poly-HEMA with inhibitors of caspases (data not shown; see below). Together, the results of our immunostaining and cross-linking studies indicate that Bax is normally present in the cytosol but that its BH3 domain is con- cealed. Loss of mammary epithelial cell interactions with the ECM result in a rapid translocation of Bax to mito- chondria, coinciding with a conformational change to ex- pose its BH3 domain. Bax Redistribution Occurs before Commitment to Apoptosis and Is Reversible The rapidity of Bax redistribution after detachment sug- gested that it was an event upstream of the execution phase of apoptosis. Treating cells on poly-HEMA with the broad spectrum caspase inhibitor zVAD-fmk, or the caspase 3–like inhibitor DEVD, did not prevent the redistribution of Bax to the insoluble compartment of FSK-7 cells (Fig. 4 a) or the appearance of the BH3 proximal epitope (Fig. 4 b). Caspase inhibitors did, however, reduce detachment-induced apop- tosis and inhibit the activation of caspase 3 (Fig. 4, c and d). We also examined the distribution of cytochrome c. Bax can induce cytochrome c release from isolated mito- chondria (Jurgensmeier et al., 1998; Narita et al., 1998), and overexpression of Bax can induce cytochrome c release in fibroblasts and melanoma cells (Rosse et al., 1998). How- ever, in hematopoietic cells, Bax association with mito- chondria does not result in cytochrome c release, but does result in mitochondrial dysfunction and apoptosis (Wolter et al., 1997; Gross et al., 1998). At 3 h after detachment from ECM, cytochrome c was still associated with the in- soluble fraction, even though Bax had redistributed from the cytosol (Fig. 4 e). This also provided further evidence that mitochondrial integrity had not been affected during homogenization. The results indicate that the adhesion- dependent relocation of Bax occurs before caspase activa- tion. Bax translocation occurred upstream of the execution phase of apoptosis. To determine if it was reversible, FSK-7 cells were detached and maintained on poly-HEMA for 1 h, after which a portion were then replated onto laminin be- fore they had committed to apoptosis. After 1 h in suspen- sion, Bax relocated to the insoluble fraction (Fig. 5 a) and its BH3 domain became exposed (Fig. 5 b). After replating for 4 h, 94.3% (63.9 SEM, n 5 3) of the cells reattached and spread, and in those cells Bax redistributed back to the soluble fraction. The extent of BH3 domain exposure determined by Bax immunostaining, diminished after re- plating for 3 h and was absent after 18 h. To confirm that the reattached cells had not committed to apoptosis, bro- modeoxyuridine (BrdU) was included in the growth me- dium for 24 h, and the cells were fixed and stained with a monoclonal antibody to BrdU. There was no difference between the percentage of replated cells that incorporated BrdU with those constantly maintained on ECM (data not shown). Together, these results indicate that detachment- induced translocation of Bax to the insoluble fraction oc- curs upstream of commitment to apoptosis and is reversible. Since the adhesion of mammary epithelial cells to laminin is mediated by b1 integrins (Klinowska et al., 1999), our re- sults also demonstrate that Bax translocation and BH3 do- main epitope exposure are integrin dependent. Gilmore et al. Integrin Regulates Apoptosis through Bax Activation 437 Signaling Pathways Associated with Cell Adhesion Regulate Bax Translocation The tyrosine phosphorylation of a number of integrin- associated signaling proteins has been shown to be central to adhesion-mediated signal transduction (Howe et al., 1998). We therefore determined whether either the subcellular distribution of Bax or mammary cell apoptosis was regulated by tyrosine kinases. The protein tyrosine kinase inhibitor, herbimycin A prevents adhesion-induced tyrosine phosphor- ylation of focal adhesion proteins, including pp125FAK and its targets (Burridge et al., 1992). Herbimycin A treatment Figure 5. Bax relocalization and exposure of its BH3 domain is reversible. (a) Adherent FSK-7 cells were detached from ECM and maintained on poly-HEMA for 1 h. Half were then homoge- nized and fractionated. The other half were replated onto lami- nin for 4 h before homogenization. Insoluble and cytosol frac- tions were analyzed by immunoblotting for Bax. (b) FSK-7 cells were immunostained for Bax. Cells were adherent, detached from ECM for 1 h, or detached for 1 h and then replated onto laminin for 3 or 18 h. Note that Bax staining is diminished 3 h af- ter plating on ECM and is virtually absent after 24 h.