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PubMed1989

Proliferation-associated expression of DNA methyltransferase in human embryonic lung cells.

The cell cycle-dependent and proliferation-associated expression of the enzyme DNA methyltransferase has been evaluated immunocytochemically in synchronized L-132 human embryonic lung cells, using the anti-DNA methyltransferase monoclonal antibody M1F6D7/5C10. DNA methyltransferase-reactivity was firstly seen in mid-G1 cells. An intense and granular reaction in the cell nuclei with a sparing of the nucleoli was observed in addition to a homogenous and faint cytoplasmic staining. The staining intensity in the cell nuclei increased progressively up to mitosis. In early mitotic cells an intense perichromosomal staining was observed in addition to a homogenous staining of cyto- and karyoplasm after the resolving of the core membrane. In late mitosis the staining intensity decreased rapidly. Early G1 cells and density inhibited, resting G0 cells showed no DNA methyltransferase reactivity at all. Our results indicate that anti-DNA methyltransferase monoclonal antibodies could become valuable tools to detect proliferating cells in cell cultures and tissues.

Cell Cycle↗
PubMedDate not supplied

Sea urchin DNA methyltransferases.

DNA methyltransferase activities have been partially purified from unfertilized eggs and blastula nuclei of sea urchin embryos. Comparative studies, using different DNAs as substrates, show that the two preparations are most active on hemimethylated and single-strand DNA, but they methylate, though at a lower rate, also on double-strand DNA. The two activities show distinctive efficiencies in methylating plasmid DNAs and marked differences in the rate of methyl transfer to DNAs in different structural states: linear, relaxed, or supercoiled. The ratio of the apparent specific activity of the two preparations depends on the particular DNA used as substrate and its structure. Methylation analysis of the restriction fragments of methylated plasmid DNAs shows a linear correlation between introduced methyl groups and the percent of CpG of each particular fragment, indicating that methylation is substantially random and sequence is less relevant than conformation in determining enzyme efficiency. The data do not permit us to decide if the two activities are different enzymes or the same enzyme with different modulating factors.

Animals↗
PubMed2006

Hypermethylation of Cyclin D2 is associated with loss of mRNA expression and tumor development in prostate cancer.

D-type cyclins play a pivotal role in cell cycle regulation and their abnormal expression was associated with several human malignancies. To assess Cyclin D2 promoter methylation status and expression levels in prostate tissues, quantitative methylation-specific PCR and quantitative reverse transcription PCR assays were performed in a large series of prostate carcinomas, high-grade prostatic intraepithelial neoplasias (HGPIN), benign prostate hyperplasias (BPH), normal prostate tissue (NPT) samples, and prostate cancer (PCa) cell lines (before and after demethylating treatment). Methylation levels were correlated with mRNA expression levels and key clinicopathologic parameters. Cyclin D2 promoter methylation was found in 117/118 PCa, 38/38 HGPIN, 24/30 BPH, 11/11 NPT, and 4/4 cell lines. Methylation levels were significantly higher in PCa compared with HGPIN, NPT, and BPH (P<0.0001), correlating with tumor stage and Gleason score (r=0.29, P=0.0014; and r=0.32, P=0.0005, respectively). Conversely, Cyclin D2 mRNA levels were significantly lower in PCa (P<0.01) and a significant inverse correlation between Cyclin D2 methylation and expression levels was found in prostatic tissues (r=-0.61, P<0.000001). Demethylating treatment induced a substantial increase in Cyclin D2 mRNA in LNCaP cells whereas decreased levels were observed in DU-145 and PC-3 cells. We concluded that Cyclin D2 promoter methylation downregulates gene transcription and occurs with high frequency at low levels in normal, hyperplastic, and preneoplastic prostate tissues. Conversely, high Cyclin D2 methylation levels characterize invasive prostatic carcinoma, correlating with clinicopathologic features of tumor aggressiveness.

