ROLE OF SUPEROXIDE IN APOPTOSIS INDUCED BY GROWTH-FACTOR WITHDRAWAL

We have examined the role of reactive oxygen species (ROS) in apoptosi s induced by growth factor deprivation in primary cultures of mouse pr oximal tubular (MPT) cells. When confluent monolayers of MPT cells are deprived of all growth factors, the cells die by apoptosis over a 10- and 14-day period. Both epidermal growth factor (EGF) and high-dose i nsulin directly inhibit apoptosis of MPT cells deprived of growth fact ors. Growth factor deprivation results in an increase in the cellular levels of superoxide anion while apoptosis of MPT cells induced by gro wth factor withdrawal is inhibited by a number of antioxidants and sca vengers of ROS. Growth factor deprivation also results in activation o f caspase activity, which is inhibited by EGF and high-dose insulin as well as by the ROS scavengers and antioxidants that inhibit apoptosis . The cell-permeant caspase inhibitor, z-Val-Ala-Asp-CH2F (zVAD-fmk), prevents the increase in caspase activity and markedly inhibits apopto sis induced by growth factor deprivation. However, zVAD-fmk had no eff ect on the increased levels of superoxide associated with growth facto r deprivation. Thus we provide novel evidence that ROS play an importa nt role in mediating apoptosis associated with growth factor deprivati on. ROS appear to act upstream of caspases in the apoptotic pathway. W e hypothesize that oxidant stress, induced by growth factor withdrawal , represents a signaling mechanism for the default pathway of apoptosi s.