5-aza-2'-deoxycytidine therapy in patients with acute leukemia inhibits DNA methylation

The effect of 5-aza-2'-deoxycytidine (5-AZA-dCyd) on methylation of DNA in blood leukemic cells from patients with lymphoid and myeloid leukemia was investigated. After treatment with continuous infusion of 5-AZA-dCyd (1.0 mg/kg/h) the blood leukemic cells were isolated and incubated in vitro with [6-3H]deoxycytidine and the incorporation of radioactivity into 5-methylcytosine and cytosine of DNA determined. 5-AZA-dCyd produced greater than 70% inhibition of DNA methylation in the leukemic cells. The antileukemic action of 5-AZA-dCyd may be related to the inhibition of DNA methylation.     

DNA methylation changes after 5-aza-2'-deoxycytidine therapy in patients with leukemia

5-Aza-2'-deoxycytidine (decitabine) is postulated to have clinical activity in myeloid leukemias via its ability to inhibit DNA methylation. To study this, we examined DNA methylation in patients with leukemia treated with decitabine. Five days after the treatment, total genomic 5-methylcytosine/cytosine decreased on average by 14% (from 4.3% to 3.7%), whereas methylation of repetitive DNA elements showed a mean decrease of 9% and 16% for Alu and long interspersed nucleotide elements, respectively. Methylation decreased linearly with increasing doses between 5 and 20 mg/m(2)/d (r = 0.88; P = 0.05) but showed a plateau above that. Hypomethylation correlated with response in patients with acute myelogenous leukemia treated with low doses (5-20 mg/m(2)/d), but patients with chronic myelogenous leukemia treated with high doses (100-180 mg/m(2)/d) showed no such correlation. Aberrant methylation of p15 (>10%) was found in 27% of patients, and 80% of these showed a decrease by at least one third, but this did not correlate with response. The imprinted gene H19 showed little change in methylation after decitabine. In conclusion, we show dose-dependent hypomethylation after decitabine at low doses. Increasing the dose, which has been shown previously to result in a reduced response rate, was not accompanied by further hypomethylation.     

De novo CpG island methylation in human cancer cells

A major obstacle toward understanding how patterns of abnormal mammalian cytosine DNA methylation are established is the difficulty in quantitating the de novo methylation activities of DNA methyltransferases (DNMT) thought to catalyze these reactions. Here, we describe a novel method, using native human CpG island substrates from genes that frequently become hypermethylated in cancer, which generates robust activity for measuring de novo CpG methylation. We then survey colon cancer cells with genetically engineered deficiencies in different DNMTs and find that the major activity against these substrates in extracts of these cells is DNMT1, with minor contribution from DNMT 3b and none from DNMT3a, the only known bona fide de novo methyltransferases. The activity of DNMT1 against unmethylated CpG rich DNA was further tested by introducing CpG island substrates and DNMT1 into Drosophila melanogaster cells. The exogenous DNMT1 methylates the integrated mammalian CpG islands but not the Drosophila DNA. Additionally, in human cancer cells lacking DNMT1 and DNMT3b and having nearly absent genomic methylation, gene-specific de novo methylation can be initiated by reintroduction of DNMT1. Our studies provide a new assay for de novo activity of DNMTs and data suggesting a potential role for DNMT1 in the initiation of promoter CpG island hypermethylation in human cancer cells.     

5-Azacytidine and 5-aza-2'-deoxycytidine as inhibitors of DNA methylation: mechanistic studies and their implications for cancer therapy

5-Azacytidine was first synthesized almost 40 years ago. It was demonstrated to have a wide range of anti-metabolic activities when tested against cultured cancer cells and to be an effective chemotherapeutic agent for acute myelogenous leukemia. However, because of 5-azacytidine's general toxicity, other nucleoside analogs were favored as therapeutics. The finding that 5-azacytidine was incorporated into DNA and that, when present in DNA, it inhibited DNA methylation, led to widespread use of 5-azacytidine and 5-aza-2'-deoxycytidine (Decitabine) to demonstrate the correlation between loss of methylation in specific gene regions and activation of the associated genes. There is now a revived interest in the use of Decitabine as a therapeutic agent for cancers in which epigenetic silencing of critical regulatory genes has occurred. Here, the current status of our understanding of the mechanism(s) by which 5-azacytosine residues in DNA inhibit DNA methylation is reviewed with an emphasis on the interactions of these residues with bacterial and mammalian DNA (cytosine-C5) methyltransferases. The implications of these mechanistic studies for development of less toxic inhibitors of DNA methylation are discussed.     

