Decreased DNA Methyltransferase Activity Research Articles

Theaflavin-Containing Black Tea Extract: A Potential DNA Methyltransferase Inhibitor in Human Colon Cancer Cells and Ehrlich Ascites Carcinoma-Induced Solid Tumors in Mice

Tea is the most popularly consumed beverage in the world. Theaflavin and thearubigins are the key bioactive compounds of black tea that have anticarcinogenic properties as reported in several studies. However, the epigenetic potential of these compounds has not yet been explored. DNA methyltransferase (DNMT) enzymes induce methylation of DNA at cytosine residues and play a significant role in epigenetic regulation and cancer therapy. The present study has explored the role of black tea as a DNMT inhibitor in the prevention of cancer. Herein, the effect of theaflavin has been studied in colon cancer cell line (HCT-116) and EAC-induced solid tumors in mice. It was found that theaflavin prevented cell proliferation and inhibited tumor progression as well. In silico study showed that theaflavin interacted with DNMT1 and DNMT3a enzymes and blocked their activity. Theaflavin also decreased DNMT activity In Vitro and In Vivo as evident from the DNMT activity assay. Results of immunohistochemistry revealed that theaflavin reduced DNMT expression in the tumors of mice. Taken together, our findings showed that theaflavin has a potential role as a DNMT inhibitor in HCT-116 cell line and EAC induced solid tumors in mice.

Spermine and gene methylation: a mechanism of lifespan extension induced by polyamine-rich diet.

The polyamines spermidine and spermine are synthesized in almost all organisms and are also contained in food. Polyamine synthesis decreases with aging, but no significant decrease in polyamine concentrations were found in organs, tissues, and blood of adult animals and humans. We found that healthy dietary patterns were associated with a preference for polyamine-rich foods, and first reported that increased polyamine intake extended the lifespan of mice and decreased the incidence of colon cancer induced by repeated administration of moderate amounts of a carcinogen. Recent investigations have revealed that changes in DNA methylation status play an important role in lifespan and aging-associated pathologies. The methylation of DNA is regulated by DNA methyltransferases in the presence of S-adenosylmethionine. Decarboxylated S-adenosylmethionine, converted from S-adenosylmethionine by S-adenosylmethionine decarboxylase, provides an aminopropyl group to synthesize spermine and spermidine and acts to inhibit DNMT activity. Long-term increased polyamine intake were shown to elevate blood spermine levels in mice and humans. In vitro studies demonstrated that spermine reversed changes induced by the inhibition of ornithine decarboxylase (e.g., increased decarboxylated S-adenosylmethionine, decreased DNA methyltransferase activity, increased aberrant DNA methylation), whose activity decreases with aging. Further, aged mice fed high-polyamine chow demonstrated suppression of aberrant DNA methylation and a consequent increase in protein levels of lymphocyte function-associated antigen 1, which plays a pivotal role on inflammatory process. This review discusses the relation between polyamine metabolism and DNA methylation, as well as the biological mechanism of lifespan extension induced by increased polyamine intake.

Curcumin Modulates DNA Methyltransferase Functions in a Cellular Model of Diabetic Retinopathy.

Hyperglycaemia-induced oxidative stress appears to be involved in the aetiology of diabetic retinopathy (DR), a major public health issue, via altering DNA methylation process. We investigated the effect of hyperglycaemia on retinal DNA methyltransferase (DNMT) expression in diabetic mice, using Gene Expression Omnibus datasets. We also evaluated the effect of curcumin both on high glucose-induced reactive oxygen species (ROS) production and altered DNMT functions, in a cellular model of DR. We observed that three months of hyperglycaemia, in insulin-deficient Ins2Akita mice, decrease DNMT1 and DNMT3a expression levels. In retinal pigment epithelium (RPE) cells, we also demonstrated that high glucose-induced ROS production precedes upregulation of DNMT expression and activity, suggesting that changes in DNMT function could be mediated by oxidative stress via a potential dual effect. The early effect results in decreased DNMT activity, accompanied by the highest ROS production, while long-term oxidative stress increases DNMT activity and DNMT1 expression. Interestingly, treatment with 25 μM curcumin for 6 hours restores ROS production, as well as DNMT functions, altered by the exposure of RPE to acute and chronic high glucose concentration. Our study suggests that curcumin may represent an effective antioxidant compound against DR, via restoring oxidative stress and DNMT functions, though further studies are recommended.

