CHARACTERIZATION OF CPP32-LIKE PROTEASE ACTIVITY FOLLOWING APOPTOTIC CHALLENGE IN SH-SY5Y NEUROBLASTOMA-CELLS

We characterized the activation of interleukin-1 beta-converting enzym e (ICE)-like proteases (caspases) in human neuroblastoma cells (SH-SY5 Y) following challenge with staurosporine, an established agent known to induce apoptosis. Time course analyses of lactate dehy drogenase re lease detected a significant increase in cell death as early as 6 h th at continued at least until 24 h following staurosporine treatment. We stern blot analyses using anti-poly(ADP-ribose) polymerase(anti-PARP) and anti-CPP32 antibodies revealed proteolytic processing of CPP32 (an ICE homologue) as well as fragmentation of PARP as early as 3 h follo wing staurosporine challenge. Furthermore, the hydrolysis of the CPP32 substrate acetyl-DEVD-7-amido-4-methylcoumarin was detected as early as 3 h and became maximal at 6 h after staurosporine challenge, sugges ting a delayed and sustained period of CPP32-like activation. In addit ion, we used the first immunohistochemical examination of CPP32 and PA RP in cells following an apoptotic challenge. The localization of CPP3 2 in untreated SH-SY5Y cells was exclusively restricted to the cytopla sm. Following staurosporine challenge there was a condensing of CPP32 immunofluorescence from the cytoplasm to a region adjacent to the plas ma membrane. In contrast, PARP immunofluorescence was evenly distribut ed in the nucleus in untreated SH-SY5Y cells and on staurosporine chal lenge was found to be associated with condensed chromatin. It is impor tant that a pan ICE inhibitor [carbobenzoxy-Asp-CH2OC(O)-2,6-dichlorob enzene] was able to attenuate lactate dehydrogenase release and PARP a nd CPP32 cleavage and altered immunohistochemical staining patterns fo r PARP and CPP32 following staurosporine challenge.