Adenocarcinoma↗
PubMed2003

Identification and functional analysis of six mycolyltransferase genes of Corynebacterium glutamicum ATCC 13032: the genes cop1, cmt1, and cmt2 can replace each other in the synthesis of trehalose dicorynomycolate, a component of the mycolic acid layer of the cell envelope.

By data mining in the sequence of the Corynebacterium glutamicum ATCC 13032 genome, six putative mycolyltransferase genes were identified that code for proteins with similarity to the N-terminal domain of the mycolic acid transferase PS1 of the related C. glutamicum strain ATCC 17965. The genes identified were designated cop1, cmt1, cmt2, cmt3, cmt4, and cmt5 ( cmt from corynebacterium mycolyl transferases). cop1 encodes a protein of 657 amino acids, which is larger than the proteins encoded by the cmt genes with 365, 341, 483, 483, and 411 amino acids. Using bioinformatics tools, it was shown that all six gene products are equipped with signal peptides and esterase domains. Proteome analyses of the cell envelope of C. glutamicum ATCC 13032 resulted in identification of the proteins Cop1, Cmt1, Cmt2, and Cmt4. All six mycolyltransferase genes were used for mutational analysis. cmt4 could not be mutated and is considered to be essential. cop1 was found to play an additional role in cell shape formation. A triple mutant carrying mutations in cop1, cmt1, and cmt2 aggregated when cultivated in MM1 liquid medium. This mutant was also no longer able to synthesize trehalose di coryno mycolate (TDCM). Since single and double mutants of the genes cop1, cmt1, and cmt2 could form TDCM, it is concluded that the three genes, cop1, cmt1, and cmt2, are involved in TDCM biosynthesis. The presence of the putative esterase domain makes it highly possible that cop1, cmt1, and cmt2 encode enzymes synthesizing TDCM from trehalose monocorynomycolate.

Arabidopsis Proteins↗
PubMed2005

Butyrates and decitabine cooperate to induce histone acetylation and granulocytic maturation of t(8;21) acute myeloid leukemia blasts.

Core histones are proteins organized in octamers, to which DNA is wrapped more or less tightly, depending on their acetylation status. Gene transcription is regulated by a complex series of epigenetic modifications, i.e., histone modification such as methylation and acetylation, events determined by the enzymatic activity of histone methyltransferases, and histone acetyltransferases, respectively, the latter counterbalanced by histone deacetylases (HDAC). Acetylation of histones facilitates destabilization of DNA-nucleosome interaction and renders DNA more accessible to transcription factors. Methylation of different specific lysine residues of histones is differently linked to euchromatin (transcripted DNA) or heterochromatin (silenced DNA). On the other hand, methylation of the promoter regions of some genes by DNA methyltransferases (DNMT) leads to transcriptional silencing and is a common mechanism to regulate gene expression. In normal eukaryotic cells, DNA methylation and histone acetylation are interdependent and maintain equilibrium, allowing temporal expression of genes. In neoplastic cells, this balance is frequently disrupted. In leukemic cells, hypermethylation of CpG islands in the promoter region of genes critical for cell cycle and maturation is frequent, and DNMTs were found to be overexpressed, findings paralleled by evidence of transcriptional repression of downstream genes. Therefore, the combination of HDAC and DNMT inhibitors has been considered to be a possible therapeutic approach to restore normal gene expression in acute myeloid leukemia (AML) and other diseases. Human AML1/ETO Kasumi cells were exposed to the HDAC inhibitor D1 (O-n-butanoil-2,3-O-isopropylidene-alpha-D: -mannofuranoside) and 5-aza-deoxycytidine (decitabine) alone and in combination. Histone acetylation as measured by flow cytometry was increased following treatment with D1 and the combination of D1 and decitabine. Addition of D1 alone or in combination with decitabine also led to inhibition of cell proliferation and induction of apoptosis. Thus, treatment of AML with HDAC inhibitors such as D1 and DNMT inhibitors such as decitabine might have clinical benefit for patients, especially these presenting subtypes of AML, like AML1/ETO, in which the leukemogenic mechanism involves corepressor protein complexes containing HDAC and DNMT.

