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Results for “DNA Modification Methylases”

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PubMed2025

High MGMT expression identifies aggressive colorectal cancer with distinct genomic features and immune evasion properties.

INTRODUCTION: The epigenetic silencing of O6-methylguanine DNA methyltransferase (MGMT) is associated with reduced DNA repair capacity, carcinogenesis and increased sensitivity to alkylating chemotherapy. However, the biological role and clinical significance of MGMT overexpression in cancer remains poorly understood. METHODS: Using multiplexed quantitative immunofluorescence we measured the localized levels of MGMT protein, γH2AX and CD8+ T cells in multiple retrospective colorectal cancer (CRC) cohorts. Genomic and transcriptomic features of selected cases were also studied with whole exome DNA sequencing and genome-wide methylation analysis. MGMT-methylated human CRC cells SW620 were transfected with an MGMT-containing plasmid and co-cultured with allogeneic peripheral blood mononuclear cells. RESULTS: A subset of CRCs showed MGMT protein upregulation associated with lower γH2AX, reduced CD8+ tumor infiltrating lymphocytes (TILs), mismatch repair proficient (pMMR) status and shorter survival. CD8+ TILs were more distant from MGMT-expressing cells than MGMT-negative cells and the MGMT promoter methylation status did not highly correlate with MGMT protein levels in CRC. In genomic/transcriptomic analysis, high MGMT expression was associated with a lower nonsynonymous somatic mutational burden, higher transition-to-transversion mutation ratio, increased deleterious TP53 variants and distinct transcriptomic profiles. The exogenous expression of MGMT in SW620 CRC cells reduced the number of spontaneous nonsynonymous mutations, reproduced mutational features of MGMT-high CRC and limited the in vitro T-cell-mediated killing of malignant cells induced by proinflammatory cytokines in tumor/immune cell co-cultures. CONCLUSIONS: MGMT overexpression identifies a previously undescribed subset of CRCs with distinct biological and clinical properties including reduced mutagenesis, adaptive immune evasion, predominantly pMMR phenotype and aggressive clinical course. Direct, quantitative assessment of MGMT protein expression using spatially resolved analysis is more reliable than inference of MGMT expression by promoter methylation status in CRC.

Humans↗

PubMed1998

Base flipping.

Base flipping is the phenomenon whereby a base in normal B-DNA is swung completely out of the helix into an extrahelical position. It was discovered in 1994 when the first co-crystal structure was reported for a cytosine-5 DNA methyltransferase binding to DNA. Since then it has been shown to occur in many systems where enzymes need access to a DNA base to perform chemistry on it. Many DNA glycosylases that remove abnormal bases from DNA use this mechanism. This review describes systems known to use base flipping as well as many systems where it is likely to occur but has not yet been rigorously demonstrated. The mechanism and evolution of base flipping are also discussed.

Base Pair Mismatch↗

PubMed2004

A mouse skin multistage carcinogenesis model reflects the aberrant DNA methylation patterns of human tumors.

Whereas accepted models of tumorigenesis exist for genetic lesions, the timing of epigenetic alterations in cancer is not clearly understood. We have analyzed the profile of aberrations in DNA methylation occurring in cells lines and primary tumors of one of the best-characterized mouse carcinogenesis systems, the multistage skin cancer progression model. Initial analysis using high-performance capillary electrophoresis and immunolocalization revealed a loss of genomic 5-methylcytosine associated with the degree of tumor aggressiveness. Paradoxically, this occurs in the context of a growing number of hypermethylated CpG islands of tumor suppressor genes at the most malignant stages of carcinogenesis. We have observed this last phenomenon using two approaches, a candidate gene approach, studying genes with well-known methylation-associated silencing in human tumors, and a mouse cDNA microarray expression analysis after treatment with DNA demethylating drugs. The transition from epithelial to spindle cell morphology is particularly associated with major epigenetic alterations, such as E-cadherin methylation, demethylation of the Snail promoter, and a decrease of the global DNA methylation. Analysis of data obtained from the cDNA microarray strategy led to the identification of new genes that undergo methylation-associated silencing and have growth-inhibitory effects, such as the insulin-like growth factor binding protein-3. Most importantly, all of the above genes were also hypermethylated in human cancer cell lines and primary tumors, underlining the value of the mouse skin carcinogenesis model for the study of aberrant DNA methylation events in cancer cells.

