of caspase activation, indicating that it is an early event af- ter inhibition of adhesion-dependent survival signals. Survival Signaling in Mammary Epithelial Cells Is Mediated through pp125FAK Most cells require adhesion to ECM for survival. This has now been demonstrated for primary cultures of endothe- lial, epithelial, fibroblastic, mesangial, muscle, and neu- ronal cells. Furthermore, survival signaling in these cell systems is mediated through integrins (Meredith et al., 1993; Frisch and Francis, 1994; Vachon et al., 1996; Ilic et al., 1998; Farrelly et al., 1999; Frost et al., 1999; Mooney et al., 1999). Here we show that mammary cells undergo ex- tremely rapid apoptosis after complete detachment from the ECM. Our previous work has shown that long-term survival of mammary epithelial cells in culture requires in- tegrin-mediated interactions with a laminin-rich basement membrane. Monolayers of cells on collagen I or on tissue culture plastic undergo apoptosis over a period of several days (Pullan et al., 1996; Farrelly et al., 1999). Thus, even though collagen I does not provide as efficient survival sig- nals as laminin, it does afford some protection to apoptosis compared with complete detachment from ECM. In both Figure 8. The p110 subunit of PI 3-kinase and pp60src act downstream of pp125FAK in adhesion-mediated apop- tosis suppression. (a) FSK-7 cells were transfected with 2 mg pCDNA.3 (mock), 2 mg pCDNA.3/DN-FAK (DN- FAK), 2 mg pCDNA.3/DN- FAK 1 1 mg pCDNA.3/ p110CAAX (DN-FAK1 p110CAAX), 2 mg pCDNA.3/ DN-FAK 1 1 mg pCDNA.3/ src (DN-FAK1src), and 2 mg pCDNA.3/p85DSH2 (p85DSH2). 24 h after trans- fection the adherent cells were stained with Hoechst 33258 and either anti-GST or anti-p85 (for the p85DSH2- transfected cells), and the percentage of adherent trans- fected cells were scored for apoptosis. Detached cells were cytospun and the num- ber of cells expressing DN- FAK or p85DSH2 were counted. (b) FSK-7 cells transfected with pCDNA.3/ p85DSH2 were immuno- stained 24 h posttransfection with anti-p85 and anti-Bax 62M, as well as Hoechst 33258 to visualize nuclei. P85DSH2- expressing cells were positive for Bax immunoreactivity, as well as showing apoptotic nu- clei, in contrast to nonex- pressing cells which were Bax negative and had healthy nuclei (examples of healthy cells are indicated by arrows). Detached apoptotic cells were cytospun before staining, and all showed p85 and Bax BH3 epitope immu- noreactivity. (c) FSK-7 cells were cotransfected with DN- FAK and p110CAAX or src as in a, 24 h later, cells were fixed and immunostained for GST and Bax, as well as Hoechst 33258. Gilmore et al. Integrin Regulates Apoptosis through Bax Activation 441