Global analysis of DNA methylation in young (J1) and senescent (J2) Gossypium hirsutum L. cotyledons by MeDIP-Seq.

DNA methylation is an important epigenetic modification regulating gene expression, genomic imprinting, transposon silencing and chromatin structure in plants and plays an important role in leaf senescence. However, the DNA methylation pattern during Gossypium hirsutum L. cotyledon senescence is poorly understood. In this study, global DNA methylation patterns were compared between two cotyledon development stages, young (J1) and senescence (J2), using methylated DNA immunoprecipitation (MeDIP-Seq). Methylated cytosine occurred mostly in repeat elements, especially LTR/Gypsy in both J1 and J2. When comparing J1 against J2, there were 1222 down-methylated genes and 623 up-methylated genes. Methylated genes were significantly enriched in carbohydrate metabolism, biosynthesis of other secondary metabolites and amino acid metabolism pathways. The global DNA methylation level decreased from J1 to J2, especially in gene promoters, transcriptional termination regions and regions around CpG islands. We further investigated the expression patterns of 9 DNA methyltransferase-associated genes and 2 DNA demethyltransferase-associated genes from young to senescent cotyledons, which were down-regulated during cotyledon development. In this paper, we first reported that senescent cotton cotyledons exhibited lower DNA methylation levels, primarily due to decreased DNA methyltransferase activity and which also play important role in regulating secondary metabolite process.

Abstract 4437: DNA methylation analysis in cisplatin resistant ovarian cancer cells and recurrent ovarian cancer patient samples

Abstract Acquisition of platinum resistance following first line platinum therapy is commonly observed in ovarian cancer patients and is a major obstacle to clinical effectiveness. Currently there are no options available to prevent platinum resistance; however, studies show that demethylating agents may resensitize patients to platinum therapy thereby demonstrating DNA methylation as a critical contributor to the development of platinum resistance. Using a cellular model of platinum resistance we have previously shown that resistant cells have increased DNA methyltransferase (DNMT) activity as well as increased global DNA methylation. These observations were dependent on cisplatin induced activation of the Epidermal Growth Factor Receptor (EGFR) which we previously identified as a novel a regulator of DNMT activity and DNA methylation. Inhibition of the EGFR conferred during a platinum resistance paradigm decreased DNMT activity, decreased global methylation and attenuated resistance in our platinum resistant model. We hypothesized that further genome wide analysis of DNA methylation in resistant, parental and EGFR inhibited cell lines at specific CG sites would reveal targets of methylation important for the development of platinum resistance. Using the Illumina 450K methylation array, we found few significant alterations in DNA methylation for given genes between control and cisplatin resistant (CPR) cells. However, additional DNA methylation analysis with the 450K methylation array of patient tumor samples at initial diagnosis as well as after relapse, while not significant, showed similar trends in the data for certain genes namely CSRNP3, LOC400904, LYPD6, MTMR9L, NIPAL4 and RBP7. We are still evaluating the effects of EGFR inhibition in vitro on DNA methylation patterns. We suggest that while the in vitro model of platinum resistance corresponds to patient samples in some aspects, ovarian cancer cells display a great deal of heterogeneity in DNA methylation as described by others and a much larger sample size is necessary to draw concrete conclusions. Current studies are evaluating gene expression in control and resistant cells to validate whether alterations in DNA methylation coincide with changes in expression. Citation Format: Michaela L. Granados, Laurie G. Hudson, Li Luo, Sabrina L. Samudio-Ruiz. DNA methylation analysis in cisplatin resistant ovarian cancer cells and recurrent ovarian cancer patient samples. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4437.

