Granzyme B-induced loss of mitochondrial inner membrane potential (Delta Psi(m)) and cytochrome c release are caspase independent

CTLs kill targets by inducing them to die through apoptosis, A number of mo rphological and biochemical events are now recognized as:characteristic fea tures of the apoptotic program. Among these, the disruption of the inner mi tochondrial transmembrane potential (Delta Psi(m)) and the release of cytoc hrome c into the cytoplasm appear to be early events in many systems, leadi ng to the activation of caspase-3 and, subsequently, nuclear apoptosis, We show here that; in Jurkat targets treated in vitro with purified granzyme B and perforin or granzyme B and adenovirus, Delta Psi(m) collapse, reactive oxygen species production, and cytochrome c release from mitochondria were observed. Loss of Delta Psi(m) was also detected in an in vivo System wher e green fluorescent protein-expressing targets were attacked by a cytotoxic T cell line that kills predominantly through the granzyme pathway,DNA frag mentation, phosphatidylserine externalization, and reactive oxygen species Production were inhibited in the presence df the caspase inhibitors benzylo xycarbonyl-Val-Ala-Asp-fluoromethyl ketone (zVAD-fmk) and benzyloxycarbonyl -Asp-Glu-Val-Asp-fluoromethyl ketone (zDEVD-fmk) in our in vitro system. Im portantly, in either the in vitro or in vivo systems, these inhibitors at c oncentrations up to 100 mu M did not prevent Delta Psi(m) collapse. In addi tion, cytochrome c release was observed in the in vitro system in the absen ce or presence of zVAD-fmk, Thus the granzyme B;dependent killing pathway i n Jurkat targets involves mitochondrial alterations that occur independentl y of caspases.