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PubMed1996

DMI-2 and DMI-3, DNA methyltransferase inhibitors produced by Streptomyces sp. strain no. 560.

Streptomyces sp. strain No. 560 produces several types of DNA methyltransferase inhibitors in the culture filtrate. Two of them, DMI-2 and DMI-3, were distinguished from the previously reported DMI-1 by their inhibitory spectrum and inhibition characteristics against DNA methyltransferase. The molecular weights of DMI-2 and DMI-3 were 854 and 435, respectively. The structure of DMI-2 was determined to be 4"'R,6aR,10S,10aS-8-acetyl-6a, 10a-dihydroxy-2-methoxy-12-methyl-10-[4'-[3"-hydroxy-3",5"-dimethyl-4" (Z-2"',4"'-dimethyl-2"'-heptenoyloxy) tetrahydropyran-1"-yloxy]-5'-methylcyclohexan-1'-yloxy ]-1,4,6, 7,9-pentaoxo-1,4,6,6a,7,8,9,10,10a,11-decahydronaphthacene. The chemical structure of DMI-2 was established as a tautomer of dutomycin which is an antitumor antibiotic produced by Streptomyces sp. 1725. DMI-2 and DMI-3 showed strong inhibition against N6-methyladenine-DNA methyltransferase (M. Eco RI). DMI-2 inhibited M. Eco RI in a competitive manner with respect to plasmid pUC19 used as DNA substrate and in an uncompetitive manner with respect to S-adenosylmethionine (SAM) used as methyl donor. DMI-3 inhibited M. Eco RI in a competitive manner with respect to plasmid pUC19 and SAM. The inhibitory activities of both inhibitors depended upon the pH and temperature in the assay media.

DNA Methylation↗
PubMed1996

Inhibition of DNA methyltransferase by microbial inhibitors and fatty acids.

Streptomyces sp. strain No. 560 produces four kinds of DNA methyltransferase inhibitors in the culture filtrate. One of them, DMI-4 was distinguished from DMI-1, -2 and -3 previously reported with respect to certain properties, DMI-4 is considered to be a triglyceride consisting of the fatty acids anteisopentadecanoic acid (C15:0), isopalmitic acid (C16:0) and isostearic acid (C18:0) from the results of gas chromatography analysis. Since DMI-4 contains three molecules of fatty acid, and the previously reported DMI-1, 8-methylpentadecanoic acid, is analogous to a fatty acid, the inhibitory activity has been examined of various fatty acids and their methyl esters against Eco RI DNA methyltransferase (M. Eco RI). Oleic acid (C18:1) was found to be a potent inhibiton of M. Eco RI. The inhibitory activity of oleic acid was shown to be pH- and temperature-dependent and inhibited M. Eco RI in a noncompetitive manner with respect to DNA or S-adenosylmethionine (SAM). The number of carbon atoms and double bonds in the fatty acid molecule affected the inhibitory activity, but their methyl esters were not inhibitors. Our results suggest that the length of the carbon chain, the number of double bonds and the presence of a carboxyl group and branched methyl group in the fatty acid molecule may play an important role in the inhibition of DNA methyltransferase.

DNA Modification Methylases↗
PubMed2001

Demethylation by 5-aza-2'-deoxycytidine (5-azadC) of p16INK4A gene results in downregulation of vascular endothelial growth factor expression in human lung cancer cell lines.