A Specific Mechanism By Which NPM1 mutations Impede Myeloid Differentiation Also Explains The Link With DNMT3A Mutation

NPM1 and DNMT3A mutations are highly recurrent (∼30% of cases) in de novo acute myeloid leukemia (AML). Besides aberrant cytoplasmic localization of mutated NPM1, and decreased DNA methyltransferase activity of mutated DNMT3A, the molecular mechanisms of pathogenesis are mysterious. Here, novel specific molecular mechanisms are demonstrated. These results build on and are separate from data presented (poster) at ASH 2012.LC-MSMS analysis of murine hematopoietic cells was used to analyze the protein interaction network of the key monocyte/macrophage differentiation-driving transcription factor Pu.1. Npm1 was noted to interact with Pu.1 in these analyses, confirmed by bidirectional co-immunoprecipitation-Western blot assays. This interaction suggested that PU.1 might be dragged into the cytoplasm by mutated NPM1 in AML. Accordingly, in the OCI-AML3 cell line that contains an NPM1 mutation, but not in OCI-AML2 cells with wild-type NPM1, PU.1 was co-dislocated into the cytoplasm together with NPM1, obvious by Western analysis of cellular fractions, and by immunofluorescence (IF) assays. IF analysis of primary AML cells from patients with NPM1 mutated AML confirmed this cytoplasmic dislocation of both NPM1 and PU.1 (n=3), with strong cytoplasmic instead of nuclear staining of both proteins. In contrast, primary AML cells with wild-type NPM1demonstrated the expected strong staining of these proteins in the nucleus but not in the cytoplasm (n=3).NPM1 movement into the cytoplasm is mediated by CRM1 (exportin). Thus, we hypothesized that antagonizing mutated NPM1 interaction with CRM1 would retain mutated NPM1 and PU.1 in the nucleus. Decoy peptides based on NPM1 C-terminal sequences, to hopefully minimize disruption to CRM1 interactions with other proteins, were designed to antagonize NPM1 binding to CRM1. These decoy peptides were combined with different nuclear delivery sequences including the TAT peptide, and with fluorescent tags for tracking. All six peptides entered AML-OCI3 cells (shown by IF), with the strongest signals observed for peptides fused to TAT and TAT-NES (nuclear export signal). The decoy peptides significantly and substantially inhibited cell growth of AML-OCI3 cells (>3-fold reduction), accompanied by an increase in expression of the monocyte differentiation marker CD14 (quantified by flow-cytometry). In contrast, TAT peptide alone, as a control, did not inhibit cell growth or induce monocytic differentiation. Western and IF analyses was used to study PU.1 and NPM1 localization: decoy peptide treatment clearly increased nuclear presence of both PU.1 and NPM1, although these proteins remained detectable in cytoplasmic fractions also.At ASH 2012, we showed that OCI-AML3 cells have high nuclear CEBPA and retain granulocytic differentiation potential, readily induced by ATRA. Thus, we hypothesized that the NPM1-induced differentiation block, that is specific for the monocytic lineage, creates selective pressure for cooperative mutations that derepress monocyte-commitment. DNMT3A interacts with polycomb proteins that repress lineage-programs, and in NPM1/DNMT3A double mutant versus NPM1 mutant AML, double mutation was significantly associated with M4/M5 (14/23 [61%]) versus M1 morphology (4/13 [30%], p<0.001), higher peripheral blood absolute monocyte counts (median 3.96 v 0.37, p=0.04) and bone marrow non-specific esterase positivity (14/21 [67%] v 7/19 [37%], p=0.06)(TCGA). Chromatin-immunoprecipitation (ChIP) analyses in OCI-AML3 (NPM1/DNMT3A double mutant) and THP1 cells (NPM1/DNMT3A wild-type) that have similar DNMT3A protein expression, demonstrated DNMT3A localization at all (n=7) PU.1 proximal promoter and distal enhancer elements quantified by QRT-PCR (2-40 fold enrichment over ChIP with IgG). Consistent with decreased methyl-transferase activity of the recruited mutated DNMT3A in OCI-AML3, methylation levels at 9 CpG in the PU.1proximal promoter (chr11: 47399783–47400031, pyrosequencing) were significantly lower in OCI-AML3 than in THP1 cells (30-60% of methylation level in THP1). PU.1 mRNA expression was strikingly higher (>40-fold, p<0.001) in OCI-AML3 cells compared to THP1, although THP1 cells also have M5 morphology.Thus, specific molecular mechanisms explain the lineage-associations and coincidence of NPM1 and DNMT3A mutations in AML. These mechanisms provide guidance for novel treatment methods. Disclosures:Maciejewski:NIH: Research Funding; Aplastic anemia&MDS International Foundation: Research Funding.