Enhanced sensitivity of prostate cancer DU145 cells to cisplatinum by 5-aza-2'-deoxycytidine

During the oncopathogenic process aberrant DNA methylation frequently occurs, leading to silencing of sets of genes involved in cell cycle and apoptosis control pathways and other important biological functions. Targeting such a change has been suggested as a novel strategy for cancer prevention and therapy. In the present study, we examined whether suppression of DNA methylation was capable of enhancing sensitivity of prostate cancer DU145 cells to cisplatinum. 5-aza-2'-deoxycytidine (5-aza), a specific DNA methylation inhibitor, when added into DU145 cell culture alone, did not induce significant apoptosis. However, a combination of 5-aza with the chemotherapeutic agent cisplatinum showed great synergy in triggering apoptotic death of DU145 cells. The present finding provides a rationale to evaluate therapeutic effects of the DNA methylation inhibition and chemotherapy in patients with prostate cancer.     

Analysis of gene induction in human fibroblasts and bladder cancer cells exposed to the methylation inhibitor 5-aza-2'-deoxycytidine

Hypermethylation of the promoters of cancer-related genes is often associated with their inactivation during tumorigenesis. Several preclinical and clinical trials have been developed to use DNA methylation inhibitors, such as 5-aza-2'-deoxycytidine (5-Aza-CdR) in attempts to reactivate silenced genes in human cancers. We used high-density oligonucleotide gene expression microarrays to examine the effects of 5-Aza-CdR treatment on human fibroblast cells (LD419) and a human bladder tumor cell line (T24). Data obtained 8 days after recovery from 5-Aza-CdR treatment showed that more genes were induced in tumorigenic cells (61 genes induced; >or=4-fold) than nontumorigenic cells (34 genes induced; >or= 4-fold). Approximately 60% of induced genes did not have CpG islands within their 5' regions, suggesting that some genes activated by 5-Aza-CdR may not result from the direct inhibition of promoter methylation. Interestingly, a high percentage of genes activated in both cell types belonged to the IFN signaling pathway, confirming data from other tumor cell types.     

CpG island methylation is a common finding in colorectal cancer cell lines

Tumour cell lines are commonly used in colorectal cancer (CRC) research, including studies designed to assess methylation defects. Although many of the known genetic aberrations in CRC cell lines have been comprehensively described, no studies have been performed on their methylation status. In this study, 30 commonly used CRC cell lines as well as seven primary tumours from individuals with hereditary nonpolyposis colorectal cancer (HNPCC) were assessed for methylation at six CpG islands known to be hypermethylated in colorectal cancer: hMLH1, p16, methylated in tumour (MINT-)-1, -2, -12 and -31. The cell lines were also assessed for microsatellite instability (MSI), ploidy status, hMLH1 expression, and mutations in APC and Ki-ras. Methylation was frequently observed at all examined loci in most cell lines, and no differences were observed between germline-derived and sporadic cell lines. Methylation was found at MINT 1 in 63%, MINT 2 in 57%, MINT 12 in 71%, MINT 31 in 53%, p16 in 71%, and hMLH1 in 30% of cell lines. Overall only one cell line, SW1417, did not show methylation at any locus. Methylation was found with equal frequency in MSI and chromosomally unstable lines. MSI was over-represented in the cell lines relative to sporadic CRC, being detected in 47% of cell lines. The rate of codon 13 Ki-ras mutations was also over three times that expected from in vivo studies. We conclude that CpG island hypermethylation, whether acquired in vivo or in culture, is a ubiquitous phenomenon in CRC cell lines. We suggest that CRC cell lines may be only representative of a small subset of real tumours, and this should be taken into account in the use of CRC cell lines for epigenetic studies.     

Delivery of 5-aza-2'-deoxycytidine to cells using oligodeoxynucleotides

The major goal of epigenetic therapy is to reverse aberrant promoter hypermethylation and restore normal function of tumor suppressor genes by the use of chromatin-modifying drugs. Decitabine, or 5-aza-2'-deoxycytidine (5-aza-CdR), is a well-characterized drug that is now Food and Drug Administration approved for the treatment of myelodysplastic syndrome. Although 5-aza-CdR is an extremely potent inhibitor of DNA methylation, it is subject to degradation by hydrolytic cleavage and deamination by cytidine deaminase. We show that short oligonucleotides containing a 5-aza-CdR can also inhibit DNA methylation in cancer cells at concentrations comparable with 5-aza-CdR. Detailed studies with S110, a dinucleotide, showed that it works via a mechanism similar to that of 5-aza-CdR after incorporation of its aza-moiety into DNA. Stability of the triazine ring in aqueous solution was not improved in the S110 dinucleotide; however, deamination by cytidine deaminase was dramatically decreased. This is the first demonstration of the use of short oligonucleotides to provide effective delivery and cellular uptake of a nucleotide drug and protection from enzymatic degradation. This approach may pave the way for more stable and potent inhibitors of DNA methylation as well as provide means for improving existing therapeutics.     