Vascular endothelial growth factor (VEGF) plays a pivotal role in tumor progression via angiogenesis. Recently, gene transduction of wild-type p16INK4A, tumor suppressor gene, has been shown to result in downregulation of VEGF expression in p16INK4A-deleted glioma cells. Because expression of p16INK4A is regulated by methylation of the p16INK4A gene, we examined whether demethylation of the p16INK4A gene by 5-aza-2'-deoxycytidine (5-azadC) could cause the protein expression of VEGF as well as of p16INK4A in human lung cancer cells. For this, five different lung cancer cell lines with or without loss of p16 activity were used. H841 and Ma-10 cells had the methylated p16INK4A gene without expression of p16INK4A protein, whereas Ma-1 and H209 cells had the unmethylated p16INK4A gene with constitutive expression of p16INK4A protein. Neither the p16INK4A gene nor p16INK4A protein was detected in A549 cells. Treatment with 5-azadC caused demethylation of the p16INK4A gene with reexpression of p16INK4A protein in H841 and Ma-10 (methylated p16INK4A gene dominant) cell, but not in other cell lines such as Ma-1, H209 (unmethylated p16INK4A gene dominant), or A549 (p16INK4A gene deleted). In a parallel experiment, 5-azadC inhibited production of VEGF protein by H841 and Ma-10 cells, especially in the later hypermethylated cells, but not Ma-1, H209, or A549 cells. RT-PCR analysis showed that Ma-10 cells expressed VEGF isoforms 121, 165, and 189, all of which were inhibited by 5-azadC. These findings indicate that the methylation status of the p16INK4A gene plays an important role in the regulation of angiogenesis associated with progression of lung cancer, through regulation of VEGF expression.

Antimetabolites, Antineoplastic↗
PubMed2000

Methylation of class II trans-activator promoter IV: a novel mechanism of MHC class II gene control.

Inhibition of class II trans-activator (CIITA) expression prevents embryonic trophoblast cells from up-regulating MHC class II genes in response to IFN-gamma. This is thought to be one mechanism of maternal tolerance to the fetal allograft. The CIITA gene is regulated by four distinct promoters; promoter III directs constitutive (B cell) expression, and promoter IV regulates IFN-gamma-inducible expression. Using in vivo genomic footprinting, promoter-reporter analysis, Southern blot analysis, and RT-PCR, we have examined the cause of CIITA silencing in a trophoblast-derived cell line. We report here that methylation of promoter IV DNA at CpG sites in Jar cells prevents promoter occupancy and IFN-gamma-inducible transcription. The inhibition of CpG methylation in Jar cells by treatment with 5-aza-2'-deoxycytidine restores IFN-gamma inducibility to CIITA. This is the first description of an epigenetic mechanism involved in regulation of CIITA and MHC class II gene expression.

Antimetabolites, Antineoplastic↗
PubMed2002

Crucial role of DNA methylation in determination of clonally distributed killer cell Ig-like receptor expression patterns in NK cells.

Human NK cells are characterized by the expression of surface receptors of the killer cell Ig-like receptor (KIR) family, which are involved in the specific recognition of pathogenic target cells. Each NK cell expresses and maintains an individual subset of inhibitory and stimulatory KIR and in this way contributes to a diversified NK cell repertoire. To date, the molecular basis for generation of clonally distributed KIR expression patterns has been elusive. Here, analyses of DNA methylation patterns of KIR genes in NK cell lines as well as in NK cells, freshly isolated from peripheral blood, demonstrated that a small CpG island surrounding the transcriptional start site of each KIR gene is consistently demethylated in expressed KIR and methylated in unexpressed KIR. DNA-demethylating treatment resulted in a rapid and stable induction of transcription and cell surface expression of all formerly unexpressed KIR in NK cell lines, NK cell clones, and freshly isolated NK cells, but not in other cell types. In vitro methylation of KIR CpG islands repressed reporter gene expression in NK cells. We conclude that clonal patterns of KIR expression are mainly epigenetically determined and maintained through DNA methylation.

5' Untranslated Regions↗
PubMed2004

Epigenetic regulation of protein phosphatase 2A (PP2A), lymphotactin (XCL1) and estrogen receptor alpha (ER) expression in human breast cancer cells.

Absence of the estrogen receptor alpha (ER) in human breast cancer cells is an indicator of poor prognosis, and predictive of lack of response to hormonal therapy. Previous studies in our laboratory and others have shown that epigenetic regulation, including DNA methylation and histone deacetylation, are common mechanisms leading to ER gene silencing. Through the use of pharmacologic inhibitors, 5-aza 2'deoxycytidine (AZA) and Trichostatin A (TSA), we have shown that alterations in both of these mechanisms results in synergistic reexpression of ER mRNA and functional protein. These alterations may play a larger role in stimulation of cell signaling pathways leading to ER expression. We have utilized newly developed genome wide screening microarray techniques to identify gene(s) contributing to the hormone independent phenotype and AZA/TSA mediated ER expression. From this screen, we identified and confirmed expression of 4 candidate genes (PP2A, XCL1, THY1 and NBC4) as potential regulators of the hormone independent phenotype. Expression of two genes, XCL1 and PP2A, appeared to be correlated with ER expression. PP2A expression was not changed with ER degradation using ICI 182,780 whereas XCL1 expression decreased in the presence of AZA/TSA and ICI 182,780. This suggests that PP2A may be a determinant of ER expression while XCL1 appears to be ER responsive and downstream of ER expression. These gene products may be novel targets to be further explored in the development of new therapeutics for ER negative breast cancer.