FOLIC ACID PREVENTS LINE-1 HYPOMETHYLATION IN RATS WITH HYPERHOMOCYSTEINEMIA

ObjectivesLINE-1 (Long Interspersed Nuclear Elements-1) hypomethylation has been associated with risk of cardiovascular disease. Increased plasma homocysteine (Hcy) is an independent risk factor for cardiovascular disease. However, it is still...

EFFECTS OF ATORVASTATIN ON METHYLATION AND MRNA EXPRESSION OF BCL-2 IN HYPERLIPIDAEMIA WISTAR RATS

ObjectivesTo investigate effects of atorvastatin on methylation status of bcl-2 gene promoter and mRNA expression of bcl-2 in aortic tissue of hyperlipidaemia Wistar rats.Methods66 Wistar rats were equally randomised into...

Mechanistic aspects of cell type and drug specific inhibition of DNA methyltransferase 1

The notion that epigenetic processes may be crucially implicated in the development of psychiatric diseases raised the question whether psychoactive drugs may impact on the epigenetic machinery. Here we explored specificity and mode of action of the inhibitory effect of the tricyclic antidepressant (AD) amitriptyline (AMI) on DNA methyltransferase (DNMT) activity in cortical astrocytes. We found that another tricyclic antidepressant, imipramine, as well as the SSRI paroxetine, but not venlafaxine, carbamazepine or valproic acid decreased DNMT activity, while a trend was observed for citalopram. Surprisingly, DNMT activity in primary cortical neurons and subventricular neural stem cells (NSCs) was not changed upon AD treatment, indicating a cell type specific effect. Among the established mammalian DNMTs we identified DNMT1 as target of psychoactive drugs. The reduction of enzymatic DNMT1 activity was not caused by diminished expression, and also not by a direct drug action on DNMT1. Therefore, we tested various modulators of DNMT1 activity, including S-adenosyl homocysteine, NAA10 and CDKL5 levels as well as sumoylation and phosphorylation dependent interaction with PCNA and UHRF1. Altogether, our study presents a model where ADs impact on DNMT1 activity in a cell-type dependent and drug-specific fashion. Hence, it contributes to the understanding of antidepressants mode of action and may add to the development of novel treatment regimes.

Effect of Maternal and Postweaning Folic Acid Supplementation on Mammary Tumor Risk in the Offspring