DNA alkali-labile sites induced by incorporation of 5-aza-2'-deoxycytidine into DNA of mouse leukemia L1210 cells

The effects of 5-aza-2'-deoxycytidine on DNA in mouse L1210 leukemia cells were investigated using the alkaline elution technique. By comparing the DNA elution rate at pH 12.1 and 12.6, it was found that the drug produced DNA alkali-labile lesions. Alkali-labile sites were present only in DNA strands that were synthesized in the presence of the drug. They persisted for at least 48 h after drug treatment, and only after 72 h did the number of alkali-labile sites decline, thus suggesting a slow repair process. The production of alkali-labile sites was found to be concentration dependent and observable at concentrations which were effective in inhibiting the clonogenic viability of L1210 cells and which are attainable in vivo. 5-Aza-2'-deoxycytidine did not cause other DNA lesions such as DNA double-strand breaks or DNA-protein cross-links. Two hypotheses were considered to explain the origin of alkali-labile lesions in DNA that has incorporated 5-aza-2'-deoxycytidine: (a) the production of apyrimidinic sites by a glycosylase that recognizes and removes aza-cytosine from DNA and (b) the alkali-catalyzed decomposition of azacytosine residues to ring-opened products which could lead to alkali-induced DNA strand scission through a beta-elimination mechanism. The second hypothesis was considered to be the more probable and suggests that the alkali lability may be a means by which one could determine the extent of substitution and precise location of azacytosine residues or their ring-opened products in DNA.     

Differences in DNA damage produced by incorporation of 5-aza-2'-deoxycytidine or 5,6-dihydro-5-azacytidine into DNA of mammalian cells

The effects of 5-aza-2'-deoxycytidine (aza-dCyd) and 5,6-dihydro-5-azacytidine (H2-aza-Cyd) on the integrity of DNA from several mammalian cell lines were compared using the alkaline elution technique. While both compounds have been shown to inhibit DNA methylation, a direct comparison of their effects on DNA structure has not previously been reported. Exposure of L1210 cells to H2-aza-Cyd (1-100 micrograms/ml) and simultaneous labeling with [14C]thymidine for 24 h resulted in the production of single-strand breaks in DNA, which were significantly repaired when cells were incubated in drug-free medium for an additional 24 h. This differed from our previous findings for aza-dCyd, confirmed here in parallel experiments, which showed that this compound produces alkali-labile lesions that persist for 48 h. The DNA effects of both drugs were significantly reduced when cells were prelabeled with [14C]thymidine, indicating that production of DNA lesions requires incorporation of the anomalous base. Studies utilizing pulse-labeled DNA indicated that aza-dCyd has little effect on the rate of DNA elongation, whereas H2-aza-Cyd produced a complete inhibition for at least 6 h after drug removal. The contrasting pattern of DNA damage induced by these compounds in L1210 was also observed in two human lymphoblastoid cells lines, one of which was derived from a patient with xeroderma pigmentosum. We had previously concluded that alkali-labile sites in DNA from aza-dCyd-treated cells probably arise due to the chemical instability of aza-dCyd. In contrast, incorporated H2-aza-Cyd is chemically stable. The single-strand breaks produced in H2-aza-Cyd treated cells were not of the alkali-labile type, and may represent an accumulation of DNA replication fragments and/or intermediates in an excision repair process. Thus, the DNA lesions produced by the two drugs have markedly different characteristics, and H2-aza-Cyd should not be considered to be merely a stable pharmacological congener of aza-dCyd.     

5-aza-2'-deoxycytidine sensitizes hepatoma and pancreatic cancer cell lines

Hepatocellular carcinoma (HCC) and pancreatic cancer are at the forefront of chemotherapy-resistant tumors with poor prognosis. Even with innovative treatment regimens, response rates remain low and the duration of response is short. We examined whether the suppression of DNA methylation was capable of enhancing the sensitivity of hepatoma and pancreatic cancer cell lines to 5-fluorouracil (5-FU). 5-aza-2'-deoxycytidine (5-aza-dC) at 2 microM, a specific DNA methylation inhibitor, did not induce cell death in Huh-7 with or without HCV, HLE, HepG2 and MIA PaCa-2 cells. However, a combination of 5-aza-dC with 5-FU showed a reduction in cell viability and induction of apoptosis in these cell lines to a greater degree than with 5-FU only. These findings underline the fact that DNA methylation plays a key role in conferring chemoresistance to hepatoma and pancreatic cancer, and the combination of DNA methylation inhibitor with chemotherapy could be a novel and highly effective tool for future targeted therapy of chemoresistant tumors.     