Acetylation↗
PubMed2004

MDR1 promoter hypermethylation in MCF-7 human breast cancer cells: changes in chromatin structure induced by treatment with 5-Aza-cytidine.

Resistance to the cytotoxic actions of antineoplastic drugs, whether intrinsic or acquired, remains a barrier to the establishment of curative chemotherapy regimens for advanced breast cancer. Over-expression of P-glycoprotein (P-gp), encoded by the MDR1 gene and known to mediate resistance to many antineoplastic drugs, may contribute to poor breast cancer treatment outcome. Nonetheless, the precise molecular mechanisms responsible for high or low level P-gp expression in breast cancer cells have not been established. We assessed the role of DNA hypermethylation near the MDR1 transcriptional regulatory region in MDR1 expression in MCF-7 breast cancer cells, which fail to express MDR1 mRNA, and MCF-7/ADR cells, known to express high MDR1 mRNA levels. When compared to MCF-7/ADR cells, MCF-7 cells manifested markedly diminished MDR1 transcription rates by nuclear run-off assay, but equivalent MDR1 promoter trans-activation activity in transient transfection experiments, indicating that cis factors were most likely responsible for the differences in MDR1 transcription between MCF-7/ADR cells and MCF-7 cells. Bisulfite genomic sequencing analyses revealed substantially less extensive MDR1 promoter methylation in MCF-7/ADR cells than in MCF-7 cells, suggesting that CpG dinucleotide methylation might contribute to the observed MDR1 transcription differences. Chromatin immunoprecipitation analyses indicated an inactive MDR1 chromatin conformation in MCF-7 cells, with a paucity of acetylated histones and the presence of 5-mC-binding proteins MeCP2 and MBD2, and an active MDR1 chromatin conformation in MCF-7/ADR cells, with an abundance of acetylated histones and the presence of the transcriptional trans-activator YB-1. Stable MCF-7 sublines which had been treated with the DNA methyltransferase inhibitor 5-azacytidine, exhibited a reduction in MDR1 promoter methylation and a complex MDR1 chromatin configuration, characterized by the simultaneous presence of transcriptional activators and repressors. In this state, MDR1 expression was markedly sensitive to treatment with the histone deacetylase inhibitor trichostatin A.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed2006

Six (or more) drugs in search of a mechanism: DNA methyltransferase and histone deacetylase inhibitors in the treatment of myelodysplastic syndromes.

The clinical activity of the DNA methyltransferase inhibitors 5-azacitidine and 2'-deoxy-5-azacytidine in myelodysplastic syndromes (MDS) suggests that epigenetic modulation of gene transcription may play an important pathogenetic role in the development and expression of these diseases. Approximately 50% of patients treated with these compounds experience hematologic improvement, making these the most active single agents for unselected patients with MDS. Responses include complete and partial hematologic responses. Two randomized trials have shown that the use of these drugs significantly alters the natural history of MDS compared with supportive care. Histone deacetylase inhibitors, which may also impact the expression of genes through epigenetic mechanisms, seem to have measurable activity in MDS in preliminary studies. Histone deacetylase inhibitors are most likely used in combination with other agents, including DNA methyltransferase inhibitors. Despite the clinical activity of these classes of drugs, there is no conclusive evidence that their clinical activity is attributable to their impact on the epigenome. Such information will be critical in the development of more effective congeners and drug combinations in ongoing attempts to improve the outcome of patients with MDS.

Antimetabolites, Antineoplastic↗
PubMed1991

The effect of sinefungin and synthetic analogues on RNA and DNA methyltransferases from Streptomyces.

