Collagen gel overlay induces two phases of apoptosis in MDCK cells

We previously demonstrated that collagen gel overlay induced cell remodelin g to form lumen and apoptosis in Madin-Darby canine kidney cells. In the pr esent study, we established that collagen gel overlay-induced apoptosis was initiated at areas exclusive of cell remodeling within 24 h (first phase) and extended into areas of cell remodeling within 48 h (second phase). Coll agen gel overlay-induced apoptosis was accompanied by selective proteolysis of focal adhesion kinase (FAK), talin, p130(cas), and c-src. Upon collagen gel overlay, FAK was initially degraded into a 90-kDa product during the f irst phase and subsequently into a 80-kDa product during the second phase. Collagen gel overlay-induced apoptosis of focal adhesion complex proteins a nd apoptosis of the first phase could be blocked only by a protease inhibit or cocktail. In addition, we found that both DEVD-fmk and ZVAD-fmk inhibite d secondary proteolysis of FAK, but only ZVAD-fmk blocked collagen gel over lay-induced apoptosis of the second phase. Finally, collagen gel overlay-in duced apoptosis and proteolysis of focal adhesion complex proteins were com pletely inhibited by the combination of protease inhibitor cocktail and ZVA D-fmk. Taken together, collagen gel overlay induces two phases of apoptosis ; the first phase is dependent on proteolysis of focal adhesion complex pro teins, and the second phase on activation of caspases.