RETRACTION: 5-aza-Cytidine Is a Potent Inhibitor of DNA Methyltransferase 3a and Induces Apoptosis in HCT-116 Colon Cancer Cells via Gadd45- and p53-Dependent Mechanisms

Abstract

Methyltransferase inhibitors commonly used in clinical trials promote tumor cell death, but their detailed cytotoxic action is not yet fully understood. A deeper knowledge about their apotosis-inducing mechanisms and their interaction with DNA methyltransferases (DNMTs) DNMT1, DNMT3a, and DNMT3b might allow the design of more effective drugs with lower cytotoxicity. 5-aza-cytidine (5-aza-CR), a potent inhibitor of DNMT1, is known to induce demethylation and reactivation of silenced genes. In this study, we investigated the p53 dependence of apoptotic, cell cycle, and growth inhibitory effects of 5-aza-CR, as well as the influence on the expression level of DNMT1, DNMT3a, and DNMT3b in the colon cancer cell line HCT-116. Exposure to 5-aza-CR induced the up-regulation of genes promoting cell cycle arrest and DNA repair (p21 WAF1 and GADD45) or apoptosis (p53, RIPK2, Bak1, caspase 5, and caspase 6). In parallel, there was a down-regulation of antiapoptotic Bcl2 protein and the G 2 /M-mediator cyclin B1. Coincubation with pifithrin-alpha (PFT-␣), a selective p53 inhibitor, restored GADD45, Bcl2, cyclin B1, and p21 WAF1 expression levels and almost completely reversed the growth inhibitory, cell cycle, and apoptotic effects of 5-aza-CR. 5-aza-CR treatment caused global demethylation and reactivation of p16 expression. There was a marked decrease in DNMT1 and DNMT3a mRNA expression, with PFT-␣ reversing these effects. However, 5-aza-CR treatment did not modulate DNMT3b expression. Our data demonstrate that 5-aza-CR action in HCT-116 is mediated by p53 and its downstream effectors p21 WAF1 and GADD45. This is the first report to show a link between p53 and regulation of DNMT1 and de novo methyltransferase DNMT3a.

Association of the DNMT3A −448A>G polymorphism with genetic susceptibility to colorectal cancer

“…Given that the HIF-1α SNP C1772T (9), the DNA repair gene XRCC3 (10) SNP and the E-cadherin gene SNP -347 G→GA (11) and -160 C→A (12) are associated with the genetic susceptibility to CRC, DNMT3A is responsible for DNA methylation as a de novo methyltransferase in the tumorigenesis of numerous types of cancer, including CRC. Previous studies have suggested that ectopic DNMT3A expression plays a significant role in CRC progression (13,14). The restriction of Dnmt3a overexpression showed hypermethylation-mediated transcriptional silencing of the Wnt antagonist Sfrp5, and to a lesser extent, Sfrp1 (4).…”

Global analysis of DNA methylation and transcription of human repetitive sequences.

“…In addition, 5-azadC was also demonstrated to be a potent inhibitor of DNMT1 and DNMT3a, but not of DNMT3b, expression in HCT116. 58 It remains thus possible that the de novo methyltransferases of DNMT3 family proteins function redundantly to maintain the methylation state.…”

Growth inhibition of human cancer cells by 5-aza-2′-deoxycytidine does not correlate with its effects on INK4a/ARF expression or initial promoter methylation status

“…Yet, although it is possible that the specific cell system (and associated variations in integrity of p53-dependent apoptosis; refs. [29][30][31] contribute to the confusion in the literature, our findings raise alternative hypotheses relevant to more rational future use of hypomethylating agents.…”