5-Methylcytosine↗

PubMed2006

Genotoxic stress induces coordinately regulated alternative splicing of the p53 modulators MDM2 and MDM4.

The tumor suppressor protein p53 is a transcription factor that induces G(1) arrest of the cell cycle and/or apoptosis. The murine double-minute protein MDM2 and its homologue MDM4 (also known as MDMX) are critical regulators of p53. Altered transcripts of the human homologue of mdm2, MDM2, have been identified in human tumors, such as invasive carcinoma of the breast, lung carcinoma, and liposarcoma. MDM2 alternate forms act to negatively regulate the normal MDM2 gene product, thus activating p53. Although many reports have documented a plethora of tumor types characterized by MDM2 alternative transcripts, few have investigated the signals that might initiate alternative splicing. We have identified a novel role of these alternative MDM2 transcripts in the normal surveillance mechanism of the cell and in DNA damage response. We report that alternate forms of MDM2 are detected after UV irradiation. Furthermore, we show that mouse cells treated with UV are also characterized by alternative transcripts of mdm2, suggesting that this is an important and evolutionarily conserved mechanism for regulating the expression of MDM2/mdm2. An additional p53 regulator and mdm2 family member, MDM4, is likewise alternatively spliced following UV irradiation. By activating alternative splicing of both MDM2 and MDM4, yet another layer of p53 regulation is initiated by the cells in response to damage. A stepwise model for malignant conversion by which alternate forms of MDM2 and MDM4 place selective pressure on the cells to acquire additional alterations in the p53 pathway is herein proposed.

Alternative Splicing↗

PubMed2005

Hypermethylation of 18S and 28S ribosomal DNAs predicts progression-free survival in patients with ovarian cancer.

PURPOSE: Repetitive ribosomal DNA (rDNA) genes are GC-rich clusters in the human genome. The aim of the study was to determine the methylation status of two rDNA subunits, the 18S and 28S genes, in ovarian tumors and to correlate methylation levels with clinicopathologic features in a cohort of ovarian cancer patients. EXPERIMENTAL DESIGN: 18S and 28S rDNA methylation was examined by quantitative methylation-specific PCR in 74 late-stage ovarian cancers, 9 histologically uninvolved, and 11 normal ovarian surface epithelial samples. In addition, methylation and gene expression levels of 18S and 28S rDNAs in two ovarian cancer cell lines were examined by reverse transcription-PCR before and after treatment with the demethylating drug 5'-aza-2'-deoxycytidine. RESULTS: The methylation level (amount of methylated rDNA/beta-actin) of 18S and 28S rDNAs was significantly higher (P < 0.05) in tumors than in normal ovarian surface epithelial samples. Methylation of 18S and 28S rDNA was highly correlated (R2= 0.842). Multivariate analysis by Cox regression found that rDNA hypermethylation [hazard ratio (HR), 0.25; P < 0.01], but not age (HR, 1.29; P = 0.291) and stage (HR, 1.09; P = 0.709), was independently associated with longer progression-free survival. In ovarian cancer cell lines, methylation levels of rDNA correlated with gene down-regulation and 5'-aza-2'-deoxycytidine treatment resulted in a moderate increase in 18S and 28S rDNA gene expressions. CONCLUSION: This is the first report of rDNA hypermethylation in ovarian tumors. Furthermore, rDNA methylation levels were higher in patients with long progression-free survival versus patients with short survival. Thus, rDNA methylation as a prognostic marker in ovarian cancer warrants further investigation.

Adult↗

PubMed2006

In vitro drug response and molecular markers associated with drug resistance in malignant gliomas.

PURPOSE: Drug resistance in malignant gliomas contributes to poor clinical outcomes. We determined the in vitro drug response profiles for 478 biopsy specimens from patients with the following malignant glial histologies: astrocytoma (n = 71), anaplastic astrocytoma (n = 39), glioblastoma multiforme (n = 259), oligodendroglioma (n = 40), and glioma (n = 69). EXPERIMENTAL DESIGN: Samples were tested for drug resistance to 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU), cisplatin, dacarbazine, paclitaxel, vincristine, and irinotecan. Biomarkers associated with drug resistance were detected by immunohistochemistry, including multidrug resistance gene-1, glutathione S-transferase pi (GSTP1), O(6)-methylguanine-DNA methyltransferase (MGMT), and mutant p53. RESULTS: In vitro drug resistance in malignant gliomas was independent of prior therapy. High-grade glioblastomas showed a lower level of extreme drug resistance than low-grade astrocytomas to cisplatin (11% versus 27%), temozolomide (14% versus 27%), irinotecan (33% versus 53%), and BCNU (29% versus 38%). A substantial percentage of brain tumors overexpressed biomarkers associated with drug resistance, including MGMT (67%), GSTP1 (49%), and mutant p53 (41%). MGMT and GSTP1 overexpression was independently associated with in vitro resistance to BCNU, whereas coexpression of these two markers was associated with the greatest degree of BCNU resistance. CONCLUSIONS: Assessment of in vitro drug response and profiles of relevant tumor-associated biomarkers may assist the clinician in stratifying patient treatment regimens.