Sinefungin is an antibiotic structurally related to S-adenosylmethionine. It has been described as an inhibitor of RNA transmethylation reactions in viruses and eukaryotic organisms, but not in bacteria. We show here that sinefungin strongly inhibits RNA methyltransferase activity, but not the biosynthesis of these enzymes in Streptomyces. All the methylated bases found in Streptomyces RNA (1-methyladenine, N6-methyladenine, N6,N6-dimethyladenine and 7-methylguanine) are inhibited by this antibiotic. Experiments with sinefungin analogues show that specific changes in the ornithine radical of the molecule still preserve its inhibitory capability. The substitution of the adenine radical by uridine causes the loss of the inhibitory effect. These results and our former studies on Streptomyces DNA methylation, suggest that nucleic acid modification is the main target of sinefungin in Streptomyces.

Adenosine↗
PubMed1999

Relationship between O6-methylguanine-DNA methyltransferase levels and clinical response induced by chloroethylnitrosourea therapy in glioma patients.

BACKGROUND: Adjuvant nitrosourea chemotherapy fails to prolong survival significantly as many tumors demonstrate resistance to these drugs. It has been documented in cell lines that O6-methylguanine DNA methyltransferase (MGMT) plays an important role in chloroethylnitrosourea (CENU) drug resistance. METHODS: We evaluated MGMT expression in 22 glioma specimens by using an immunofluorescence assay and compared the results with clinical responses of the patients to CENU-based chemotherapy. RESULTS: Eight tumor samples had no detectable MGMT, whereas other samples had from 9,989 to 982,401 molecules/nucleus. In one group (12 patients), the tumor decreased in size or was stable (effective group), whereas in the other group (10 patients), the tumor demonstrated continuous growth during chemotherapy (progressive group). The Mer- patients (MGMT < 60,000 molecules/nucleus) appeared to have more chance of stable disease or response to CENU therapy than the Mer+ patients (MGMT > 60,000 molecules/nucleus) (X2 = 4.791, p = 0.0286). In patients with glioblastomas multiforme (GBMs), the median time to progression (TTP) of Mer+ patient was shorter than that of Mer- patient (t = 2.04, p = 0.049). As a corollary, the MGMT levels were significantly higher in GBM tumors from the progressive group than those from the effective group (t = 2.26, p = 0.029). However, there was no significant correlation between MGMT levels and either the survival time (r = 0.04, p = 0.8595) or TTP (r = 0.107, p = 0.6444). CONCLUSION: This study suggests that being MGMT positive is indicative of a more aggressive disease that progresses more rapidly with CENU therapy. However, MGMT negative tumors are not always sensitive to CENU agents, suggesting that other factors are also important.

Adult↗
PubMed1999

DNA methylation.

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Animals↗
PubMed1999

Hypermethylation of the DAP-kinase CpG island is a common alteration in B-cell malignancies.

Death-associated protein kinase (DAP-Kinase) is a novel serine/threonine kinase whose expression is required for gamma interferon-induced apoptosis. A previous study suggested that DAP-Kinase expression may be lost epigenetically in cancer cell lines, because treatment of several nonexpressing cell lines with 5-aza-2'-deoxycytidine resulted in the expression of DAP-Kinase. Using methylation-specific polymerase chain reaction (MSP), we examined the DAP-Kinase CpG island for hypermethylation in cancer. Normal lymphocytes and lymphoblastoid cell lines are unmethylated in the 5' CpG island of DAP-Kinase. However, in primary tumor samples, all Burkitt's lymphomas and 84% of the B-cell non-Hodgkin's lymphomas were hypermethylated in the DAP-Kinase CpG island. In contrast, none of the T-cell non-Hodgkin's lymphoma samples and 15% or less of leukemia samples examined had hypermethylated DAP-Kinase alleles. U937, an unmethylated, DAP-Kinase-expressing leukemia cell line, was treated with gamma interferon and underwent apoptosis; however, Raji, a fully methylated, DAP-Kinase nonexpressing Burkitt's lymphoma cell line, only did so when treated with 5-aza-2'-deoxycytidine followed by gamma interferon. Our findings in cell lines and primary tumors suggest that hypermethylation of the DAP-Kinase gene and loss of gamma interferon-mediated apoptosis may be important in the development of B-cell malignancies and may provide a promising biomarker for B-cell-lineage lymphomas.