Antineoplastic Combined Chemotherapy Protocols↗
PubMed2005

Quantification of outpatient management and hospitalization of patients with high-risk myelodysplastic syndrome treated with low-dose decitabine.

Intravenous low-dose 5-aza-2'-deoxycytidine (decitabine) in patients with advanced myelodysplastic syndrome (MDS) yields an approximately 50% overall response rate, including 20-25% complete remission. Decitabine-treated MDS patients can be managed as outpatients after completion of a 3-day infusion schedule. In-hospital nights (IHNs), overall survival (OS), and remaining life spent in hospital were evaluated and compared to a matched control group receiving different standard treatments. Between July 1992 and September 2001, 99 high-risk MDS patients, median age 70 years (range 49-86), were treated with low-dose decitabine. Durations of all hospitalizations were recorded. For matched-pair analysis, 44 decitabine-treated patients were matched to 44 MDS patients according to International Prognostic Scoring System classification, period of diagnosis, age, French-American-British classification, and gender. Median number of IHN across all patients was 56 and survival was 481 days, resulting in 84% of remaining life spent at home. In the matched-pair analysis, the median number of IHN was 57 in the decitabine group vs. 50 in the control group. Median survival was 400 vs. 371 days for the decitabine and control groups, respectively. Median number of remaining life spent at home was identical (83% for both groups). Matched patients who received only best supportive care (n=12) had a shorter median survival than the decitabine patients (234 vs. 400 days), and the proportion of remaining life spent at home was slightly greater (82 vs. 77%). Interestingly, matched patients with induction therapy showed comparable IHN, OS, and remaining life spent at home. In conclusion, high-risk MDS patients treated with low-dose decitabine have better survival, and spend comparable time in hospital than patients treated with supportive treatment.

Aged↗
PubMed2005

Decitabine: a historical review of the development of an epigenetic drug.

The development of decitabine from its synthesis in 1964 to the submission of a registration file has been described. Although the unique DNA-demethylating capacity of decitabine is known for a long time, its application is under continuing investigation. The use of decitabine in MDS, AML, CML, stem cell transplant, sickle cell anemia and thalassemia looks promising. The epigenetic dose seems lower than the cytotoxic dose. Whereas most drugs have matured after 40 years, decitabine is only at the beginning of a new development phase in epigenesis.

Anemia, Sickle Cell↗
PubMed2005

An epigenetic approach to the treatment of advanced MDS; the experience with the DNA demethylating agent 5-aza-2'-deoxycytidine (decitabine) in 177 patients.

During the last 10 years, three European phase II studies were performed to investigate the treatment of elderly patients with myelodysplastic syndrome (MDS) with low-dose 5-aza-2'-deoxycytidine (decitabine, DAC). All these European trial data were reviewed on the basis of the International Prognostic Scoring System (IPSS) risk criteria and the response criteria as recently published by an international working group. To investigate the results in a larger cohort of patients and to determine risk factors, all data were pooled with some observations from the PCH 95-06 US phase II study. The response rate in the 177 patients evaluated (median age 70 years) was 49%. The median response duration was 36 weeks, and the median survival was 15 months. Analysis of the data according to sex, age, French-American-British classification, percentage of blasts in the bone marrow, IPSS risk group, lactate dehydrogenase and cytogenetics did not reveal any factor predictive of response. Overall, 69% of patients benefited, including those with stable disease during therapy. Response duration was significantly shorter with increasing risk (according to the IPSS classification). Haemoglobin level and neutrophil count showed an inverse correlation to the IPSS classification. Univariate analysis showed a significantly inferior survival for elderly patients (>75 years of age) and for those with high levels of serum lactate dehydrogenase (LDH) (more than two times the normal values). Patients with high-risk cytogenetic abnormalities according to the IPSS risk criteria showed better overall survival than those with intermediate-risk abnormalities. When analysed according to the IPSS risk classification, high-risk patients had worse survival prospects following decitabine therapy than those with intermediate risk; however, compared to the originally reported IPPS outcomes for high-risk patients, they probably showed better survival. During the treatment period, 18% of the patients progressed towards acute leukaemia. Decitabine showed a rather low toxicity profile in this elderly patient group. In conclusion, low-dose decitabine is an active drug for the treatment of MDS patients, even for those older than 75 years with bad prognostic characteristics.

