title: Arsenic trioxide inhibits DNA methyltransferase and restores expression of methylation-silenced CDKN2B/CDKN2A genes in human hematologic malignant cells.
abstract: Cyclin-dependent kinase inhibitors CDKN2B and CDKN2A are tumor suppressor genes that are frequently dysregulated in a variety of cancers. Aberrant regulation via DNA hypermethylation causes gene silencing. Arsenic trioxide has been successfully used to treat malignant, hematopoietic diseases and is known to act by induction of apoptosis and inhibition of cellular proliferation. However, arsenic trioxide has been recently reported to act via inhibition of DNA hypermethylation in some solid tumors. The goal of this study was to explore the mechanism of arsenic trioxide induced demethylation of the CDKN2B and CDKN2A promoters in the hematologic malignant cell lines Molt4, MUTZ-1, U937, U266 and CA46. We used bisulphate modification and nested-methylation specific PCR to determine the levels of methylated and unmethylated promoter sequences in untreated and As2O3-treated cells. We used semi-quantitative RT-PCR and immunoblotting to quantify CDKN2B and CDKN2A mRNA and protein levels, respectively. We measured DNMT activity in nuclear extracts of untreated and treated cells using radiolabeled SAM as a methyl donor. The CDKN2B promoter was hypermethylated in Molt4 and MUTZ-1 cells, while the CDKN2A promoter was hypermethylated in U937, U266 and CA46 cells. As2O3 treatment caused demethylation associated with an increase in mRNA levels of the CDKN2B and CDKN2A genes. We also demonstrated a concomitant inhibition in DNMT activity and DNMT mRNA levels in As2O3-treated cells. In summary, As2O3 restored expression levels of tumor suppressor genes in hematologic malignant cells by causing promoter demethylation along with an inhibition of DNMTs 1, 3a and 3b.
authors.affiliation: Fujian Institute of Hematology, Union Hospital, Fujian Medical University, Fuzhou, Fujian 350001, PR China.
authors.forename: Hai-Ying
authors.initials: HY
authors.lastname: Fu
authors.forename: Jian-Zhen
authors.initials: JZ
authors.lastname: Shen
authors.forename: Yong
authors.initials: Y
authors.lastname: Wu
authors.forename: Song-Fei
authors.initials: SF
authors.lastname: Shen
authors.forename: Hua-Rong
authors.initials: HR
authors.lastname: Zhou
authors.forename: Li-Ping
authors.initials: LP
authors.lastname: Fan
mesh_terms: D000970:Antineoplastic Agents / Q000494:pharmacology; D000077237:Arsenic Trioxide; D001152:Arsenicals / Q000494:pharmacology*; D050762:Cyclin-Dependent Kinase Inhibitor p15 / Q000235:genetics* / Q000378:metabolism; D019175:DNA Methylation / Q000187:drug effects*; D015254:DNA Modification Methylases / Q000037:antagonists & inhibitors* / Q000378:metabolism; D004353:Drug Evaluation, Preclinical; D004791:Enzyme Inhibitors / Q000494:pharmacology; D015972:Gene Expression Regulation, Neoplastic / Q000187:drug effects; D020868:Gene Silencing / Q000187:drug effects; D019942:Genes, p16 / Q000187:drug effects*; D019337:Hematologic Neoplasms / Q000235:genetics* / Q000473:pathology; D006801:Humans; D010087:Oxides / Q000494:pharmacology*; D011401:Promoter Regions, Genetic / Q000187:drug effects; D014407:Tumor Cells, Cultured; D020298:U937 Cells
chemical_list: D000970:Antineoplastic Agents; D001152:Arsenicals; C493966:CDKN2B protein, human; D050762:Cyclin-Dependent Kinase Inhibitor p15; D004791:Enzyme Inhibitors; D010087:Oxides; D015254:DNA Modification Methylases; D000077237:Arsenic Trioxide