title: [Arsenic trioxide reverses hypermethylation of p16 and activates its transcription in malignant lymphoma cell line CA46].
abstract: This study was aimed to investigate the reversing effect of arsenic trioxide (As2O3) on methylation status and the regulatory effect on transcription of malignant lymphoma cell line CA46 p16 gene as well as their possibe mechanisms. The hypermethylated malignant lymphoma cell line CA46 was used as a subject of experiment for studying relation of gene methylation with expression. The effect of As2O3 on the proliferation and viability of CA46 was detected by SRB method, the change of p16 methylation status after exposure to As2O3 was determined by nMSP, the expressions of p16, DNMT1, DNMT3A, DNMT3B mRNA were assayed by RT-PCR, the influence of As2O3 on CA46 cell cycle was analyzed by flow cytometry using analytical method for DNA ploidy. The results showed that the methylation level of p16 gene was obviously reduced after treatment with As2O3 for 72 hours and the hypermethylation of p16 gene was successfully reversed; the expression of p16 gene in untreated (control) group was low while it was enhanced in treated groups; the gray scale ratios of p16 gene to beta-actin in groups treated with As2O3 of concentration 0.5, 1.0 and 2.0 micromol/L were 0.33+/-0.10, 0.57+/-0.11 and 0.67+/-0.09 respectively, exhibiting a significant difference in comparison with 0.73+/-0.13 of positive control (p<0.01); as compared with the untreated group, the expression of DNMT3A and DNMT3B in treated groups was obviously down-regulated in a concentration-dependent manner, while expression of DNMT1 was nearly unchanged; as compared with control, all the 3 different concentrations of As2O3 could inhibit the proliferation of CA46 cells and increase the cell number in G0/G1 phase. It is concluded that the As2O3 may up-regulate the expression of p16 gene, recover the activity of p16 gene, thereby promote the regulatory function on cell cycle resul-ting in arrest of cells in G0/G1 phase and inhibit growth of tumor cells through depressing the expression of DNMT3A and DNMT3B and/or directly reversing the methylation status of p16 gene.
authors.affiliation: Fujian Institute of Hematology, Union Hospital, Fujian Medical University, Fuzhou 350001, Fujian Province, China.
authors.forename: Hua-Rong
authors.initials: HR
authors.lastname: Zhou
authors.forename: Jian-Zhen
authors.initials: JZ
authors.lastname: Shen
authors.forename: Hai-Yin
authors.initials: HY
authors.lastname: Fu
authors.forename: Song-Fei
authors.initials: SF
authors.lastname: Shen
authors.forename: Li-Ping
authors.initials: LP
authors.lastname: Fan
mesh_terms: D000077237:Arsenic Trioxide; D001152:Arsenicals / Q000494:pharmacology*; D045744:Cell Line, Tumor; D019941:Cyclin-Dependent Kinase Inhibitor p16 / Q000235:genetics* / Q000378:metabolism; D019175:DNA Methylation / Q000187:drug effects*; D019942:Genes, p16; D006801:Humans; D008223:Lymphoma / Q000235:genetics*; D010087:Oxides / Q000494:pharmacology*; D015533:Transcriptional Activation
chemical_list: D001152:Arsenicals; D019941:Cyclin-Dependent Kinase Inhibitor p16; D010087:Oxides; D000077237:Arsenic Trioxide