Apoptosis↗
PubMed2000

Heterogeneous expression of the SSX cancer/testis antigens in human melanoma lesions and cell lines.

The SSX genes, located on the X chromosome, encode a family of highly homologous nuclear proteins. The SSX1 and SSX2 genes were initially identified as fusion partners of the SYT gene in t(X;18)-positive synovial sarcomas. Recently, however, it was found that these two genes, as well as the highly homologous SSX4 and SSX5 genes, are aberrantly expressed in different types of cancers, including melanomas. Because normal SSX expression has been detected only in the testis and, at very low levels, the thyroid, these proteins are considered as new members of the still growing family of cancer/testis antigens. These antigens are presently considered as targets for the development of cancer immunotherapy protocols. In the present study, we developed a monoclonal antibody found to recognize SSX2, SSX3, and SSX4 proteins expressed in formaldehyde-fixed and paraffin-embedded tissues. This antibody was used to investigate SSX expression in normal testis and thyroid, benign melanocytic lesions, melanoma lesions, and melanoma cell lines. SSX nuclear expression in the testis was found to be restricted to spermatogenic cells, mainly spermatogonia. Of 18 melanoma cell lines analyzed, 9 showed SSX RNA and protein expression, although heterogeneously and at variable levels. Treatment of an SSX-negative cell line with 5-aza-2'-deoxycytidine, a demethylating agent, led to SSX RNA and protein expression, indicating a role for methylation in transcription regulation. Thirty-four of 101 primary and metastatic melanoma cases and 2 of 24 common nevocellular and atypical nevus cases showed SSX nuclear staining. Again, SSX expression was heterogeneous, ranging from widespread to scarce. Our findings stress the importance of assessing the a priori SSX expression status of melanoma cases that may be selected for immunotherapeutic trials.

Adult↗
PubMed2000

[Overall genomic DNA methylation in relation to estrogen].

Overall genomic DNA methylation was analyzed using enzymatic digestion into nucleotides, 32P postlabeling, two-dimensional thin-layer chromatography on cellulose plates and phosphobioimaging quantitation, in relation to immunohistochemically measured estrogen (ER) and progesterone receptor (PR) status of 15 uterine cancers. Mean 5-methyldeoxycytosine (m5dC) content did not differ between ER-positive and ER-negative neoplasms. Highest values of m5dC were noted both in ER-negative and ER-positive tumors. Additionally, there was no low DNA methylation in ER negative uterine cancer tissues. Decrease of the overall genomic DNA methylation could be related to the increase of ER/PR ratio, however it was not significant in our investigation. The potential role of steroid receptors status in uterine cancer tissue is discussed.

Aged↗
PubMed2001

DNA methyltransferase inhibition enhances apoptosis induced by histone deacetylase inhibitors.

Histone acetylation has long been associated with transcriptional activation, whereas conversely, deacetylation of histones is associated with gene silencing and transcriptional repression. Here we report that inhibitors of histone deacetylase (HDAC), depsipeptide and trichostatin A, induce apoptotic cell death in human lung cancer cells as demonstrated by DNA flow cytometry and Western immunoblot to detect cleavage of poly(ADP-ribose) polymerase. This HDAC inhibitorinduced apoptosis is greatly enhanced in the presence of the DNA methyltransferase inhibitor, 5-aza-2'-deoxycytidine (DAC). The HDAC inhibitor-induced apoptosis appears to be p53 independent, because no change in apoptotic cell death was observed in H1299 cells that expressed exogenous wild-type p53 (H1299 cells express no endogenous p53 protein). To further investigate the mechanism of DAC-enhanced, HDAC inhibitor-induced apoptosis, we analyzed histone H3 and H4 acetylation by Western immunoblotting. Results showed that depsipeptide induced a dose-dependent acetylation of histones H3 and H4, which was greatly increased in DAC-pretreated cells. By analyzing the acetylation of specific lysine residues at the amino terminus of histone H4 (Ac-5, Ac-8, Ac-12, and Ac-16), we found that the enhancement of HDAC inhibitor-induced acetylation of histones in the DAC-pretreated cells was not lysine site specific. These results demonstrate that DNA methylation status is an important determinant of apoptotic susceptibility to HDAC inhibitors.

