Detection And Analysis Of Apoptosis Related Genes Of NB4 Cells Induced By As₂O₃ By DNA Microarray

【Objective】To investigate alterations in cell growth, apoptosis, and cell cycle induced by arsenic trioxide on the acute promyelocytic leukemia NB4 cell line. Apoptosis related genes and the molecular mechanisms of arsenic trioxide therapy for APL will be explored.【Methods】NB4 cells were cultured in RPMI-1640 medium with 0～8μmol/L As₂O₃ for 12, 24, 36 and 48 hours. The growth inhibition rates of NB4 cells by various concentrations of As₂O₃ were detected by MTT assay. Apoptosis was assessed by flow cytometry (FCM) with Annexin V/PI double staining and cell cycle was detected by FCM with Propidium iodide DNA staining. The data were analyzed by SPSS13.0 software. Total RNA was isolated from cells, treated and 24 hours by 4μmol/L As₂O₃, and reverse-transcribed into a cDNA probe labeled with Bio-16-dUTP. cDNA was hybridized to the Human Apoptosis and Cell Cycle Gene Array of Superarray Bioscience, containing 269 key genes involved in apoptosis, cell cycle and oxidative stress, et al. The gene expression profile was analyzed by GEArray Analyzer software. The microarray results were confirmed by quantity real time polymerase chain reaction. The genes with altered expression level were analyzed by bioinformatics to explore the molecular mechanisms of arsenic trioxide inducing cell apoptosis.【Results】①1～8μmol/L As₂O₃ significantly inhibited the proliferation of NB4 cells in a time and dose dependent manner in vitro.â‘¡2,4,8μmol/L As₂O₃ could obviously induce NB4 cell apoptosis. The apoptosis rate of NB4 cells treated by As₂O₃ for 12 and 24 hours showed a linear relationship with the concentration of As₂O₃; at 36 and 48 hours the NB4 cells mainly showed late apoptosis. Flow cytometry analysis showed that the number of G2/M phase cells increased gradually with raised concentrations treatment of As₂O₃.â‘¢4μmol/ L As₂O₃ treatment of NB4 cells for 24 hours altered the expression level of 100 genes (37.2% of total genes on microarray) related to cell apoptosis, cell cycle, stress and toxicity. Among them, 97 (97%,97/100) genes were up-regulated and 3 (3%,3/100) genes were down-regulated. The up-regulated genes mainly belonged to the tumor necrosis factor (TNF) ligand and receptor superfamily, Caspase family, Bcl-2 family, DNA damage checkpoint/P53 and ATM pathways, cell division cycle proteins and kinases, cyclin, cyclin-dependent kinases and inhibitors. The down-regulated genes were TRAF2, TNFSF10 and XRCC3.â‘£The microarray results were confirmed and consistent with the real time polymerase chain reaction results.【Conclusion】1) As₂O₃ might inhibit the proliferation of NB4 cells and the mechanism was probably through blocking of the cell cycle G2/M phase and showed the ability to induce NB4 cells apoptosis in a time and dose dependent manner;2) The analysis of the genes with alerted expression level suggested: the apoptosis process of NB4 cells induced by As₂O₃ involves many genes and pathways,and the genes among Fas and Casepase pathways and cell cycle regulated genes may play pivotal role.3) The expression level of 3 genes(BAI1,BAI2 and VEGI) with potential anti-angiogenesis function, were up-regulated induced by As₂O₃. BAI1 expresses specifically in brain, which suggest that As₂O₃ may be used to therapy brain tumors.4) As₂O₃ can up-regulated some pro-apoptosis genes and pro-survival genes, this may partly explain why As₂O₃ not only as anti-cancer drug, but also a carcinogen. This result provided the theatrical foundment of As₂O₃ combining with other drugs in clinic.