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

Abstract

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...

p21WAF1/CIP1 induction by 5-azacytosine nucleosides requires DNA damage

“…However, in this study, treatment of ML-1 cells with DAC in vitro does not lead to detectable changes in histone acetylation neither globally nor at the promoter region of p21 WAF1/CIP1 as demonstrated by chromatin immunoprecipitation using acetylated H3 antibody (data not shown). p21 WAF1/CIP1 induction by DAC was also observed in p53-mutated leukemia cell lines and was dependent on reversal of methylation of the tumor suppressor p73 (Schmelz et al, 2005b;Tamm et al, 2005). However, in ML-1 cells, we could not detect p73 promoter methylation by MSP (data not shown).…”

The role of p73 in hematological malignancies

“…Although P73 hypermethylation is rare event in AML, 5-Aza-CdR occurred to induce p21 WAF1 expression via demethylation of P73, leading to p53-independent apoptosis in ex vivo AML cells. 76 Moreover, 5-Aza-CdR enhanced p73 and p15 expression, inhibiting growth and DNA synthesis in KG1a myeloid leukemic cell line in a dosedependent manner. 88 As presented in this review, it seems to be quite a well-documented rationale for the use of hypomethylating agents also in tumors derived from lymphoid cell lineage, including NHL, MG or ALL, which show a high rate of P73 hypermethylation.…”

“…These findings indicate that methylation of P73 gene is rare in AML, and this epigenetic mechanism has no impact on its pathogenesis. However, most recently, Schmelz et al 76 reported hypermethylation of P73 promoter in both AML cell lines and in primary AML cells, as a relevant target for methylation-dependent proapoptotic activity of 5-Aza-2 0 -deoxycytidine (5-Aza-CdR).…”

Nucleoside analogs: molecular mechanisms signaling cell death

“…In contrast, deacetylation of these residues is associated with a repressive state (Rice and Allis, 2001). Consequently, combinations of histone deacetylase inhibitors with hypomethylating agents results in reactivation of gene expression (Richon and O'Brien, 2002), cell cycle arrest and apoptosis (Zhu et al, 2001b;Tang et al, 2004;Schmelz et al, 2005;Walton et al, 2008). The azanucleosides analogs have also shown synergistic activity with conventional nucleoside analog chemotherapeutic agents such as 5-fluorouracil, which is based on their ability to reactivate previously silenced proapoptotic genes (Kanda et al, 2005;Morita et al, 2006).…”