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

Abstract

Decitabine (DAC) and 5-azacitidine have recently been approved for the treatment of myelodysplastic syndrome. The pharmacodynamic effects of DAC and 5-azacitidine outside their known activity as inhibitors of DNA methyltransferases (DNMTs) require further investigation. The purpose of this study was to investigate the effect of DAC on the expression of p21^WAF1/CIP1, a gene with a putative CpG island surrounding its promoter region. Promoter methylation analysis of p21^WAF1/CIP1 in leukemia cells revealed the absence of CpG methylation. However, DAC upregulated p21^WAF1/CIP1 expression in a dose-dependent manner (ED₅₀=103.34 nM) and induced G2/M cell cycle arrest in leukemia cells. Sequential application of DAC followed by different histone deacetylase inhibitors induced expression of p21^WAF1/CIP1 synergistically. Upregulation of p21^WAF1/CIP1 paralleled DAC-induced apoptosis (ED₅₀=153 nM). Low doses of DAC induced γ-H2AX expression (ED₅₀=16.5 nM) and upregulated p21^WAF1/CIP1 in congenic HCT 116 colon cancer cells in a DNMT-independent and p53-dependent fashion. Inhibition of p53 transactivation by pifithrin-α or the kinase activity of ATM by either the specific ATM inhibitor KU-5593 or caffeine abrogated p21^WAF1/CIP1 upregulation, indicating that DAC upregulation of p21^WAF1/CIP1 was p53- and ATM-dependent in leukemia cells. In conclusion, DAC upregulates p21^WAF1/CIP1 in DNMT-independent manner via the DNA damage/ATM/p53 axis.