Effects of B cell lymphoma/leukemia-2 associated X protein gene over-expression on expression of second mitochondria-derived activator of caspase/direct IAP-binding protein with low pI in human glioblastoma U251 cells

Abstract

Objective To explore the effects of B cell lymphoma/leukemia-2 associated X protein (bax) gene over-expression on the expression of second mitochondria-derived activator of caspase (Smac)/direct IAP-binding protein with low pI (DIABLO) in human glioma cell line.Methods The plasmid of expression vector pEGFP-bax was transfected into human glioblastoma U251 cells with lipofectamine2000,and meanwhile the non-transfected cells and the pEGFP-transfected U251 cells were taken as controls.Cellular bax and Smac/DIABLO expression was determined by Western boltting.Cellular distribution of Smac/DIABLO protein was evaluated by immunofluorescence (IF) assays and observed under inverted fluorescence microscope.Results The bax protein expression level in pEGFP-bax-transfected U251 cells was (0.82 ±0.12),significantly higher than in non-transfected group (0.35 ±0.05) and pEGFP-transfected control group (0.32 ± 0.09,P ＜ 0.05).The expression level of Smac/DIABLO protein in pEGFP-bax-transfected group was (1.09 ± 0.16),significantly higher than in non-transfected group (0.62 ± 0.10) and pEGFP-transfected control group (0.70 ± 0.20) (P ＜ 0.05).There was no significant difference in the expression of bax and Smac/DIABLO between non-transfected and pEGFP-transfected groups (P ＞0.05).IF revealed that Smac/DIABLO was mainly localized in the cytoplasm.Conclusion Transfection of extrinsic bax gene results in its over-expression in U251 cells,which could up-regulate the expression of Smac/DIABLO protein. Key words: Glioma; B cell lymphoma/leukemia-2 associated X protein; Second mitochondria-derived activator of caspase/direct IAP-binding protein with low pI