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Inhibitory effect on the mitochondrial changes of FAS-mediated apoptosis in Jurkat cells by the actin-regulatory protein gelsolin: Richard Chikara KOYA, Makoto OHTSU, Hisakazu FUJITA, Masato TAKIMOTO, Noboru KUZUMAKI (Div.Gene Reg.,Cancer Inst.,Hokkaido Univ. Sch. of Med.)

[Introduction] Gelsolin is an actin-regulatory protein that modulates the organization of the cytoskeleton. We have shown previously that apoptosis triggered by anti-Fas antibody, C2-ceramide or dexamethasone treatment was strongly inhibited in T-cell lymphoma Jurkat cells overexpressing gelsolin. DEVD-specific caspase activity was also suppressed and pro-caspase-3 remained uncleaved in gelsolin transfectant cells (EMBO J 16;4650,1997). To advance in the understanding of the inhibitory mechanism, the mitochondrial system which has been implicated with growing evidence in the execution of apoptosis was analysed.
[Materials and Methods] Jurkat cells transfected with gelsolin or control vectors were stimulated by anti-Fas treatment (100 ng/ml) to induce apoptosis. Flow cytometry utilizing the cationic dye Rhodamine 123 was used to measure the mitochondrial transmembrane potential. To analyse the release of cytochrome C from mitochondria into the cytosol, cells submitted to the treatment above were lysed and fractions containing heavy membranes and cytosol (S100 fraction) were produced followed by immunoblotting.
[Results] The drop of the mitochondrial transmembrane potential, an early event in the apoptotic process, was markedly suppressed in gelsolin transfectant cells. Cytochrome C, a mitochondrial protein released into the cytosol in apoptotic cells where it results in activation of caspase-3, presumably requiring the proteins Apaf-1 and Apaf-3/caspase-9, was not released in the gelsolin transfectants in contrast to the rapid rise and high levels of cytosolic Cytochrome C in the control cells.
[Conclusion] Overall, the findings here indicate that gelsolin overexpression affects a pathway upstream of caspase-3 activation, as demonstrated by its effects which results in inhibition of the mitochondrial step in apoptosis .