contributed equally to this workThe release of cytochrome c from mitochondria results in the formation of an Apaf-1±caspase-9 apoptosome and induces the apoptotic protease cascade by activation of procaspase-3. The present studies demonstrate that heat shock protein 90 (Hsp90) forms a cytosolic complex with Apaf-1 and thereby inhibits the formation of the active complex. Immunodepletion of Hsp90 depletes Apaf-1 and thereby inhibits cytochrome c-mediated activation of caspase-9. Addition of puri®ed Apaf-1 to Hsp90-depleted cytosolic extracts restores cytochrome cmediated activation of procaspase-9. We also show that Hsp90 inhibits cytochrome c-mediated oligomerization of Apaf-1 and thereby activation of procaspase-9. Furthermore, treatment of cells with diverse DNA-damaging agents dissociates the Hsp90± Apaf-1 complex and relieves the inhibition of procaspase-9 activation. These ®ndings provide the ®rst evidence for a negative cytosolic regulator of cytochrome c-dependent apoptosis and for involvement of a chaperone in the caspase cascade.

The release of mitochondrial cytochrome c by genotoxic stress induces the formation of a cytosolic complex with Apaf-1 (mammalian CED4 homolog) and thereby the activation of procaspase-3 (cas-3) and procaspase-9 (cas-9). Here we demonstrate that heat-shock protein 27 (Hsp27) inhibits cytochrome c (cyt c)-dependent activation of cas-3. Hsp27 had no e ect on cyt c release, Apaf-1 and cas-9 activation. By contrast, our results show that Hsp27 associates with cas-3, but not Apaf-1 or cas-9, and inhibits activation of cas-3 by cas-9-mediated proteolysis. Furthermore, the present results demonstrate that immunodepletion of Hsp27 depletes cas-3. Importantly, treatment of cells with DNA damaging agents dissociates the Hsp27/cas-3 complex and relieves inhibition of cas-3 activation. These ®ndings de®ne a novel function for Hsp27 and provide the ®rst evidence that a heat shock protein represses cas-3 activation.

The ubiquitously expressed c-Abl protein tyrosine kinase localizes to both the nucleus and cytoplasm. The nuclear form of c-Abl is activated in the cellular response to genotoxic stress. Here we show that cytoplasmic c-Abl is activated by oxidative stress. The results also demonstrate that mitochondrial cytochrome c is released in the cellular response to H 2 O 2 and that this effect is mediated by a c-Abl-dependent mechanism. In concert with these results, we show that H 2 O 2 -induced apoptosis is attenuated in c-Abl-deficient cells. These findings demonstrate that cytoplasmic c-Abl is involved in the apoptotic response of cells to oxidative stress.Normal cellular metabolism is associated with the production of reactive oxygen species (ROS) 1 and, as a consequence, damage to DNA and proteins (1, 2). The generation of ROS is also known to induce apoptosis; however, the molecular mechanisms responsible for ROS-induced apoptosis are unclear. Studies have indicated that ROS induce activation of topoisomerase II-mediated cleavage of chromosomal DNA and thereby apoptosis (3). Other work has suggested that ROSinduced apoptosis is p53-dependent (4, 5) and that p53-induced apoptosis is mediated by . In addition, the p66 shc adaptor protein (5) and the p85 subunit of phosphatidylinositol 3-kinase (PI3K) (4) have been implicated in the apoptotic response to oxidative stress.The nuclear form of the c-Abl tyrosine kinase is activated in the cellular response to genotoxic stress (9). Nuclear c-Abl has been implicated in the apoptotic response to DNA damage by mechanisms in part dependent on p53 and its homolog, p73 (10 -14). c-Abl also functions as an upstream effector of the proapoptotic SAPK/JNK and p38 mitogen activated protein kinase (MAPK) pathways in the genotoxic stress response (9,15,16). Other studies have demonstrated that c-Abl phosphorylates p85 and thereby inhibits PI3K activity in the apoptotic response to DNA damage (17). Additional evidence supporting a role for c-Abl in apoptosis has been provided by the findings that cells deficient in c-Abl or expressing a dominant-negative c-Abl mutant exhibit an attenuated apoptotic response to genotoxic agents (18,19).Recent work has shown that c-Abl phosphorylates protein kinase C (PKC) ␦ in cells treated with H 2 O 2 (20). The present results demonstrate that the cytoplasmic, and not the nuclear, form of c-Abl is activated in the cellular response to H 2 O 2 . We also show that H 2 O 2 induces mitochondrial cytochrome c release and apoptosis by a c-Abl-dependent mechanism. MATERIALS AND METHODSCell Culture-COS7 cells and MEFs derived from wild-type and c-Abl Ϫ/Ϫ mice (21) were cultured in Dulbecco's modified Eagle's medium containing 10% heat-inactivated fetal calf serum, 2 mM L-glutamine, 100 units/ml penicillin, and 100 g/ml streptomycin. DLD1 cells were grown as described (7). Cells were treated with H 2 O 2 (Sigma), 30 mM N-acetyl-L-cysteine (NAC; Sigma), or 10 M cis-platinum (Sigma).Analysis of Kinase Activity-Cell lysates were prepared in lysis buffer (10 mM Tr...