DNA Methyltransferase Inhibition Induces the Transcription of the Tumor Suppressor p21

Abstract

Previous lines of evidence have shown that inhibition of DNA methyltransferase (MeTase) can arrest tumor cell growth; however, the mechanisms involved were not clear. In this manuscript we show that out of 16 known tumor suppressors and cell cycle regulators, the cyclin-dependent kinase inhibitor p21 is the only tumor suppressor induced in the human lung cancer cell line, A549, following inhibition of DNA MeTase by a novel DNA MeTase antagonist or antisense oligonucleotides. The rapid induction of p21 expression points to a mechanism that does not involve demethylation of p21 promoter. Consistent with this hypothesis, we show that part of the CpG island upstream of the endogenous p21 gene is unmethylated and that the expression of unmethylated p21 promoter luciferase reporter constructs is induced following inhibition of DNA MeTase. These results are consistent with the hypothesis that the level of DNA MeTase in a cell can control the expression of a nodal tumor suppressor by a mechanism that does not involve DNA methylation.DNA methylation of cytosine residues located at the dinucleotide sequence CpG can suppress expression of genes either by directly interfering with the binding of transcription factors (1) or by attracting methylated-DNA binding proteins such as MeCP2 (2-4). To ascertain that the epigenetic information encoded in the methylation pattern is faithfully maintained, the expression of the maintenance DNA MeTase 1 enzyme DNMT1, which catalyzes the transfer of a methyl group from S-adenosyl-methionine to the 5Ј position on cytosines residing at the dinucleotide CpG (5), is tightly coordinated with DNA replication (6) and the state of growth of the cell (7). Different protooncogenic pathways can up-regulate dnmt1 expression (8), and high levels of dnmt1 mRNA have been observed in many cancer cells (9, 10). It has been proposed that increased DNA MeTase levels are a downstream component of oncogenic programs (11) and that they play a causal role in cellular transformation (12, 13). This hypothesis has been supported by the observation that a reduction in DNA MeTase levels by either 5-aza-deoxycytidine (13, 14), DNA MeTase antisense mRNA or antisense oligonucleotides can reverse tumor growth in vivo and in vitro (13-15).The mechanisms responsible for cellular transformation by DNMT1 and the reversal of transformation by DNA MeTase inhibition are unknown. One attractive hypothesis is that high levels of DNA MeTase lead to ectopic methylation and inactivation of tumor suppressor genes such as p16 (16). Similarly, the inhibition of DNA MeTase could result in demethylation and lead to activation of p16 (17, 18). However, because both the increase in DNA MeTase levels and its inhibition by pharmacological inhibitors are global processes, it is difficult to understand how they could predictably result in a discrete change of the methylation state of specific sites. A more likely explanation is that ectopic methylation of tumor suppressors in tumor cells is a slow and stochastic process, but the aberrant meth...

Effects of demethylating agent 5-aza-2′-deoxycytidine and histone deacetylase inhibitor FR901228 on maspin gene expression in oral cancer cell lines

“…Kondo et al [20] have reported that 5Aza-dC dramatically decreases histone H3 Lys-9 methylation, slightly increases Lys-9 acetylation, and moderately increases histone H3 Lys-4 methylation and reactivated gene expression. These findings could explain the previously reported 5Aza-dC induced activation of the un-methylated gene [21,22] and could also explain the maspin activation in our results.…”

Induction of gene expression by 5-Aza-2′-deoxycytidine in acute myeloid leukemia (AML) and myelodysplastic syndrome (MDS) but not epithelial cells by DNA-methylation-dependent and -independent mechanisms

“…This methylation-independent effect of 5-Aza-CdR is in agreement with other studies, indicating that silenced genes with unmethylated CpG islands or without CpG islands can be induced by 5-Aza-CdR. 15,20,36,37 In addition, effects of 5-Aza-CdR upon histone methylation and RB phosphorylation have been recently described. 38,39 In contrast to this, we found DNA methylation in the 5 0 region of the MPO gene in AML cells.…”

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

“…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.…”