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

Christman, Judith K.

doi:10.1038/sj.onc.1205699

Cited by 1,269 publications

(1,021 citation statements)

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“…Consistent with our previous microarray study (Karpf et al, 2004), we observed that treatment with DAC, a classical DNMT inhibitor (Christman, 2002), elicited robust dose-dependent induction of CG-X antigen genes in the colorectal cancer cell lines HCT116 and RKO (Supplemental Figure 1a-c). Based on previous reports (Cameron et al, 1999;Weiser et al, 2001), we investigated whether treatment with the histone deacetylase (HDAC) inhibitor Trichostatin A (TSA) enhances the ability of DAC to activate CG-X antigen gene expression.…”

Section: Activation Of Cg-x Antigen Gene Expression By Dac Treatmentsupporting

confidence: 91%

Epigenetic regulation of X-linked cancer/germline antigen genes by DNMT1 and DNMT3b

2006

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We examined the function of two key DNA methyltransferase (DNMT) enzymes in epigenetic regulation of Xlinked cancer/germline (CG-X) antigen genes in human cancer cells, using MAGE-A1, NY-ESO-1, and XAGE-1 as models. In HCT116 cells, genetic knockout of DNMT1 caused moderate activation of CG-X genes, DNMT3b knockout had a negligible effect, and double knockout of both enzymes caused robust gene induction. Similarly, dual DNMT knockout caused dramatic hypomethylation of the MAGE-A1 and NY-ESO-1 promoters, DNMT1 knockout showed moderate hypomethylation, and DNMT3b knockout elicited only slight methylation changes. In contrast, both single and double knockout cells showed significant hypomethylation of the XAGE-1 promoter. RNA interference (RNAi) targeting of DNMT1 in HCT116 cells validated the results seen using genetic knockout cells; however, RNAi targeting of DNMT1 in a different colorectal cancer cell line revealed a greater independent role for DNMT1 in mediating CG-X gene repression and promoter methylation in other cell types. Notably, the histone H3 modification pattern at CG-X promoters was altered following DNMT knockout. DNMT1 or DNMT3b knockout reduced dimethylated lysine-9 (diMe-H3K9) levels, but did not significantly affect dimethylated lysine-4 (diMe-H3K4) or acetylated lysine-9 (Ac-H3-K9) levels. In contrast, dual DNMT1/3b knockout reduced the level of diMe-H3K9 and dramatically increased the levels of diMe-H3K4 and Ac-H3K9 at CG-X gene loci. In summary, DNMT1 and DNMT3b were found to perform both redundant and independent functions in epigenetic regulation of CG-X antigen genes in human cancer cells.

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Section: Activation Of Cg-x Antigen Gene Expression By Dac Treatmentsupporting

confidence: 91%

Epigenetic regulation of X-linked cancer/germline antigen genes by DNMT1 and DNMT3b

2006

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“…10,11 In addition, 5-Aza-CdR's ability to bind DNMTs has been attributed to its cytotoxic activity as a methylation-independent function. 12 The cellular events induced by 5-Aza-CdR that occur downstream of DNA methylation inhibition and DNA adduct formation are less well characterized. We therefore examined the DNA methylation inhibitory effect of 5-Aza-CdR with regard to its ability to induce apoptosis.…”

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confidence: 99%

5‐Aza‐2′‐deoxycytidine induces p21^WAF expression by demethylation of p73 leading to p53‐independent apoptosis in myeloid leukemia

