β1-MEDIATED Adhesion to fibronectin protects cells from CD95-mediated apoptosis

Abstract

Damiano et al recently demonstrated that interactions between fibronectin (FN) and β1 integrins mediate cellular resistance to doxorubicin melphalan (Damiano et al Blood. 1999). To determine if this anti-apoptotic phenomenon also protected cells from physiological programmed cell death (PCD), we examined the effects of FN adhesion on CD95-mediated apoptosis. We demonstrate that cellular adhesion to FN suppresses both agonist antibody and FasL-induced apoptosis in the U937cell line. Annexin V-FITC staining demonstrated that cellular adhesion, for either two or 24 hours, was able to attenuate CD95-induced PCD. Analysis of caspase activation demonstrated that the cleavage and activity of the effector caspases, caspase-3 and caspase-7 was also reduced. Moreover, we observed a similar attenuation of cleavage and activity the CD95 proximal procaspase-8, suggesting that the mechanism by which cellular adhesion protects cells from CD95-mediated apoptosis may effect a step upstream of procaspase-8 activation. We recently demonstrated that CD95 expression directly correlates with sensitivity to Fas-mediated apoptosis (Shain et al. Leukemia. 2000). To determine if Fn adhesion mediated down-regulated CD95 surface expression, we examined surface levels using indirect immuno-staining by flow cytometry (FCM). FCM analysis revealed no alterations in CD95 expression following adhesion to FN. We also demonstrated that cellular adhesion to FN did not alter the ability of agonist antibody to bind to CD95 on the cell surface. The reduced caspase-8 activity, in the face of the unchanged agonist antibody/receptor binding, suggests that the protective effect(s) of adhesion to FN target a point at or upstream of caspase-8 activation, but downstream of the receptor.