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Acta haematologica

Arsenic trioxide inhibits DNA methyltransferase and restores TMS1 gene expression in K562 cells.


PMID 24993472

Abstract

Gene silencing associated with aberrant methylation of promoter region CpG islands is an acquired epigenetic alteration that serves as an alternative to genetic defects in the inactivation of tumor suppressor genes in human cancers. The demethylating, dose-dependent effect of arsenic trioxide (As2O3) on several tumor-related genes has already been postulated. However, whether such a demethylating effect also applies to the TMS1 gene in chronic myeloid leukemia cell line K562 cells has not been studied so far. The aim of the present study was to detect the methylation status of the TMS1 gene in K562 cells and the demethylation effect of As2O3 on TMS1 as well as TMS1 apoptosis-associated protein Bcl-2/Bax and DNA methyltransferase (DNMT) expression. TMS1 mRNA expression in K562 cells and normal bone marrow was determined by reverse transcription (RT) polymerase chain reaction (PCR), and the DNA methylation status of the TMS1 promoter in K562 cells treated with different concentrations of As2O3 for 48 h was determined by methylation-specific PCR. RT-PCR and Western blot were used to detect TMS1 and DNMT expression. We also assessed TMS1-associated apoptosis protein Bcl-2/Bax expression by Western blot and apoptosis rates by flow cytometry using annexin V/propidium iodide double staining. In K562 cells, TMS1 was completely methylated and both TMS1 mRNA and protein showed a low expression, but 2 μmol/l As2O3 could significantly restore the expression of the TMS1 gene both at mRNA and protein level (p < 0.01) by fully reversing DNA methylation. As2O3 decreased mRNA and protein expression of DNMT1 (p < 0.05) in a dose-dependent manner. Flow cytometry showed that in the experimental group (2 μmol/l As2O3), cell apoptosis was significantly increased compared with the control group (no As2O3; p < 0.05). In the experimental group, Western blot showed that the expression of the anti-apoptotic protein Bcl-2 was significantly decreased; however, the proapoptotic protein Bax was markedly increased and the Bcl-2/Bax ratio was markedly reduced (p < 0.01). As2O3 could restore the expression of TMS1 by inhibiting DNMT to reverse the hypermethylation and induced apoptosis of K562 cells by downregulation of Bcl-2/Bax expression.