Adult↗
PubMed2005

Clinical experience with decitabine in North American patients with myelodysplastic syndrome.

Recent evidence demonstrates that epigenetic silencing of genes is associated with myelodysplasia and that a worse prognosis may be correlated with hypermethylation of certain genes, such as the cyclin-dependent kinase inhibitor p15. 5-Aza-2'-deoxycytidine (decitabine, DAC) is a nucleoside analog, which, at low doses, acts as a hypomethylating agent and is fivefold to tenfold more active than 5-azacytidine (azacitidine, Vidaza)--currently the only approved drug for treatment of myelodysplastic syndrome (MDS). Clinical studies have demonstrated that decitabine has activity in patients with MDS. Preliminary results of a phase III multicenter North American trial comparing low-dose decitabine to supportive care verified that therapy with decitabine resulted in higher response rates, improved quality of life, and prolonged time to leukemic transformation and/or death. However, further elucidation of its mechanism of action is required, as clinical response to decitabine does not correlate with demethylation of the p15 gene promoter or the repetitive DNA element LINE. Decitabine appears to upregulate both hypermethylated and nonmethylated genes. Ongoing studies aim to determine the optimal dose, schedule, and route of administration of decitabine, and to evaluate whether efficacy can be improved by using it in combination with other agents, such as histone deacetylase inhibitors.

Antineoplastic Combined Chemotherapy Protocols↗
PubMed2005

The hematopathological basis for studying effects of the demethylating agent 5-aza-2'-deoxycytidine (decitabine) in myelodysplasia.

The myelodysplastic syndromes have, since their first recognition decades ago, been considered notoriously difficult with regard to their proper classification, determination of prognosis, and optimal treatment. With the advent of the French-American-British (FAB) classification, now aided but not superseded by the World Health Organization classification, distinct biological entities have been delineated, which in turn are very useful for stratification to different, established and experimental treatment modalities. However, precise subclassification of different types of myelodysplastic syndrome (MDS) is only possible with hematopathological studies based on the analysis of peripheral blood, bone marrow smear, and bone marrow biopsy, backed by appropriate clinical information. Bone marrow cytogenetics are also essential for any risk stratification since they still provide the second most powerful prognostic parameter after bone marrow blast enumeration. This paper will review the most important aspects of hematopathological diagnostics in MDS, risk scoring, and their application to the inclusion and stratification of patients into the European Organization for Research and Treatment of Cancer (EORTC)/German MDS Study Group Phase III multicenter trial of low-dose decitabine in patients more than 60 years old with high-risk MDS. Emphasis is placed on itemizing the broad spectrum of cytologic and histologic stigmata defining the myelodysplastic categories that are to be considered in this study.

Antimetabolites, Antineoplastic↗
PubMed2006

Different susceptibility of increased DNMT1 expression by exposure to tobacco smoke according to histology in primary non-small cell lung cancer.

PURPOSE: DNA methyltransferase 1 (DNMT1) is known to play an important role in the development of cancers. However, the underlying mechanisms responsible for the altered expression of DNMT1 in non-small cell lung cancers (NSCLCs) remain to be elucidated. METHODS: We investigated the relationships of mRNA expression levels of DNMT1 to the altered expression of retinoblastoma (Rb) and p53 and to the clinicopathological variables in 153 NSCLCs. The expression of DNMT1 was determined by quantitative real-time PCR, and the altered expressions of p53 and Rb were assessed by immunohistochemistry. RESULTS: The increased expression of DNMT1 was found in 47 (31%) of 153 NSCLC patients examined. The prevalence of increased DNMT1 expression was significantly different between adenocarcinoma and squamous cell carcinoma (42% vs. 19%, respectively; P = 0.004). Patients who had smoked more than 65 packyears showed a 4.17 times [95% confidence interval (CI) = 1.17-69.49; P = 0.007] higher risk of increased DNMT1 expression compared to those who had smoked less than 45 packyears in adenocarcinoma. The expressions of Rb and p53 proteins were not associated with the increased expression of DNMT1 in 153 NSCLCs (P = 0.18 and 0.54, respectively). CONCLUSIONS: The present study suggests that the susceptibility of increased DNMT1 expression by exposure to tobacco smoke may be different according to histologic subtypes in NSCLC.

