| IntroductionArsenic trioxide ( As2O3) has recently been shown to be effective in the treatment of acute promyelocytic leukemia ( APL) including relapsed or retinoic acid - resistant APL. In vitro, As2O3 exerts a dose -dependent dual effect; high dose (1.0 -2. 0uM) triggering apop-tosis and low dose (0. 1 -0. 5uM) inducing partial differentiation. The dual effect may be mediated through modulation of the oncogenic PML/RARofusion protein specific to APL cells. While some clinical observations suggest that inducing differentiation may play a major role in As2O3 - induced remission in patients with APL. But, its serious clinical side effects such as hepatic damage, cardiac dysfunctions, leukocytosis, as well as symptoms similar to those of retinoid acid syndrome hampered extensive clinical application of As2O3. Y Shen et al recently reported that low - dose As2O3 is as effective as conventional dosage in remission induction with reduction of toxic effects. However , its efficacy and longer follow - up are still unclear. Therefore, further observation and study will be needed to clarify the more detailed molecular mechanisms of As2O3 - induced remission of APL patients, and it's very important to search for a new and safer and more effective approach to As2O3 treatment of APL.It is well known that GM - CSF and G - CSF have a potential to stimulate proliferation and induce the granulocytic differentiation ofmyeloid leukemia cells. Muto et al have reported that As2O3 alone could induce apoptosis of retinoic acid - resistant APL cell line UF -1 cells, while As2O3 could induce differentiation of UF - 1 cells into mature granulocytes in the presence of GM - CSF, but not apoptosis. However, whether the two agents exert the similar effect in APL cells in vivo still remains unclear.To determine the effects of As2O3 on differentiation of APL cells, and the effects of combination As2O3 and GM - CSF on APL cells particularly primary cells from APL patients, we examined the effects of As2O3 alone and a combination of As2O3 and GM - CSF on NB4 cells and APL primary cells from 12 patients with APL.Materials and methodsReagentsAs2O3 was purchased from Harbin of China. ATRA, NBT, TPA, MTT, DMSO, NaN3 from Sigma Chemical Co,rhGM - CSF from KI-RIN,RPMI - 1640 from GIBCO BRL USA,CDllb - FITC from Coulter Immunology Co.Cells and cell culturePrimary APL cells were obtained from BM of 12 cases of de novo APL and 1 relapsed APL. Fresh cells were separated by centrifugation on Ficoll's solution. Only specimens containing at least 70% leukemia cells were studied and were seeded at 1 x 106/ml. NB4 cells were kindly provided by Dr Muto (Japan) and were seeded at 1. 0 x 105 / ml. All cells were cultured in RPMI - 1640 medium supplemented with 10% -20% fetal bovine serum a humidified atmosphere of 95% air/5% CO2 at 37C.Assays for cellular proliferation and viabilityCellular proliferation was determined by counting Bttrker chamber and cell viability was determined by typan blue exclusion or MTT assay.Assays for apoptosisApoptotic cells were determined by morphological analysis.Assays for differentiation1. Maturation was assessed by changes in cellular morphology stained with May - Griinwald and Giemsa solutions.2. Function was assessed by NET reduction.3. Expression of the granulocyte/macrophage - specific antigen CDllb was analyzed by monoclonal antibody labeling and flow cytom-etry.Statistical analysisThe data were analyzed by SPSS. The Wilcoxon signed rank test was used to assess the significance of differences of the results.Results(一) Effects of As2O3 and GM - CSF in NB4 cells 1.Inhibition cellular proliferation by As2O3After incubated with 0. 1 - 2. 0uM of As2o3for 24,48 and 72 hours, the number of viable cells of NB4 was inhibited in a time -and dose - dependent manner. And the IC50 of 72 hours was 0. 75 uM. 2.Induction cell apoptosis in a time - and dose - dependent mannerMorphologically, after treatment with 1.0 -2. 0uM of As2O3for 24 - 72 hours, NB4 cells pres...
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