5-Aza-2′-deoxycytidine Activates the p53/p21Waf1/Cip1 Pathway to Inhibit Cell Proliferation

Abstract

In addition to its demethylating function, 5-aza-2-deoxycytidine (5-aza-CdR) also plays an important role in inducing cell cycle arrest, differentiation, and cell death. However, the mechanism by which 5-aza-CdR induces antineoplastic activity is not clear. In this study, we found that 5-aza-CdR at limited concentrations (0.01-5 M) induces inhibition of cell proliferation as well as increased p53/p21Waf1/Cip1 expression in A549 cells (wild-type p53) but not in H1299 (p53-null) and H719 cells (p53 mutant). The p53-dependent p21 As demethylating agents, 5-aza-cytidine and 5-aza-2Ј-deoxycytidine (5-aza-CdR) 1 have been extensively used for epigenetic research (1-4). Both demethylating agents are incorporated into DNA where they bind DNA methyltransferase (DNMT) in an irreversible, covalent manner, thus sequestering the enzyme and preventing maintenance of the methylation state (5-7). Consequently, silenced genes induced by hypermethylation are re-expressed after treatment with these demethylating agents.Originally, 5-aza-cytidine and 5-aza-CdR were developed as anticancer agents (5, 8) and have been shown to have significant cytotoxic and antineoplastic activities in many experimental tumors (9 -12). 5-Aza-CdR is reported to be noncarcinogenic and incorporates into DNA but not RNA or protein (13,14). 5-Aza-CdR has been found empirically to have more potent therapeutic effects than 5-aza-cytidine in cell culture and animal models of human cancers. However, 5-aza-CdR-induced cytotoxicity may not be linked to its demethylating function (3,(15)(16)(17). In addition, the therapeutic effects of 5-aza-CdR in the treatment of different human cancer cells are conflicting. 5-Aza-CdR appears to be beneficial in the treatment of human leukemias (9,18,19), myelodysplastic syndromes (20, 21), and hemoglobinopathies (22, 23). On the other hand, there has been less positive experience in the effectiveness of 5-aza-CdR for the treatment of human solid tumors (10, 24). Therefore, it is possible that one or more critical factors may be involved in regulating the cellular response to 5-aza-CdR treatment that vary in different cell types.p53 is a very important tumor suppressor gene and is reported to be abnormal in more than 50% of human cancers (25). Chemotherapeutic agents frequently act through the mechanism of DNA damage, and p53 plays an important role in the induction of cell cycle arrest and apoptosis in response to DNA damage (26). 5-Aza-CdR has also shown anticancer activity that may be related to its ability to induce DNA damage (15,27). Based on the scenario mentioned above, it is hypothesized that 5-aza-CdR may induce DNA damage, thereby activating p53, which in turn increases p21Waf1/Cip1 expression, leading to the inhibition of cell proliferation.To confirm the role of p53 in the 5-aza-CdR-induced inhibition of cell proliferation, human lung cancer cells with different p53 status were selected as the targets for this study. As an important downstream target of p53 activation, p21Waf1/Cip1 plays a critical role in inhibit...

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

“…The inhibition of p21 WAF1/CIP1 upregulation by Pifithrin-a and the lack of p21 WAF1/CIP1 upregulation in the p53-null HL-60 cells support a major role of the DNA damage/p53 axis in this effect, which is consistent with the effect of DAC on human lung cancer cells (Zhu et al, 2004). Also, p21 WAF1/CIP1 upregulation in HCT116 cells in a DNMT1-and DNMT3b-independent manner further supports the hypothesis of DNA damage-induced upregulation of p21 WAF1/CIP1 .…”

“…DAC induced p21 WAF1/CIP1 expression in other systems. DAC induction of p21 WAF1/CIP1 expression in lung and colon cancer cells was p53 dependent and not associated with chromatin remodeling events (Karpf et al, 2001;Zhu et al, 2004). On the other hand, chromatin immunoprecipitation experiments suggested that treatment of KG-1 and KG-1a leukemia cells with DAC was associated with an increase in H3 acetylation at the unmethylated p21 WAF1/CIP1 promoter (Scott et al, 2006).…”

Growth delay of human pancreatic cancer cells by methylase inhibitor 5-aza-2′-deoxycytidine treatment is associated with activation of the interferon signalling pathway

“…The tumour suppressor genes p16 INK4 and p53, which are critically involved in pancreatic cancer tumorigenesis, are also implicated in the response to 5-aza-CdR treatment (Bender et al, 1998;Suh et al, 2000;Nieto et al, 2004;Zhu et al, 2004). The analysis of the expression of p16 protein confirmed its upregulation after the treatment in both PaCa44 and CFPAC1.…”

Combinations of DNA Methyltransferase and Histone Deacetylase Inhibitors Induce DNA Damage in Small Cell Lung Cancer Cells: Correlation of Resistance with IFN-Stimulated Gene Expression

“…5-AZA-dC and HDAC inhibitors can reactivate aberrantly hypermethylated or acetylated genes, thereby inhibiting tumor cells by inducing apoptosis, cell cycle arrest, and differentiation (32)(33)(34)(35). In addition to its demethylating function, 5-AZA-dC may also play an antineoplastic role that is methylation independent (36,37).…”

“…Reduction of cell viability by both 5-AZA-dC and HDAC inhibitors could also result from their genotoxic effects (10,15,37,41,42). 5-AZA-dC is incorporated into DNA and covalently binds with DNMTs with the 5-AZA-DNMT-DNA adducts causing DNA damage (43).…”