Acetylation↗
PubMed2001

Activation of the p53 DNA damage response pathway after inhibition of DNA methyltransferase by 5-aza-2'-deoxycytidine.

Transcriptional silencing of tumor suppressor genes by DNA methylation occurs in cancer cell lines and in human tumors. This has led to the pursuit of DNA methyltransferase inhibition as a drug target. 5-Aza-2'-deoxycytidine [5-aza-CdR (decitabine)], a potent inhibitor of DNA methyltransferase, is a drug currently in clinical trials for the treatment of solid tumors and leukemia. The efficacy of 5-aza-CdR may be related to the induction of methylation-silenced tumor suppressor genes, genomic hypomethylation, and/or enzyme-DNA adduct formation. Here, we test the hypothesis that 5-aza-CdR treatment is perceived as DNA damage, as assessed by the activation of the tumor suppressor p53. We show that 1) colon tumor cell lines expressing wild-type p53 are more sensitive to 5-aza-CdR mediated growth arrest and cytotoxicity; 2) the response to 5-aza-CdR treatment includes the induction and activation of wild-type but not mutant p53 protein; and 3) the induction of the downstream p53 target gene p21 is partially p53-dependent. The induction of p53 protein after 5-aza-CdR treatment did not correlate with an increase in p53 transcripts, indicating that hypomethylation at the p53 promoter does not account for the p53 response. It is relevant that 5-aza-CdR has shown the greatest promise in clinical trials for the treatment of chronic myelogenous leukemia, a malignancy in which functional p53 is often retained. Our data raise the hypothesis that p53 activation may contribute to the clinical efficacy and/or toxicity of 5-aza-CdR.

Antimetabolites, Antineoplastic↗
PubMed2001

Reversal of GSTP1 CpG island hypermethylation and reactivation of pi-class glutathione S-transferase (GSTP1) expression in human prostate cancer cells by treatment with procainamide.

Among the many somatic genome alterations present in cancer cells, changes in DNA methylation may represent reversible "epigenetic" lesions, rather than irreversible "genetic" alterations. Cancer cell DNA is typically characterized by increases in the methylation of CpG dinucleotides clustered into CpG islands, near the transcriptional regulatory regions of critical genes, and by an overall reduction in CpG dinucleotide methylation. The transcriptional "silencing" of gene expression associated with such CpG island DNA hypermethylation presents an attractive therapeutic target: restoration of "silenced" gene expression may be possible via therapeutic reversal of CpG island hypermethylation. 5-Aza-cytidine (5-aza-C) and 5-aza-deoxycytidine (5-aza-dC), nucleoside analogue inhibitors of DNA methyltransferases, have been widely used in attempts to reverse abnormal DNA hypermethylation in cancer cells and restore "silenced" gene expression. However, clinical utility of the nucleoside analogue DNA methyltransferase inhibitors has been limited somewhat by myelosuppression and other side effects. Many of these side effects are characteristic of nucleoside analogues that are not DNA methyltransferase inhibitors, offering the possibility that nonnucleoside analogue DNA methyltransferase inhibitors might not possess such side effects. Human prostate cancer (PCA) cells characteristically contain hypermethylated CpG island sequences encompassing the transcriptional regulatory region of GSTP1, the gene encoding the pi-class glutathione S-transferase (GSTP1), and fail to express GSTP1 as a consequence of transcriptional "silencing." Inactivation of GSTP1 by CpG island hypermethylation, the most common somatic genome alteration yet reported for human PCAs, occurs early during human prostatic carcinogenesis and results in a loss of GSTP1 "caretaker" function, leaving prostate cells with inadequate defenses against oxidant and electrophile carcinogens. We report here that the drug procainamide, a nonnucleoside inhibitor of DNA methyltransferases, reversed GSTP1 CpG island hypermethylation and restored GSTP1 expression in LNCaP human PCA cells propagated in vitro or in vivo as xenograft tumors in athymic nude mice.

Animals↗