ATP Binding Cassette Transporter, Subfamily B↗

PubMed2006

The high frequency of de novo promoter methylation in synchronous primary endometrial and ovarian carcinomas.

PURPOSE: The methylation status of hMLH1, CDKN2A, and MGMT was investigated in a panel of synchronous cancers of the ovary and endometrium, fulfilling the clinicopathologic criteria for independent primary tumors to define the possible role of epigenetic mechanisms in the development of these cancers. EXPERIMENTAL DESIGN: Bisulfite-converted DNA from 31 tumors (13 endometrial and 18 ovarian carcinomas) and from matched normal tissue of 13 patients was analyzed by a methylation-specific PCR assay at the CpG-rich 5' regions of all three genes. In all tumors, we also investigated the presence of microsatellite instability and hMLH1 immunohistochemical expression in relation to hMLH1 hypermethylation status. RESULTS: Methylation of hMLH1, CDKN2A, and MGMT was detected in 39%, 41%, and 48% of endometrial and ovarian tumors, respectively. hMLH1 hypermethylation was observed in all tumors of five patients, and it was invariably associated with loss of hMLH1 protein and presence of microsatellite instability. CDKN2A and MGMT methylation was randomly detected among both endometrial (45% and 24% of cases, respectively) and ovarian carcinomas (39% and 39% of cases, respectively). Concordant methylation at two or three genes was observed in 35% of cases. CONCLUSIONS: Epigenetic inactivation of hMLH1, CDKN2A, and MGMT may be a common and early event in the development of synchronous primary endometrial and ovarian carcinomas and may qualify as a marker of a field cancerization encompassing the ovary and endometrium. Detection of MGMT hypermethylation may be useful to define a set of gynecologic malignancies with a specific sensitivity to alkylating chemotherapy.

Adaptor Proteins, Signal Transducing↗

PubMed2006

Implications of methylation patterns of cancer genes in salivary gland tumors.

PURPOSE: We investigated the methylation status and protein expression of four tumor suppressor genes to determine their role in salivary gland tumorigenesis. EXPERIMENTAL DESIGN: We performed methylation-specific PCR and protein analyses of 29 normal salivary glands, 23 benign, and 79 malignant salivary gland neoplasms to determine the pattern and potential diagnostic and/or biological role of the RASSF1, RARbeta2, DAPK, and MGMT tumor suppressor gene methylation in these tumors. RESULTS: No methylation was detected in the normal tissues. Methylation occurred in 9 of 23 (39.1%) benign tumors; 3 (25.0%) pleomorphic adenomas and 6 (66.7%) Warthin's tumors at the MGMT, DAPK, or RASSF1 genes. Methylation occurred in 33 of 79 (41.8%) malignant tumors; 8 (30.8%) adenoid cystic carcinomas, 6 (33.3%) mucoepidermoid carcinomas, 6 (42.9%) acinic cell carcinomas, and 13 (62.0%) salivary duct carcinomas. RASSF1 and RARbeta2 represented 75.8% of methylation events occurring most frequently in salivary duct and acinic cell carcinomas. Overall, we found no significant correlation between protein expression and methylation status of individual genes, but observed low or absent protein expression in several methylated tumors. Significant correlations were found between methylation and aggressive malignant phenotypes (P = 0.0004) and age (P = 0.05). CONCLUSIONS: (a) Benign and malignant salivary tumors differed in the frequency and pattern of gene methylation; (b) high-grade carcinomas were significantly methylated compared with low-grade phenotypes; (c) RASSF1 and RARbeta2 were highly methylated in malignant tumors and can be targeted for therapy; and (d) methylation pattern may serve as a diagnostic and biological marker in assessing these tumors.

Adolescent↗

PubMed2006

Gene promoter hypermethylation in mouse lung tumors.

