Down-regulation of Human DNA-(Cytosine-5) Methyltransferase Induces Cell Cycle Regulators p16  and p21WAF/Cip1 by Distinct Mechanisms

Fournel, Marielle; Sapieha, Przemysław; Beaulieu, Norman; Jm, Besterman; MacLeod, A. Robert

doi:10.1074/jbc.274.34.24250

Cited by 109 publications

(92 citation statements)

References 43 publications

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“…33 In addition, reduction of DNMTase level by antisense oligonucleotides could increase p21 protein independently of the methylation status of p21/ CDKN1A in cancer cell lines. 34,35 The lack of p21 promoter methylation led us to postulate p73 as a target of 5-Aza-CdR-mediated demethylation in AML. We found time-and dose-dependent induction of p73 mRNA by 5-Aza-CdR in AML cells.…”

Section: Discussionmentioning

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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Section: Discussionmentioning

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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“…In particular, DNMT1 activity is associated with the transformed state (Wu et al, 1993;Bakin and Curran, 1999), but its relation to tumor progression is unclear. On one hand, antisense DNMT1 treatment of tumor cell lines with methylated INK4A resulted in demethylation of the locus, re-expression of p16, and cessation of cell growth (Fournel et al, 1999). On the other hand, homozygous deletion of DNMT1 in the diploid human colon cancer cell line HCT116 had no effect on the status of a methylated and silenced wildtype p16 INK4A allele (Rhee et al, 2000).…”

Section: Introductionmentioning

confidence: 99%

“…In fact, pharmacological or antisense-induced demethylation in experimental systems results in re-expression of tumor suppressor genes and restoration of growth control Otterson et al, 1995;Fournel et al, 1999). Thus, a thorough understanding of the mechanisms of regulated DNA methylation would reveal likely targets for antineoplastic agents that could inhibit these processes and reinstate tumor suppressor gene expression.…”

Section: Introductionmentioning

confidence: 99%

DNA methyltransferase 3b contributes to oncogenic transformation induced by SV40T antigen and activated Ras

2003

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Transcriptional silencing of tumor suppressor genes in association with DNA methylation contributes to malignant transformation. However, the specific DNA methyltransferases that initiate this process are unknown. Here we show that a de novo DNA methyltransferase, DNMT3b, substantially contributes to the oncogenic phenotype in a lung cancer model. Normal human bronchial epithelial (NHBE) cells expressing telomerase, SV40 large T antigen, and activated Ras were immortal, formed colonies in soft agar, and expressed DNMT3b. Antisense suppression of DNMT3b prevented soft agar growth. Furthermore, mouse embryo fibroblasts expressing T antigen and Ras formed soft agar colonies and large tumors, but fibroblasts from Dnmt3b À/À mice did not grow in soft agar and were much less tumorigenic in vivo. The tumor suppressor genes, FHIT, TSLC1, and RASSF1A were downregulated in transformed NHBE cells, and antisense DNMT3b treatment resulted in reexpression of FHIT and TSLC1. While expression of TSCL1 correlated with methylation of CpG dinucleotides in its promoter region, the expression of FHIT did not, suggesting that DNMT3b may silence genes by several mechanisms including direct DNA methylation or recruitment of proteins that modify chromatin. Regardless of mechanism, our data indicate that DNMT3b plays an important role in transformation.

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“…More recently, two alternative inhibitors of DNMT1 have been described (Bigey et al, 1999;Fournel et al, 1999). These inhibitors are of interest because they circumvent a number of the potential drawbacks associated with 5-aza-CdR treatment, including the requirement for DNA incorporation, the formation of toxic protein-DNA adducts, and the lack of selectivity for the different DNA methyltransferase enzymes.…”

Section: Reactivating Gene Expression: the Presentmentioning

confidence: 99%

“…These inhibitors are of interest because they circumvent a number of the potential drawbacks associated with 5-aza-CdR treatment, including the requirement for DNA incorporation, the formation of toxic protein-DNA adducts, and the lack of selectivity for the different DNA methyltransferase enzymes. The first of these inhibitors are antisense oligonucleotides directed against the DNMT1 mRNA (Fournel et al, 1999). Treatment of cells with DNMT1 antisense oligonucleotides leads to the loss of DNMT1 protein, demethylation of the promoter of the tumor suppressor gene p16, and expression of the p16 mRNA (Fournel et al, 1999).…”

Section: Reactivating Gene Expression: the Presentmentioning

confidence: 99%

Reactivating the expression of methylation silenced genes in human cancer

2002

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Down-regulation of Human DNA-(Cytosine-5) Methyltransferase Induces Cell Cycle Regulators p16 and p21WAF/Cip1 by Distinct Mechanisms

Cited by 109 publications

References 43 publications

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

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

DNA methyltransferase 3b contributes to oncogenic transformation induced by SV40T antigen and activated Ras

Reactivating the expression of methylation silenced genes in human cancer

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Down-regulation of Human DNA-(Cytosine-5) Methyltransferase Induces Cell Cycle Regulators p16 and p21WAF/Cip1 by Distinct Mechanisms

Cited by 109 publications

References 43 publications

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

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

DNA methyltransferase 3b contributes to oncogenic transformation induced by SV40T antigen and activated Ras

Reactivating the expression of methylation silenced genes in human 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

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