Ribozyme-Mediated Inhibition of Caspase-3 Protects Cerebellar Granule Cells from Apoptosis Induced by Serum–Potassium Deprivation

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Abstract
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 systems. One of these is a model of cerebellar granule cells (CGCs) in which apoptosis is induced by acute removal of serum and depolarizing concentrations of potassium. Previous work by several authors showed that benzyloxycarbonyl-DEVD-fluoromethylketone, a somewhat selective caspase inhibitor, significantly protected CGCs from apoptosis; however, because this molecule targets multiple caspases, it is not known whether a single caspase is primarily responsible for effecting cell death in this model. We attempted to answer this question by cotransfecting CGCs with green fluorescent protein reporter and a hammerhead ribozyme directed against caspase-3 mRNA. Maximal protection 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. Significant protection was also observed with human inhibitor of apoptosis (IAP)-like protein–X-linked IAP, a specific inhibitor of caspase-3, -7, and -9, and with p35, a general caspase inhibitor. Overexpression of bcl-2 produced almost complete protection from apoptosis after 24 hr of serum–K+ deprivation (5 ± 2 vs 44 ± 2% in control cells). These results confirm that caspases play an important role in CGC apoptosis and indicate that caspase-3 itself is a significant mediator of this process.