The mouse is a good model for evaluating the efficacy of chemopreventive agents for lung cancer. Gene silencing by promoter hypermethylation is a critical component for the development and progression of lung cancer and an emerging target for preventive intervention by demethylating agents. Genes methylated in mouse lung tumors could serve as biomarkers to evaluate the effectiveness of demethylating agents for preventing lung cancer and causing gene reexpression in vivo. The purpose of the current study was to evaluate a panel of genes inactivated by promoter hypermethylation in human lung cancer for silencing by this epigenetic mechanism in murine lung tumors induced by 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), cigarette smoke, or arising spontaneously. Cadherin-13, estrogen receptor-alpha, progesterone receptor, and runt-related transcription factor-3 were frequently methylated in mouse lung tumor-derived cell lines, whereas cadherin-1 and suppressor of cytokine signaling-1 were not. Methylation within these four genes was associated with lack of expression that could be restored after treatment with 5-aza-2'-deoxycytidine and with methylation within the CpG island of each gene. Methylation-specific PCR revealed that methylation of these four genes occurred at prevalences of 24% to 69% in primary lung tumors arising spontaneously or induced by exposure to cigarette smoke or NNK. Estrogen receptor-alpha methylation was more frequent in spontaneously occurring lung cancer than cigarette smoke-induced or NNK-induced lung cancer, whereas runt-related transcription factor-3 showed the opposite relationship. Thus, genes can be targeted for inactivation by methylation, depending on exposure history. This study indicates that methylation events frequently observed in human lung cancer are recapitulated in the mouse model and identifies four potential biomarkers for assessing intervention approaches for reversing epigenetically mediated gene silencing.

Animals↗

PubMed2000

DNA methylation and chromosome instability in lymphoblastoid cell lines.

In order to gain more insight into the relationships between DNA methylation and genome stability, chromosomal and molecular evolutions of four Epstein-Barr virus-transformed human lymphoblastoid cell lines were followed in culture for more than 2 yr. The four cell lines underwent early, strong overall demethylation of the genome. The classical satellite-rich, heterochromatic,juxtacentromeric regions of chromosomes 1, 9, and 16 and the distal part of the long arm of the Y chromosome displayed specific behavior with time in culture. In two cell lines, they underwent a strong demethylation, involving successively chromosomes Y, 9, 16, and 1, whereas in the two other cell lines, they remained heavily methylated. For classical satellite 2-rich heterochromatic regions of chromosomes 1 and 16, a direct relationship could be established between their demethylation, their undercondensation at metaphase, and their involvement in non-clonal rearrangements. Unstable sites distributed along the whole chromosomes were found only when the heterochromatic regions of chromosomes 1 and 16 were unstable. The classical satellite 3-rich heterochromatic region of chromosomes 9 and Y, despite their strong demethylation, remained condensed and stable. Genome demethylation and chromosome instability could not be related to variations in mRNA amounts of the DNA methyltransferases DNMT1, DNMT3A, and DNMT3B and DNA demethylase. These data suggest that the influence of DNA demethylation on chromosome stability is modulated by a sequence-specific chromatin structure.

Ataxia Telangiectasia↗

PubMed2006

An inverse correlation between Interleukin-6 and select gene promoter methylation in patients with gastric cancer.

BACKGROUND: Both serum IL-6 levels and CpG island methylation have been shown to have prognostic significance in gastric cancer, it was suggested that an important link existed between IL-6 and methylation of cancers. AIM: To investigate the prognostic value of IL-6 serum level and the association between serum IL-6 levels and CpG island methylation at p16, DAPK, MGMT and E-cadherin in patients with gastric cancer. PATIENTS AND METHODS: Methylation status was assessed by MSP in 75 surgical specimens of gastric adenocarcinoma. IL-6 serum levels were measured by chemiluminescent enzyme immunoassay (CLEIA). RESULTS: Methylation of p16, DAPK, MGMT, and E-cadherin were present in 53, 48, 32, and 59% of patients. Patients with tumors methylated at p16 and DAPK had lower serum levels of IL-6 compared to unmethylated tumors (1.8 vs. 4.8 pg/ml, p = 0.01 for p16; 1.5 vs. 6.2 pg/ml, p = 0.0001 for DAPK). But there was no difference with MGMT and E-cadherin methylation status. Serum IL-6 levels were also associated with TNM stage (p = 0.001), depth of tumor invasion (p = 0.002), lymphatic invasion (p = 0.01), vascular invasion (p = 0.008), metastasis (p = 0.002) and signet cell histology (p = 0.001). CONCLUSION: IL-6 is of prognostic value for patients of gastric cancer. Low serum IL-6 levels were associated with p16 or DAPK gene methylation in patients with gastric cancer.