Schmelz

Wagner

Dörken

et al. 2005

Intl Journal of Cancer

106

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The DNA methylation inhibitor 5-Aza-2 -deoxycytidine (5-AzaCdR) has significant therapeutic value for the treatment of patients with myelodysplastic syndrome (MDS), acute myeloid leukemia (AML) and chronic myeloid leukemia (CML). The demethylating effect of 5-Aza-CdR has been well characterized. In contrast, less is known about the molecular events downstream of the methylation inhibition. Here, 5-Aza-CdR induced apoptosis in AML cells (both p53 mutant and wild-type) but not in epithelial or normal PBMCs. Cell death was accompanied by activation of the mitochondrial apoptosis pathway, as shown by release of cytochrome c and AIF and loss of mitochondrial membrane potential (⌬⌿m). Activation of caspase-3 (but not ؊6 and ؊8) was detectable using Western blot analysis and measurement of caspase enzymatic activity. 5-Aza-CdR treatment resulted in the induction of p21, which correlated with the arrest of AML cells in the G 1 cell cycle phase. Induction of p21 expression was independent of its promoter methylation status but mediated by 5-Aza-CdR-induced reexpression of the tumor-suppressor p73, a known upstream regulator of p21. The p73 promoter was hypermethylated in AML cell lines and in primary AML cells but not in epithelial cells, which were resistant toward 5-Aza-CdR. Therefore, 5-Aza-CdRmediated specific killing of myeloid cells might be dependent on its ability to revert p73 promoter methylation and to reexpress p73 mRNA. In addition, exogenous expression of p73 rendered epithelial cells sensitive to apoptosis induced by 5-Aza-CdR or other cytostatic drugs. We therefore conclude that p73 is a relevant target for methylation-dependent efficacy of 5-Aza-CdR in AML cells.Key words: 5-aza-2Ј-deoxycytidine; p21 WAF ; p73; p53; myeloid leukemia; DNA methylation DNA methylation is an epigenetic mechanism of gene regulation that plays a crucial role in carcinogenesis due to silencing of cancer-related genes. 1 Transcriptional blockage occurs as a consequence of methylated CpG islands in the 5Ј region of genes, which is mediated by DNMTs during DNA replication. 2 In contrast to gene mutation, DNA methylation is a reversible event open to attack by DNA methylation inhibitors like 5-Aza-CdR. As a nucleoside analogue, 5-Aza-CdR is integrated into DNA and inhibits DNA methylation by trapping DNMTs. While other methylation inhibitors like zebularine are known, most data are available for 5-Aza-CdR in terms of its clinical utility. Two phase II clinical studies have reported promising therapeutic activity of 5-Aza-CdR (decitabine) for patients with MDS 3 or CML. 4 Two other studies, enrolling mostly patients with AML, reported good response rates after decitabine treatment. 5,6 5-Aza-CdR effectively reverts methylation-dependent gene repression, as shown, e.g., for p15 INK4b in vitro and in vivo. 6 In AML cells, promoter hypermethylation has been reported for several other genes, including the estrogen receptor, E-cadherin 7 HIC1, calcitonin 8 and MYOD. 9 The molecular effects of 5-Aza-CdR can include histone deacetylation a...

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“…Another possibility is that 5-aza-dC might activate upstream genes that are responsible for the activation of POU2F3, and the methylation status of the POU2F3 CpG island is not directly linked to the expression of this gene. The treatment of 5-aza-dC causes a variety of changes in cells, including the decondensation of chromatin and global genomic hypomethylation (Christman, 2002;Suzuki et al, 2002). Alternatively, the pattern of the DNA demethylation after 5-aza-dC treatment suggests that DNA methylation of only specific CpG is linked with expression.…”

Section: Discussionmentioning

confidence: 99%

Aberrant promoter methylation and silencing of the POU2F3 gene in cervical cancer

et al. 2006

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POU2F3 (OCT11, Skn-1a) is a keratinocyte-specific POU transcription factor whose expression is tied to squamous epithelial stratification. It is also a candidate tumor suppressor gene in cervical cancer (CC) because it lies in a critical loss of heterozygosity region on11q23.3 in that cancer, and its expression is lost in more than 50% of CC tumors and cell lines. We now report that the loss of POU2F3 expression is tied to the hypermethylation of CpG islands in the POU2F3 promoter. Bisulfite sequencing analysis revealed that methylation of specific CpG sites (À287 to À70 bp) correlated with POU2F3 expression, which could be reactivated with a demethylating agent. Combined bisulfite restriction analysis revealed aberrant methylation of the POU2F3 promoter in 18 of 46 (39%) cervical tumors but never in normal epithelium. POU2F3 expression was downregulated and inversely correlated with promoter hypermethylation in 10 out of 11 CC cell lines. Immunohistochemical analysis on a cervical tissue microarray detected POU2F3 protein in the epithelium above the basal layer. As the disease progressed, expression also decreased, especially in invasive squamous cell cancer (70% loss). Thus, aberrant DNA methylation of the CpG island in POU2F3 promoter appears to play a key role in silencing this gene expression in human CC. The results suggested that POU2F3 might be one of the CC-related tumor suppressor genes, which are disrupted by both epigenetic and genetic mechanisms.

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

Cited by 1,269 publications

References 118 publications

Epigenetic regulation of X-linked cancer/germline antigen genes by DNMT1 and DNMT3b

Epigenetic regulation of X-linked cancer/germline antigen genes by DNMT1 and DNMT3b

5‐Aza‐2′‐deoxycytidine induces p21^WAF expression by demethylation of p73 leading to p53‐independent apoptosis in myeloid leukemia

Aberrant promoter methylation and silencing of the POU2F3 gene in cervical cancer

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

Cited by 1,269 publications

References 118 publications

Epigenetic regulation of X-linked cancer/germline antigen genes by DNMT1 and DNMT3b

Epigenetic regulation of X-linked cancer/germline antigen genes by DNMT1 and DNMT3b

5‐Aza‐2′‐deoxycytidine induces p21WAF expression by demethylation of p73 leading to p53‐independent apoptosis in myeloid leukemia

Aberrant promoter methylation and silencing of the POU2F3 gene in cervical cancer

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5‐Aza‐2′‐deoxycytidine induces p21^WAF expression by demethylation of p73 leading to p53‐independent apoptosis in myeloid leukemia