Ribozyme-mediated inhibition of caspase-3 protects cerebellar granule cells from apoptosis induced by serum-potassium deprivation

Apoptosis is an important mechanism of physiological and pathological cell death. It is regulated by several gene products, including caspases and the bcl-2-like proteins, whose roles have been demonstrated in numerous system s. One of these is a model of cerebellar granule cells (CGCs) in which apop tosis is induced by acute removal of serum and depolarizing concentrations of potassium. Previous work by several authors showed that benzyloxycarbony l-DEVD-fluoromethylketone, a somewhat selective caspase inhibitor, signific antly protected CGCs from apoptosis; however, because this molecule targets multiple caspases, it is not known whether a single caspase is primarily r esponsible for effecting cell death in this model. We attempted to answer t his question by cotransfecting CGCs with green fluorescent protein reporter and a hammerhead ribozyme directed against caspase-3 mRNA. Maximal protect ion by this ribozyme was observed after 24 hr of deprivation, at which time apoptosis was 18 +/- 0.7% compared with 32 +/- 2% in control cells. Signif icant protection was also observed with human inhibitor of apoptosis (IAP)- like protein-X-linked IAP, a specific inhibitor of caspase-3, -7, and -9, a nd with p35, a general caspase inhibitor. Overexpression of bcl-2 produced almost complete protection from apoptosis after 24 hr of serum-K+ deprivati on (5 +/- 2 vs 44 +/- 2% in control cells). These results confirm that casp ases play an important role in CGC apoptosis and indicate that caspase-3 it self is a significant mediator of this process.