胃癌E-cadherin基因启动子CpG岛甲基化的研究

背景与目的:目前认为CpG岛甲基化导致转录抑制是恶性肿瘤发生的重要机制之一。E-cadherin能抑制肿瘤细胞的浸润和转移,被公认为是浸润、转移抑制基因。低分化胃癌常表现有E-cadherin基因失活。我们通过检测胃癌及癌旁组织中E-cadherin基因启动子CpG岛甲基化水平,初步探讨胃癌的发病机制。方法:采用特异性甲基化PCR(MSP)法检测51例胃癌组织及37例癌旁组织中的E-cadherin基因启动子CpG岛甲基化水平,采用免疫组织化学染色法检测E-cadherin表达。结果:健康对照组织中未检测到CpG岛甲基化,4例(10.8%)癌旁组织检测到CpG岛甲基化,32例(62.7%)胃癌组织中检测到CpG岛甲基化。低分化腺癌(72.2%,26/36)CpG岛甲基化显著高于高分化腺癌(33.3%,6/15)(P<0.01),T1/T2期(55.6%,10/18)与T3/T4期胃癌(66.7%,22/33)的CpG岛甲基化率无显著性差异(P>0.05)。32例CpG岛甲基化胃癌中27例(84.5%)E-cadherin表达下调,19例非甲基化胃癌中5例(26.3%)E-cadherin表达下调。未发现E-cadherin基因CpG岛甲基化与幽门螺杆菌感染有关。结论:胃癌、尤其是低分化腺癌广泛存在E-cadherin基因启动子CpG岛甲基化,启动子CpG岛甲基化可能参与胃癌的早期过程,E-cadherin基因CpG岛甲基化与幽门螺杆菌感染无相关关系。     

Effects of Promoter Region 5＇CpG Island Demethylation on the Biological Behavior of Human Colorectal Cancer RKO Cells in Vitro

OBJECTIVE To explore the relationship between the methylation status of the promoter 5＇CpG island region and the biological behavior of human colorectal cancer RKO cells in vitro. METHODS RKO cells were treated with a selective DNA methyltransferase inhibitor-5-aza-2＇-deoxycytidine （5-aza-CdR） for 72 h. Methylationspecific PCR （MSP）, T-A cloning and DNA sequence analysis were used to determinate the 5＇CpG island methylation status of the p16/CDKN2 tumor suppressor gene. Cell growth, morphological changes and apoptosis were analyzed by the MTT assay, flow cytometry, fluorescence staining and electron microscopy. RESULTS The 5＇CpG island of the p16/CDKN2 tumor suppressor gene in RKO cells was a typically hypermethylated. The DNA methyltransferase inhibitor （5-Aza-CdR） effectively reversed the hypermethylation status of the promoter region. With demethylation, RKO cell growth was suppressed, the cells doubling times were prolonged （P〈0.01） and apoptosis was induced, which showed a relationship. CONCLUSION A selective DNA methyltransferase （DNMT） inhibitor can inhibit proliferation by demethylation in 5＇CpG islands, and may be a potential new therapy target for colorectal cancer.     

Epigenetic therapy of cancer with 5-aza-2'-deoxycytidine (decitabine)

Epigenetic events, such as aberrant DNA methylation, have been demonstrated to silence the expression of many genes that suppress malignancy. Since the event is reversible, it is an interesting target for intervention with specific inhibitors of DNA methylation, such as 5-aza-2'-deoxycytidine (5-AZA-CdR, decitabine). 5-AZA-CdR is a prodrug that requires activation via phosphorylation by deoxcytidine kinase. The nucleotide analog is incorporated into DNA, where it produces an irreversible inactivation of DNA methyltransferase. 5-AZA-CdR is an S-phase-specific agent. The demethylation of DNA by this analog in neoplastic cells can lead to the reactivation of silent tumor-suppressor genes, induction of differentiation or senescence, growth inhibition, and loss of clonogenicity. 5-AZA-CdR was demonstrated to be a potent antineoplastic agent against leukemia and tumors in animal models. Preliminary clinical trials of 5-AZA-CdR using different dose-schedules have shown interesting antineoplastic activity in patients with leukemia, myelodysplastic syndrome (MDS), and non-small cell lung cancer (NSCLC). Pharmacokinetic studies have shown that 5-AZA-CdR has a short in vivo half-life of 15 to 25 minutes. The major toxicity produced by this analog is granulocytopenia. To exploit the full chemotherapeutic potential of 5-AZA-CdR for the treatment of cancer, its optimal dose-schedule has to be found. This will require a good understanding of the pharmacology of this analog and its action on both normal and neoplastic cells.