Abstract
Survivin, a member of IAP ( inhibitor of apoptosis proteins ), is highly expressed in tumor cells. This protein is reported to be phosphorylated at threonine 34 (T34) by CDC2 and to suppress apoptosis by inhibiting caspases. Recently, the residue of aspartic acid 53 (D53) was also demonstrated to be important for the inhibition of apoptotic signaling. In this study, we constructed a replication-deficient adenovirus encoding survivin with point mutations (T34A and D53A) and examined their effects on radiation-induced apoptosis. The cDNAs consisting of GFP and wild type (WT) survivin or survivin mutants (T34A and D53A) were inserted in pAd/CMV/V5-DEST vector. Virus was produced in 293A cells and the crude viral supernatants were used to transduce human lung carcinoma A549 cells. The expression of GFP proteins was detected in all of the cells after transfection of GFP viral vector as confirmed by fluorescence microscopy. After irradiation, the apoptosis induction was evaluated by microscopic observation. Overexpression of survivin-T34A enhanced the radiation-induced apoptosis in comparison with that of wild type survivin. The result suggested that the point mutations at T34 abolished the anti-apoptotic activity of survivin and enhanced the induction of apoptosis in X-irradiated A549 cells. Examination of the effects of survivin-D53A on radiation-induced apoptosis was under progress.