Adult↗

PubMed1992

Rescue of the adeno-associated virus 2 genome correlates with alterations in DNA-modifying enzymes in human cells.

The adeno-associated virus 2 (AAV) genome can be rescued either from a recombinant plasmid upon transfection into human cells or from cells latently infected with AAV, following subsequent infection with adenovirus. Using human diploid fibroblasts as a model for a natural AAV infection, we observed increased efficiency of rescue of the AAV genome in these cells as they traversed their limited proliferative life span in vitro. The efficiency of rescue correlated well with the augmented nuclease activity in these cells. Furthermore, rescue of the AAV genome, either from a recombinant plasmid or from the chromosomal DNA, was more efficient in cells from a patient with Bloom's syndrome, a rare autosomal recessive disease associated with increased chromosomal breakage due to DNA ligase I deficiency, as compared with normal human diploid fibroblasts. These studies suggest that alterations in DNA-modifying enzymes may play a role in rescue of the AAV genome in human cells.

Blotting, Southern↗

PubMed1989

Nonrandom X chromosome DNA methylation patterns in hemophiliac females.

Molecular X chromosome inactivation analysis was used to characterize three females (and their families) with severe hemophilia. First, the maternal and paternal X chromosomes were distinguished by restriction fragment length polymorphisms (RFLPs). Second, the patterns of methylation of X chromosome genes using methylation-sensitive restriction endonucleases were determined. Of the six X chromosome probes tested, only the phosphoglycerol-kinase (PGK) and hypoxanthine-phosphoribosyl-transferase (HPRT) clones were informative, indicating that other X chromosome probes are not useful for X inactivation analysis. After digestion with Hpa II or Hha I, the hybridization intensity of the RFLPs of all three mothers and an unaffected sister were diminished by 50%, consistent with random X chromosome inactivation. The methylation patterns of the X chromosomes of the affected females, however, were clearly nonrandom. Depending upon the probe and the patient, HPRT and PGK sequences were either completely methylated or unmethylated. These findings are extremely suggestive that nonrandom X chromosome inactivation (lyonization) is the basis for severe hemophilia in these females.

DNA Modification Methylases↗

PubMed2003

Lack of involvement of known DNA methyltransferases in familial hydatidiform mole implies the involvement of other factors in establishment of imprinting in the human female germline.

BACKGROUND: Differential methylation of the two alleles is a hallmark of imprinted genes. Correspondingly, loss of DNA methyltransferase function results in aberrant imprinting and abnormal post-fertilization development. In the mouse, mutations of the oocyte-specific isoform of the DNA methyltransferase Dnmt1 (Dnmt1o) and of the methyltransferase-like Dnmt3L gene result in specific failures of imprint establishment or maintenance, at multiple loci. We have previously shown in humans that an analogous inherited failure to establish imprinting at multiple loci in the female germline underlies a rare phenotype of recurrent hydatidiform mole. RESULTS: We have identified a human homologue of the murine Dnmt1o and assessed its pattern of expression. Human DNMT1o mRNA is detectable in mature oocytes and early fertilized embryos but not in any somatic tissues analysed. The somatic isoform of DNMT1 mRNA, in contrast, is not detectable in human oocytes. In the previously-described family with multi-locus imprinting failure, mutation of DNMT1o and of the other known members of this gene family has been excluded. CONCLUSIONS: Mutation of the known DNMT genes does not underlie familial hydatidiform mole, at least in the family under study. This suggests that trans-acting factors other than the known methyltransferases are required for imprint establishment in humans, a concept that has indirect support from recent biochemical studies of DNMT3L.

Animals↗

PubMed2006

Methylation mediated silencing of TMS1/ASC gene in prostate cancer.