Intrauterine and early life exposure to folic acid has significantly increased in North America owing to folic acid fortification, widespread supplemental use, and periconceptional supplementation. We investigated the effects of maternal and postweaning folic acid supplementation on mammary tumor risk in the offspring. Female rats were placed on a control or folic acid-supplemented diet prior to mating and during pregnancy and lactation. At weaning, female pups from each maternal diet group were randomized to the control or supplemented diet and mammary tumors were induced with 7,12 dimethylbenz[a]anthracene at puberty. At necropsy, mammary tumor parameters, genomic DNA methylation, and DNA methyltransferase activity were determined in the offspring. Both maternal and postweaning folic acid supplementation significantly increased the risk of mammary adenocarcinomas in the offspring (OR = 2.1, 95% CI 1.2-3.8, P = 0.008 and OR = 1.9, 95% CI 1.1-3.3, P = 0.03, respectively). Maternal folic acid supplementation also significantly accelerated the rate of mammary adenocarcinoma appearance (P = 0.002) and increased the multiplicity of mammary adenocarcinomas (P = 0.008) in the offspring. Maternal, but not postweaning, folic acid supplementation significantly reduced global DNA methylation (P = 0.03), whereas postweaning, but not maternal, folic acid supplementation significantly decreased DNA methyltransferase activity (P = 0.05) in nonneoplastic mammary glands of the offspring. Our findings suggest that a high intrauterine and postweaning dietary exposure to folic acid may increase the risk of mammary tumors in the offspring. Further, they suggest that this tumor-promoting effect may be mediated in part by altered DNA methylation and DNMT activity.

Abstract 946: Effects of maternal and postweaning folic acid supplementation on gene-specific DNA methylation in the offspring

Abstract Background: Folate is an important co-factor in the transfer of one-carbon moieties essential in DNA methylation reactions, aberrations of which have been implicated in several chronic diseases including cancer. Previous studies have demonstrated that in utero supplementation of methyl donors, including folic acid (FA), can affect DNA methylation with permanent and heritable phenotypic changes in the offspring. Recently, we observed that maternal and postweaning FA supplementation significantly decreased global DNA methylation in the liver of the offspring. We investigated the effect of maternal and postweaning FA supplementation on gene-specific methylation in the offspring. Methods: Female Sprague-Dawley rats were placed on a control diet (2 mg FA/kg diet) or supplemented (5 mg) diet for 3 weeks prior to breeding and throughout pregnancy and lactation. At weaning, pups were randomized to the control or supplemented diet until 14 weeks of age. At necropsy, plasma folate and homocysteine (Hcy; an accurate inverse indicator of tissue folate status), and liver folate concentrations of the pups were determined. Site-specific methylation of the promoter region of the Pparγ, p16 and ER and intraexonic regions of the p53 (exons 6 &amp; 7) and Apc (exon 15) was determined in the liver at weaning and at 14 weeks of age using methylation-sensitive PCR following restriction endonuclease digestion. DNA methyltransferase (DNMT) activity in the liver was also determined at both time points. Results: At weaning and 14 weeks of age, plasma and liver folate concentrations accurately reflected dietary FA levels and were higher in the supplemented FA groups (p&amp;lt;0.001). Plasma Hcy was significantly lower in pups supplemented with FA (p&amp;lt;0.001). At weaning, maternal FA supplementation was associated with significant hypomethylation of the Pparγ, ER, p53, and Apc genes (p&amp;lt;0.05). At 14 weeks of age, however, maternal and postweaning FA supplementation increased methylation of the Pparγ, p53, and p16 genes (p&amp;lt;0.05) and postweaning FA supplementation independently increased methylation of the ER and Apc genes (p&amp;lt;0.05). No interactions between maternal or postweaning diets on gene-specific methylation were observed. FA supplementation also decreased DNMT activity at both time points (p&amp;lt;0.05). Conclusions: Our data suggest that FA supplementation during the intrauterine, perinatal and postweaning periods significantly modulates DNA methylation of critical genes with tumor suppressor or transcription regulation functions in the liver of offspring at weaning and 14 weeks of age. Given the drastically increased FA intake and blood measurements in women at childbearing age in North America, in part due to widespread FA supplementation and mandatory fortification, whether or not FA can influence cancer risk in the offspring via epigenetic mechanisms warrants further investigation. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 946.

