Effects Of Survivin On Differentiation And Apoptosis In Human Leukemia Cells

Survivin, a novel inhibitor of apoptosis, locates on 17q25. In these years, some scientists have reported survivin is faintly detected in fetal tissues and thy-mus gland and over expresses in most human cancers, but not usually expresses in normal tissues. Sruvivin expression up-regulation predicts poor prognosis in human cancers. ATRA induced NB4 cells differentiation. Survivin protein expression was downregulated during the differentiation of NB4 cells induced by ATRA, but the alteration of survivin localization is not clear. As2O3 induced NB4 cells apoptosis The regulation of survivin induced by As2O3- is not clear. Caspase-9 and caspase-3 is protein during the trail of internal apoptosis. The relationship between survivin and caspase-9 and caspase-3 on differentiation and apoptosis of NB4 cells is not clear.In the present study, we observed the differentiation- and apoptosis- inducing action on NB4 by ATRA and As2O3, at the same time we examined the expression of survivin caspase-9 and caspase-3 protein, studied effects of ATRA and As2O3 on survivin effects of survivin on differentiation and apoptosis of NB4.Methods一 Cell cultureThe human promyelocytic leukemia cell line NB4 cells were kindly provided by Muto (Japan). AU cells were cultured in RPMI-1640 medium supplemented with 10% fetal bovine serum a humidified atmosphere of 95% air/5% CO2 at 37C Cells were used in logarithmic growth phase.二 Main materialsAs2O3 was purchased from Harbin of China. ATRA PI RNAase NBT TPA were purchased from Sigma Chemical Co. RPMI-1640 was purchased from GIB-CO BRL USA and fetal bovine serum was purchased from Tianjin blood institution.三 Assays for cellular proliferationCellular proliferation was determined by counting Burker chamber and cell viability was determined by typan blue exclusion.四 Assays for differentiation1 Differentiation was assessed by changes in cellular morphology stained with May-Griinwald and Giemsa solutions.2Differentiation of NB4 cells was assessed by the nitro blue tetrazolium ( NBT) reduction test. The percentage of cells containing formazan depoisits, corresponding to the capacity to reduce NBT, was determined by counting 200 cells.五 Assays for apoptosis1 Apoptosis was assessed by changes in cellular morphology stained with May-Griinwald and Giemsa solutions.2 Apoptosis was analyzed by flow cytometry.六 Assays for survivin caspase-9 and caspase-3 protein The expression of survivin caspase-9 and caspase-3 protein were analyzed by immunohistochemistry. 七 Statistical analysis The data were analyzed by t-test.Results一 Effects of ATRA on NB41 Inhibitory effects of ATRA on NB4 cells proliferation ATRA inhibited NB4 cells proliferation in a time-and dose-dependent manner. IC50 of 72 h was 1.71uM.2 Effects of ATRA on differentiation of NB4 cellsNB4 cells was cultured with 1uM ATRA for 4-6 days. On day 4, we can observe differentiation toward granulocytes and differential cells had the faction of NBT reduction. On day 4 and on day 6 , the percentage of NET reduction cells was 35. 2% and 72. 6% , respectively.3 The expression of survivin caspase-9 and caspase-3 protein was changed by ATRAWe cultured NB4 cells with 1u MATRA for 3-6 days. Immunohistochemis-try analysis showed the expression of survivin protein was negative in cell nuclear and was positive in cell plasm.We cultured NB4 cells with 1 uM ATRA for 24 hours. Immunohistochemis-try analysis showed the expression of caspase-9 and caspase-3 protein was upreg-ulated compared to the control.二 Effects of As2O3 on NB4 cells1 Inhibitory effects of As2O3 on NB4 cells proliferationAs2O3 inhibited NB4 cells proliferation in a time-and dose-dependent manner. IC50 of 72 h was 0. 53uM.2 Effects of As2O3 on apoptosis of NB4 cellsAfter treatment with 1. 5u,M As2O3 for 24 hours, NB4 cells exhibited apoptosis morphological changes. The percentage of G2/M phase cells increased to 21. 5% and the cycle progression was blocked in G2/M phase. The percentage of sub G0...