title: Ribozyme-mediated inhibition of caspase-3 protects cerebellar granule cells from apoptosis induced by serum-potassium deprivation.
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.
authors.affiliation: Georgetown Institute for Cognitive and Computation Sciences, Departments of Neuroscience, Neurology, and Pharmacology, Interdisciplinary Program in Neuroscience, Georgetown University Medical Center, Washington, DC 20007, USA.
authors.forename: B A
authors.initials: BA
authors.lastname: Eldadah
authors.forename: R F
authors.initials: RF
authors.lastname: Ren
authors.forename: A I
authors.initials: AI
authors.lastname: Faden
mesh_terms: D000818:Animals; D017209:Apoptosis / Q000502:physiology*; D001798:Blood Proteins / Q000494:pharmacology*; D053148:Caspase 3; D061945:Caspase Inhibitors; D002478:Cells, Cultured; D002531:Cerebellum / Q000166:cytology; D015971:Gene Expression Regulation, Enzymologic; D017930:Genes, Reporter; D049452:Green Fluorescent Proteins; D006801:Humans; D020287:In Situ Nick-End Labeling; D007202:Indicators and Reagents / Q000378:metabolism; D008164:Luminescent Proteins / Q000235:genetics; D009474:Neurons / Q000166:cytology* / Q000187:drug effects / Q000201:enzymology; D010957:Plasmids; D011188:Potassium / Q000494:pharmacology*; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000235:genetics; D016337:RNA, Catalytic / Q000235:genetics / Q000378:metabolism*; D051381:Rats; D017207:Rats, Sprague-Dawley; D014162:Transfection; D001616:beta-Galactosidase / Q000235:genetics
chemical_list: D001798:Blood Proteins; D061945:Caspase Inhibitors; D007202:Indicators and Reagents; D008164:Luminescent Proteins; D019253:Proto-Oncogene Proteins c-bcl-2; D016337:RNA, Catalytic; D049452:Green Fluorescent Proteins; D001616:beta-Galactosidase; C505407:CASP3 protein, human; C505410:Casp3 protein, rat; D053148:Caspase 3; D011188:Potassium