Abstract 942: Effects of maternal and postnatal folic acid supplementation on genomic DNA methylation in the mammary glands in a chemical rat model of mammary tumorigenesis

Abstract Background: Widespread loss of genomic DNA methylation is an early and consistent epigenetic event in cancer development including breast carcinogenesis. Folate, an essential co-factor in the transfer of one-carbon units, has been shown to modulate both cancer risk and genomic DNA methylation. We recently observed that folic acid (FA) supplementation provided in utero and in early postnatal life significantly increased risk of mammary tumors in the offspring in the dimethylbenzanthracene (DMBA) rat model. To determine a potential underlying mechanism of FA-mediated mammary tumor development, we investigated the association between global DNA methylation, DNA methyltransferase (DNMT) activity, FA supplementation, and mammary tumor status in the offspring. Methods: Female Sprague-Dawley rats were placed on a control diet (2 mg FA/kg diet) or a supplemented (5 mg) diet for 3 weeks prior to breeding and throughout pregnancy and lactation. At weaning, female pups were randomized to the control or supplemented diet. At 7 weeks of age, all pups received a single intragastric dose (5 mg) of DMBA to induce mammary tumors. At 28 weeks of age, plasma, mammary gland and liver folate and plasma homocysteine concentrations were determined and mammary tumors were enumerated and histologically confirmed. Genomic DNA methylation and DNMT activity were determined in non-neoplastic mammary glands. Results: Plasma, liver, and mammary folate and homocysteine concentrations accurately reflected dietary FA levels at weaning and 28 weeks of age (p&amp;lt;0.05). A significant interaction was observed between genomic DNA methylation and mammary adenocarcinoma status in the offspring (p&amp;lt;0.001), indicating that among tumor-free offspring, genomic DNA methylation was significantly decreased by maternal FA supplementation. In addition, in offspring bearing at least one adenocarcinoma, genomic DNA methylation was significantly decreased regardless of dietary FA intervention. Postweaning FA supplementation independently decreased DNMT activity in the offspring (p=0.05). Conclusions: Our data suggest that decreased genomic DNA methylation and DNMT activity associated with maternal and postweaning FA supplementation might be a mechanism by which maternal and postweaning FA supplementation increased mammary tumorigenesis in the offspring. Given the drastic increase in FA intake among women of childbearing age in North America, future studies are need to determine whether or not FA supplementation increases the risk of breast cancer through epigenetic mechanisms. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 942.

Altered DNA methylation is associated with docetaxel resistance in human breast cancer cells

Docetaxel is an effective chemotherapy drug to treat breast cancer but the underlying molecular mechanisms of drug resistance are not fully understood. DNA methylation is an epigenetic event, involved in the control of gene expression, which is known to play an important role in cancer and chemotherapy drug resistance. To investigate the role of DNA methylation in docetaxel resistance in breast cancer we used two human breast cancer cell lines (MCF-7 and MDA-MB-231) that were made resistant to docetaxel. Docetaxel-resistant sub-lines were treated with different concentrations of decitabine. Global methylation and DNA methyltransferase (DNMT) activity was measured using an ELISA-based assay. Quantitative real-time PCR was used to study DNMT gene expression. Cell viability was studied by MTT assay. Global methylation was increased in MCF-7 but not significantly changed in MDA-MB-231 docetaxel-resistant cells. Decreased DNMT activity and decreased DNMT1 and DNMT3b mRNA expression was associated with docetaxel resistance in both cell lines. To investigate how the components of the DNA methylation machinery may contribute towards docetaxel resistance, decitabine (5-aza-2'-deoxycytidine), an inhibitor of DNA methylation, was used. Decitabine treatment decreased global methylation, DNMT activity and DNMT1, DNMT3a and DNMT3b mRNA expression in MDA-MB-231 docetaxel-resistant cells. In contrast, decitabine-treated MCF-7 docetaxel-resistant cells showed increased DNMT1, DNMT3a and DNMT3b mRNA expression indicating a cell line specific effect of decitabine. Decitabine treatment increased resistance in MCF-7 docetaxel-resistant cells and in the parental MCF-7 and MDA-MB231 docetaxel-sensitive cell lines, however, it did not alter response to docetaxel in MDA-MB-231 docetaxel-resistant cells. This study demonstrates that changes in the DNA methylation machinery are associated with resistance to docetaxel in breast cancer cells. The use of epigenetic therapies, as a strategy to overcome drug resistance, needs to be investigated more fully to determine their effectiveness in different cancers and for different chemotherapy drugs.

