c-jun, c-fos, jun B, c-myc, and IkappaB; protein repair components Rdelta, RC10-II, C3, RC-7, HR6B ubiquitin-conjugating enzyme and ubiquitin; DNA repair components PCNA, msh2, and O-6 methylguanine DNA methyltransferase; lipid peroxide excision enzyme PLA2; and apoptogenic components TNFalpha, iNOS1 and FasL. Decreases occurred in bcl-2 (antiapoptotic), bax alpha, bad, and bok (proapoptotic) and cell cycle control elements (cyclins). Cyclin G and p14ink4b (which inhibit entry into cell cycle) were increased. Numerous signal transduction, cell membrane transport, membrane-associated receptor, and fatty acid biosynthesis and repair components were altered. Morphologic endpoints examined were number of mitotic figures, number of apoptotic cells, and antibody-specific localization of HO-1 (which demonstrated increased HO-1 protein expression). PCR analysis confirmed HO-1, p21(waf1/cip1), HSP70, GPX1, GADD45, QR, mdr1, PGHS, and cyclin D1 changes. A model for KBrO(3)-induced carcinogenicity in the F344 rat mesothelium is proposed, whereby KBrO(3) generates a redox signal that activates p53 and results in transcriptional activation of oxidative stress and repair genes, dysregulation of growth control, and imperfect DNA repair leading to carcinogenesis.
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PMID:Morphologic analysis correlates with gene expression changes in cultured F344 rat mesothelial cells. 1113 43


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