STAUROSPORINE-INDUCED APOPTOSIS/PROGRAMMED CELL DEATH

Abstract

Apoptosis is the mechanism by which cells are programmed to die under a wide range of physiological and developmental stimuli. Three phases of the apoptosis process, (a) induction phase, (b) commitment phase and (c) degradation phase, have been described in interphase cells only. This study investigated whether mitotic cells can be apoptotic, and what is the early high molecular weight genomic breakdown which could commit the cell to die. Staurosporine, an established 'universal' apoptogen was used to induce apoptosis in human Chang liver cells and human KB epidermoid carcinoma cells. While staurosporine is known to induce interphase specific cell cycle arrest at either G1/S or G2/M interphase, it has never been shown to cause mitosis specific cell cycle arrest and apoptosis. This study demonstrated (a) mitotic and interphase cells dying by apoptosis using 20 , µM staurosporine treatment for 20 hr, (b) induction of telophase-specific cell cycle arrest using 500 nM staurosporine pulse for 15 min, and (c) telophase-specific apoptosis (mitotic-apoptosis) (Swe et al., 1996; Swe and Sit, 1997). The biochemical hallmark of apoptosis has for a long time been held as the manifestation of 200 bp DNA ladder cleavages. It is now clear that this DNA ladder hallmark is a late stage event in the third or degradation phase of apoptosis. Upstream kilobase level cleavages involving 50 kb chromatin loops and 200-300 kb chromatin rosettes have for sometime been considered as preceding commitments in the apoptotic death cascade. However there have been various reports suggesting their occurrence even in necrotic cell death. The more recent suggestion of megabase level cleavage as the death commitment point have not, however, defined the critical genetic damage nor specific pattern of the cleavages. This study showed that in the early first stage (also called initial latent phase) of the apoptotic process induced by staurosporine, the genomic cleavage pattern is a specific cleavage of euchromatin megabase domain where CpG islands are concentrated; these sites have been identified cytogenetically as light G- bands in chromatin (Chen et al., I 998). These are the sites of housekeeping genes in euchromatin domains. The hallmark apoptosis manifestations have generally been suggested to be mediated by caspase cascade involving specifically the CPP32/caspase-3-like proteases. However, various caspase-dependent/-independent apoptosis and necrotic cell death pathways have been described. This study further showed that the caspase-deficient state imposed by zVAD-fmk, an established substrate-specific inhibitor or caspase-3- like proteases is apoptogenic (Swe and Sit, 1999).