Adenocarcinoma↗
PubMed1998

Mutations in the dim-1 gene of Neurospora crassa reduce the level of DNA methylation.

Mutants that show reduced DNA methylation were identified in a mutant screen based on the assumptions that (i) the nucleoside analog 5-azacytidine (5-azaC) promotes the formation of potentially lethal DNA-methyltransferase adducts; (ii) reduction in DNA methyltransferase will decrease the sensitivity of cells to 5-azaC; and (iii) this potential selective advantage will be enhanced in mutants that are deficient in the repair of 5-azaC-induced DNA damage. Of fifteen potential repair mutants screened for sensitivity to 5-azaC, five (mus-9, mus-10, mus-11, mus-18, and uvs-3) showed moderately increased sensitivity and two (mus-20, mei-3) showed highly increased sensitivity. A mus-20 mutation was used to isolate three non-complementing methylation mutants. The mutations, named dim-1 (defective in methylation), reduced female fertility, reduced methylation by 40-50%, and altered patterns of methylation. In wild-type strains hypomethylation per se fails to alter methylation specificity. We demonstrate a growth-phase-dependent change in methylation patterns, detectable only in hypomethylated DNA from dim+ cultures. This may represent a growth-phase-dependent change in the relative amounts of distinct species of methyltransferase, one of which may be encoded by the dim-1 gene.

Azacitidine↗
PubMed2005

The controversial denouement of vertebrate DNA methylation research.

The study of the biological role of DNA methylation in vertebrates has involved considerable controversy. Research in this area has proceeded well despite the complexity of the subject and the difficulties in establishing biological roles, some of which are summarized in this review. Now there is justifiably much more interest in DNA methylation than previously, and many more laboratories are engaged in this research. The results of numerous studies indicate that some tissue-specific differences in vertebrate DNA methylation help maintain patterns of gene expression or are involved in fine-tuning or establishing expression patterns. Therefore, vertebrate DNA methylation cannot just be assigned a role in silencing transposable elements and foreign DNA sequences, as has been suggested. DNA methylation is clearly implicated in modulating X chromosome inactivation and in establishing genetic imprinting. Also, hypermethylation of CpG-rich promoters of tumor suppressor genes in cancer has a critical role in downregulating expression of these genes and thus participating in carcinogenesis. The complex nature of DNA methylation patterns extends to carcinogenesis because global DNA hypomethylation is found in the same cancers displaying hypermethylation elsewhere in the genome. A wide variety of cancers display both DNA hypomethylation and hypermethylation, and either of these types of changes can be significantly associated with tumor progression. These findings and the independence of cancer-linked DNA hypomethylation from cancer-linked hypermethylation strongly implicate DNA hypomethylation, as well as hypermethylation, in promoting carcinogenesis. Furthermore, various DNA demethylation methodologies have been shown to increase the formation of certain types of cancers in animals, and paradoxically, DNA hypermethylation can cause carcinogenesis in other model systems. Therefore, there is a need for caution in the current use of demethylating agents as anti-cancer drugs. Nonetheless, DNA demethylation therapy clearly may be very useful in cases where better alternatives do not exist.

Animals↗
PubMed1989

Polypeptide composition and an immunological analysis of DNA methyltransferases from different species.

