The Effect of Mutant Inhibitor of Caspase-Activated DNase on Apoptosis Induced by Staurosporine

DNA degradation is the first biochemical hallmark of apoptotic execution to be identified, and CAD/DFF40 probably is the major endonuclease during DNA degradation. To more clearly define the role of CAD in nuclear disassembly, we have used the recombinant replication-incompetent retroviral vectors with pBabe-based constructs including inhibitor of CAD(ICAD/DFF45) and double mutations of ICAD(ICAD-DM) to transfect retrovirus packaging line phoenix. High titer retrovirus was generated and infected rat neuroglioma line C6. Undergoing apoptosis with 100 nmol/L STS, group of ICAD-DM had slight better inhibition than group of ICAD against STS-induced MTT reduction (P0.05). TEM analysis illustrated that STS induced apoptosis in both ICAD-infected C6 cells and ICAD-DM-infected C6 cells. After 6～12 h of STS treatment, DNA laddering was observed in ICAD-infected C6 cells, however no DNA fragmentation could be detected in ICAD-DM-infected C6 cells. Our results demonstrated that ICAD/DFF45 is the mayor inhibitor of CAD. Although the inhibition of the cleavage of ICAD/DFF45 can delay the nuclear changes and the death of cells, it can not resist the apoptosis induced by STS.