BACKGROUND: Transcriptional silencing associated with aberrant promoter methylation has been established as an alternate pathway for the development of cancer by inactivating tumor suppressor genes. TMS1 (Target of Methylation induced Silencing), also known as ASC (Apoptosis Speck like protein containing a CARD) is a tumor suppressor gene which encodes for a CARD (caspase recruitment domain) containing regulatory protein and has been shown to promote apoptosis directly and by activation of downstream caspases. This study describes the methylation induced silencing of TMS1/ASC gene in prostate cancer cell lines. We also examined the prevalence of TMS1/ASC gene methylation in prostate cancer tissue samples in an effort to correlate race and clinico-pathological features with TMS1/ASC gene methylation. RESULTS: Loss of TMS1/ASC gene expression associated with complete methylation of the promoter region was observed in LNCaP cells. Gene expression was restored by a demethylating agent, 5-aza-2'deoxycytidine, but not by a histone deacetylase inhibitor, Trichostatin A. Chromatin Immunoprecipitation (ChIP) assay showed enrichment of MBD3 (methyl binding domain protein 3) to a higher degree than commonly associated MBDs and MeCP2. We evaluated the methylation pattern in 66 prostate cancer and 34 benign prostatic hyperplasia tissue samples. TMS1/ASC gene methylation was more prevalent in prostate cancer cases than controls in White patients (OR 7.6, p 0.002) while no difference between the cases and controls was seen in Black patients (OR 1.1, p 0.91). CONCLUSION: Our study demonstrates that methylation-mediated silencing of TMS1/ASC is a frequent event in prostate cancer, thus identifying a new potential diagnostic and prognostic marker for the treatment of the disease. Racial differences in TMS1/ASC methylation patterns implicate the probable role of molecular markers in determining in susceptibility to prostate cancer in different ethnic groups.

Aged↗

PubMed2004

Targeted gene modification using triplex-forming oligonucleotides.

In recent years, triplex-forming oligonucleotides (TFOs) have emerged as powerful tools for site-specific gene modification. Their sequence specificity, binding affinity, and ability to provoke repair and recombination make them promising reagents for altering gene expression. This chapter highlights the binding requirements for triplex formation, identifies a number of chemical modifications that have been used with some success, and discusses studies using TFOs for inhibiting transcription. It also reviews work done using TFOs and related molecules to direct site-specific DNA damage, inducing mutagenesis or sensitizing a site to recombination. TFOs were initially used as positioning devices for nonspecific mutagens but were later discovered to have mutagenic properties of their own in cells with functional nucleotide excision repair (NER) and transcription-coupled repair (TCR) pathways. In subsequent studies triplex formation was able to induce both intramolecular and intermolecular homologous recombination, revealing its potential application for gene therapy. Recent reports demonstrate the ability of these molecules to locate and modify their cognate sites in chromosomal DNA in both cell culture and live animals, laying the foundation for triplex technology in vivo.

Animals↗

PubMed2001

Targeting hypomethylation of DNA to achieve cellular differentiation in myelodysplastic syndromes (MDS).

Considerable progress has been made recently in defining and understanding the diverse members of the group of hematologic disorders now known as the myelodysplastic syndrome (MDS). New systems of classification, based on the latest cytogenetic methodologies, have generated better prognostic data, and basic research has more closely associated molecular mechanisms with clinical subgroups. The mechanisms underlying most cases of myelodysplasia appear to be an array of chromosomal abnormalities leading to suppression of normal myeloid cell differentiation and dominance of abnormal, immature cells. The process is progressive and is mediated by a variety of cytokines, potential loss of tumor suppressor genes, aberrations in signal transduction pathways, and perhaps immune mechanisms. Hypermethylation of specific DNA sequences has been implicated in the pathogenesis of MDS. Until recently, treatment options have been few, high risk, and mostly ineffective. New discoveries, particularly in the area of stimulating remaining normal myeloid cells to resume growth and differentiation, hold promise for safer treatment regimens and improved outcomes. Among the promising new agents are nucleoside analogues, such as 5-azacytidine and decitabine, which reactivate tumor suppressor gene transcription through effects on DNA methylation.

Antimetabolites, Antineoplastic↗

PubMed2002

Determination of methylation site of DNA-methyltransferase NlaX by a hybrid method.

Using a new method based on a combination of bisulfite reaction, the repair enzyme uracil-DNA glycosylase, and synthetic oligodeoxyribonucleotides, the methylation site of DNA-methyltransferase NlaX (M.NlaX) from Neisseria lactamica was established to be the inner cytosine in the double-stranded pentanucleotide recognition sequence 5'-CCNGG-3' (where N = any nucleoside). 5-Methylcytosine (m5C) type modification by M-N1aX was confirmed by the use of oligonucleotide substrates that contain 5-fluoro-2'-deoxycytidine.

Chemistry Techniques, Analytical↗