Effects of phosphocreatine on apoptosis in a cell-free system

The characteristic morphological and biochemical changes during caspase-mediated apoptosis can be reproduced to a large extent in a Xenopus laevis egg extract cell-free system by addition of mouse liver nuclei and exogenous cytochrome c. We show that in this system phosphocreatine accelerated the apoptotic morphological changes of the nuclei, but selectively inhibited DNA fragmentation. Western blot showed that the degradation of lamins A and C is accelerated, which is possibly responsible for the nuclear changes during cell apoptosis. However, the degradation of ICAD/DFF45-like protein in the egg extracts is inhibited in a time-dependent manner. Exogenous creatine, ATP, and several organic acids have no effect on DNA fragmentation, excluding the possibility that creatine, ATP, or acidic conditions resulting from phosphocreatine are responsible for inhibiting DNA fragmentation. Lithium chloride, a kinase inhibitor, can overcome the phosphocreatine effects and can restore DNA fragmentation. Our results indicate that phosphocreatine protects ICAD/DFF45-like protein from proteolysis, probably through kinase actions, resulting in its resistance to caspase cleavage and leading to an inhibition of DNA fragmentation.