The cross-reactivity of the monoclonal anti-human placental DNA methyltransferase antibody M2B10 with DNA methyltransferases isolated from other species was investigated. This antibody immunoprecipitates DNA methyltransferases from mammalian cells, i.e., human placenta, mouse P815 cells, and rat liver cells. No cross-reactivity is observed with DNA methyltransferases from wheat germ and with bacterial DNA methyltransferases HpaII and EcoRI. The mammalian enzymes are characterized by polypeptides of molecular mass 150-190 kDa. Polypeptides smaller than 190 kDa are presumably generated by proteolysis of the native 190-kDa DNA methyltransferase. Trypsin digestion of the 190-kDa polypeptide isolated from mouse cells results in progressive appearance of DNA methyltransferase polypeptides of 150-190, 110, 100, and 52-60 kDa.

Animals↗
PubMed1991

Purification and properties of a novel DNA methyltransferase from cultured rice cells.

DNA methyltransferase activity has been observed in a total crude homogenate of rice cells grown in suspension culture using either native plant DNA or, under the conditions used, the more responsive hemimethylated poly (dI-MedC).poly(dI-dC). Using the latter substrate we have purified an enzyme fraction 380-fold by salt extraction of chromatin, DEAE cellulose and phosphocellulose. This purified fraction showed enzyme activity only with poly (dI-MedC).poly(dI-dC) thus suggesting the occurrence in plants of a DNA methyltransferase specific for hemimethylated DNA. A Mr value of 54000 was calculated on the basis of the sedimentation coefficient which was determined by sucrose density gradient centrifugation. Apparent Km values for poly (dI-MedC).poly(dI-dC) and S-adenosyl-L-methionine were found to be 17 micrograms/ml and 2.6 microM, respectively.

Cells, Cultured↗
PubMed1992

Expression of CYP2E1 during human fetal development: methylation of the CYP2E1 gene in human fetal and adult liver samples.

The expression and regulation of cytochrome P450IIE1 (CYP2E1) in adult and fetal human liver has been investigated. Three mRNA transcripts of 1.9, 2.7 and 3.8 kb were detected in all adult liver samples after hybridization with a full length cDNA to CYP2E1 whereas no expression was detected in 12 fetal liver samples studied. Similarly, expression of CYP2E1 was not detected in 11 placental samples (10-17 weeks gestational age) or in two full-term placental samples. CYP2E1 expression was not detected in fetal liver, kidney, lung, placenta (18 weeks gestational age) or liver (6 weeks gestational age) obtained at termination of pregnancy where maternal alcohol abuse had been established. Southern blot analysis of the cytosine methylation status of the CYP2E1 gene revealed substantial methylation of the 3' region of the gene in both adult and fetal human liver samples. No differences were observed in the methylation pattern of fetal liver samples between the gestational ages 12 and 17 weeks. Two small DNA fragments detected by the 5' end of the CYP2E1 cDNA were cleaved by the restriction enzyme HpaII in adult liver DNA but not in the fetal liver DNA samples. Methylation of specific 5' residues in the CYP2E1 gene may be responsible for the lack of transcription of the CYP2E1 gene in fetal liver.

Alcoholism↗
PubMed1991

Mutations that confer de novo activity upon a maintenance methyltransferase.

DNA methyltransferases are not only sequence specific in their action, but they also differentiate between the alternative methylation states of a target site. Some methyltransferases are equally active on either unmethylated or hemimethylated DNA and consequently function as de novo methyltransferases. Others are specific for hemimethylated target sequences, consistent with the postulated role of a maintenance methyltransferase in perpetuating a pattern of DNA modification. The molecular basis for the difference between de novo and maintenance methyltransferase activity is unknown, yet fundamental to cellular activities that are affected by different methylation states of the genome. The methyltransferase activity of the type I restriction and modification system, EcoK, is the only known prokaryotic methyltransferase shown to be specific for hemimethylated target sequences. We have isolated mutants of Escherichia coli K-12 which are able to modify unmethylated target sequences efficiently in a manner indicative of de novo methyltransferase activity. Consistent with this change in specificity, some mutations shift the balance between DNA restriction and modification as if both activities now compete at unmethylated targets. Two genes encode the methyltransferase and all the mutations are loosely clustered within one of them.

Bacteriophage lambda↗