～(131)I-GMCSF Induce Apoptosis Of HL-60/ADM Cells

Objective: To compare the cytotoxicity of HL60/ADM cells and HL60 cells treated with 131I-GMCSF and to investigate the mechanism of the apoptosis of HL-60/ADM cells induced by 131I-GMCSF. Methods : The GM-CSF was labeled with 131I by chloramines-T method. 131I-GMCSF was purified and verified by thin layer chromatography (TLC). An in vitro radioimmunotherapy (RAIT) model was employed. Data were collected from tetrazolium microculture (MTT) cellular cytotoxicity test, transmission electron microscopy, flow cytometric analysis and immunocytochemistry assay. Results :1. The labelling rate of 131I-GMCSF is 66%, radiochemical purity is 96%, radioactive concentration is 4.4×106Bq/ml (or 4.4×109Bq/L) and cells immune binding rate is 88.7%. 2. After exposure to 131I-GMCSF, HL-60/ADM cells and HL60 cells were induced to apoptosis. The IC50　values of HL60/ADM and HL60 cells are 17.2×108 Bq/L and 11.8×108 Bq/L .The inhibition rate increased in the radiation dose-dependent manner. 131I-GMCSF showed the similar cytotoxic effect for sensitive and resistant cells. 3. After treated 24h at the radioactive concentration of 18.5×108Bq/L , both of cells treated with 131I-GMCSF and the 131I showed S arrest and G2/M arrest and mostly G2/M arrest after 48h. 4. The apoptotic rate of HL60/ADM cells in both treated groups is higher than that of the control group (p<0.05). 5. Loss of the mitochondrial transmembrane potential (MTP), destruction of cells ultrastructure, up-regulation of Bax and down-regulation of Bcl-2 and Bcl-xl were observed in process of apoptosis of HL-60/ADM cells induced by 131I-GMCSF. 6. The expression of MRP1 in each group was: 84% (131I-GMCSF group), 83% (131I group ), 76% ( control group) (p>0.05) . The expression of GST-π in each group was: 72% (131I-GMCSF group), 68% (131I group), 64% (control group) (p>0.05) . Conclusions 1.These results show that 131I-GMCSF can induce HL-60/ADM apoptosis through opening the mitochondrial permeability transition pore and reducing MTP, up-regulating Bax ,down-regulating Bcl-2 and Bcl-xl which suggest that 131I-GMCSF may be available in therapy of AML. 2. The inhibition rate increased in the radiation dose-dependent manner when HL60/ADM cells and HL60 cells were treated with 131I-GMCSF. However, the inhibition rate was not significant at low dose radiation. 3. Expressions of MRP1 and GST-π have no effect on the apoptotic pathway induced by 131I-GMCSF in HL60/ADM cells.