Cleavage of Cdc6 by caspase-3 promotes ATM/ATR kinase-mediated apoptosis of HeLa cells

Abstract

We show that caspase-3 cleaves Cdc6 at D-290/S and D-442/G sites, producing p32tCdc6 ( truncated Cdc6) and p49-tCdc6, respectively, during etoposide- or tumor necrosis factor (TNF)-alpha-induced apoptosis. The expression of these tCdc6 proteins, p32- and p49- tCdc6, promotes etoposideinduced apoptosis. The expression of tCdc6 perturbs the loading of Mcm2 but not Orc2 onto chromatin and activates ataxia telangiectasia mutated (ATM) and ATM and Rad-3 related (ATR) kinase activities with kinetics similar to that of the phosphorylation of Chk1/2. The activation kinetics are consistent with elevated cellular levels of p53 and mitochondrial levels of Bax. The tCdc6- induced effects are all suppressed to control levels by expressing a Cdc6 mutant that cannot be cleaved by caspase- 3 (Cdc6-UM). Cdc6-UM expression attenuates the TNF-alpha induced activation of ATM and caspase- 3 activities. When ATM or ATR is down-expressed by using the small interfering RNA technique, the TNF-alpha- or tCdc6-induced activation of caspase- 3 activities is suppressed in the cells. These results suggest that tCdc6 proteins act as dominant-negative inhibitors of replication initiation and that they disrupt chromatin structure and/or induce DNA damage, leading to the activation of ATM/ATR kinase activation and p53-Bax-mediated apoptosis.