CLEAVAGE OF CYTOSKELETAL PROTEINS BY CASPASES DURING OVARIAN CELL-DEATH - EVIDENCE THAT CELL-FREE SYSTEMS DO NOT ALWAYS MIMIC APOPTOTIC EVENTS IN INTACT-CELLS

Several lines of evidence support a role for protease activation durin g apoptosis, Herein, we investigated the involvement of several member s of the CASP (cysteine aspartic acid-specific protease; CED-3- or ICE -like pro tease) gene family in fodrin and actin cleavage using mouse ovarian cells and HeLa cells combined with immunoblot analysis, Hormon e deprivation-induced apoptosis in granulosa cells of mouse antral fol licles incubated for 24 h was attenuated by two specific peptide inhib itors of caspases, zVAD-FMK and zDEVD-FMK (50-500 mu M), confirming th at these enzymes are involved in this paradigm of cell death, Proteoly sis of actin was not observed in follicles incubated in vitro while fo drin was cleaved to the 120 kDa fragment that accompanies apoptosis, F odrin, but not actin, cleavage was also detected in HeLa cells treated with various apoptotic stimuli, These findings suggest that, in contr ast to recent data, proteolysis of cytoplasmic actin may not be a comp onent of the cell death cascade, To confirm and extend these data, tot al cell proteins collected from mouse ovaries or non-apoptotic HeLa ce lls were incubated without and with recombinant caspase-1 (ICE), caspa se-2 (ICH-1) or caspase-3 (CPP32), Immunoblot analysis revealed that c aspase-3, but not caspase-1 nor caspase-2, cleaved fodrin to a 120 kDa fragment, wheres both caspases-1 and -3 (but not caspase-2) cleaved a ctin, We conclude that GASP gene family members participate in granulo sa cell apoptosis during ovarian follicular atresia, and that collapse of the granulosa cell cytoskeleton may result from caspase-3-catalyze d fodrin proteolysis. However, the discrepancy in the data obtained us ing intact cells (actin not cleaved) versus the cell-free extract assa ys (actin cleaved) raises concern over previous conclusions drawn rela ted to the role of actin cleavage in apoptosis.