Maintenance forced by a restriction-modification system can be modulated by a region in its modification enzyme not essential for methyltransferase activity.

Several type II restriction-modification gene complexes can force their maintenance on their host bacteria by killing cells that have lost them in a process called postsegregational killing or genetic addiction. It is likely to proceed by dilution of the modification enzyme molecule during rounds of cell division following the gene loss, which exposes unmethylated recognition sites on the newly replicated chromosomes to lethal attack by the remaining restriction enzyme molecules. This process is in apparent contrast to the process of the classical types of postsegregational killing systems, in which built-in metabolic instability of the antitoxin allows release of the toxin for lethal action after the gene loss. In the present study, we characterize a mutant form of the EcoRII gene complex that shows stronger capacity in such maintenance. This phenotype is conferred by an L80P amino acid substitution (T239C nucleotide substitution) mutation in the modification enzyme. This mutant enzyme showed decreased DNA methyltransferase activity at a higher temperature in vivo and in vitro than the nonmutated enzyme, although a deletion mutant lacking the N-terminal 83 amino acids did not lose activity at either of the temperatures tested. Under a condition of inhibited protein synthesis, the activity of the L80P mutant was completely lost at a high temperature. In parallel, the L80P mutant protein disappeared more rapidly than the wild-type protein. These results demonstrate that the capability of a restriction-modification system in forcing maintenance on its host can be modulated by a region of its antitoxin, the modification enzyme, as in the classical postsegregational killing systems.

Inhibition of DNA methyltransferase reverses cisplatin induced drug resistance in murine neuroblastoma cells

Background: Acquired drug resistance is a major obstacle to the successful treatment of neuroblastoma by chemotherapy. Recent studies from our laboratory have demonstrated that drug-induced alterations in DNA methylation play an important role in this process. Methods: The reversal of resistance to cisplatin in murine neuroblastoma (MNB) was induced by inhibition of DNA methyltransferase activity. MNB cells overexpressing DNA methyltransferase activity (Dnmt3a or Dnmt3b) were established by stable co-transfection of wild type MNB cells with plasmids containing Dnmt3a or Dnmt3b cDNA. Cytotoxic response (IC 50), total DNA methyltransferase activity and expression of Dnmt3a or Dnmt3b methyltransferase were determined in Dnmt3a or Dnmt3b transfected MNB cells, respectively. Results: These data demonstrated that total DNA methyltransferase activity was increased to 3-fold above controls ( P < 0.001) in cisplatin resistant MNB cells, 3-fold in Dnmt3a and 4-fold in Dnmt3b transfected MNB cells. Western blot and RT-PCR data confirmed a corresponding increase in Dnmt3a and 3b expression in cisplatin resistant and transfected cells when compared with control MNB cells ( P < 0.001). MNB clones overexpressing Dnmt3a or Dnmt3b proteins were resistant to cisplatin treatment (10 −6 M). Incubation of cisplatin resistant, Dnmt3a or Dnmt3b overexpressing MNB cells with 5′-azacytidine (5′-azaC), a methylation inhibitor (2.5 μM) significantly decreased DNA methyltransferase activity, expression of Dnmt3a and Dnmt3b proteins and mRNA levels of cisplatin resistant, Dnmt3a and Dnmt3b transfected MNB cells. Cytotoxicity studies using the MTT assay demonstrated that the sensitivity of cisplatin resistant, Dnmt3a and Dnmt3b overexpressing MNB cells to cisplatin was increased 10-fold ( P < 0.001) following 5′-azaC treatment. Conclusions: These findings suggest that the overexpression of DNA methyltransferase is associated with a cisplatin resistant phenotype in MNB cells that may or may not be true in animal studies or in the clinical setup. Thus, DNA methylation plays a central role in onset of drug resistance in cisplatin resistant neuroblastoma cells in vitro.