Activation of Caspase 3 in HL-60 Cells Exposed to Hydrogen Peroxide Anna M. DiPietrantonio, Tze-chen Hsieh, and Joseph M. Wu 1 Department of Biochemistry and Molecular Biology, New York Medical College, Valhalla, New York 10595 Received January 10, 1999 Recent studies have suggested that hydrogen perox- ide (H 2 O 2 ), a reactive compound formed endogenously in the breakdown of superoxide, may mediate the in- duction of apoptosis in various cell types in response to external stimuli. However, the role of H 2 O 2 in the apoptotic pathway has not been clearly established. The purpose of this study was to determine if H 2 O 2 treatment could induce apoptosis through the activa- tion of caspases. Doses of H 2 O 2 ranging from 10 M to 100 M, when added to HL-60 cells, resulted in the cleavage of poly(ADP-ribose) polymerase (PARP) from its native 113 Kd form to a processed 89 Kd fragment, indicative of cells undergoing apoptosis. PARP was predominantly in the fragmented form when doses of 20 M and greater were used. A time course study of changes in PARP processing in H 2 O 2 -treated cells re- vealed that 10 and 50 MH 2 O 2 required 6 and 3 h, respectively, to specifically degrade PARP, suggesting that the H 2 O 2 -induced PARP cleavage is both time and concentration dependent. Since PARP is cleaved by CPP32 (caspase-3), we next determined if H 2 O 2 was capable of effecting changes in CPP32 activity. The caspase activity was assayed using a colorimetric sub- strate, DEVD-pNa. Results of these experiments showed that H 2 O 2 increased caspase activity at 3 h, corresponding to the time of appearance of frag- mented PARP. Also, CPP32 activity and PARP pro- cessing were both significantly suppressed by caspase-3 inhibitors. Taken together, these results suggest that H 2 O 2 mediates specific cleavage of PARP and possibly apoptosis by activating caspase 3. © 1999 Academic Press Apoptosis, also referred to as programmed cell death, is an active, energy- and macromolecular synthetic activity-dependent pathway that plays a fundamental role in the development of multicellular organisms, numerous physiological processes, and the mainte- nance of homeostasis (1, 2). Imbalance in apoptosis can lead to many pathological states including cancer, characterized by inadequate cell death relative to growth, and neurodegenerative disorders such as Alz- heimer’s disease, in which apoptosis appears to occur prematurely (2). Despite intense studies on different aspects of apoptosis, the mechanism(s) that underlie the onset, propagation, and ultimate establishment of the apoptotic state remain poorly understood. The events leading to apoptosis vary according to the stimuli, however, a common feature in the pathway appears to be the activation of a family of cysteine proteases called caspases (3, 4). Caspases exist in the cytoplasm of cells as zymogens, which upon the induc- tion of apoptosis, are processed into their active form either through autocatalysis or by other caspases (5, 6). Once activated, the enzymes proceed to cleave other downstream caspases as well as a variety of proteins including PARP, U1-70 Kd, actin, and cell cycle regu- latory proteins such as Rb and mdm2 (reviewed in 7). Although many downstream substrates for caspases have been identified, the trigger for the caspase cas- cade, the manner in which the triggering signals are controlled, as well as the role of caspase substrates in apoptosis has not been clearly established. Recent studies have indicated that reactive oxygen species (ROS) including superoxide and H 2 O 2 , formed in association with a variety of oxidative stress- induced disorders, may be linked to excess cell loss and hence play an important role in apoptosis (8-10). This conclusion is based on several observations. First, in- ducers of apoptosis such as TNF and ionizing radiation lead to an elevation of intracellular peroxides (11, 12). Hydrogen peroxide has been reported to induce oxida- tive damage to membrane lipids (13), proteins (14), and DNA (15). It can also effect cell killing either by necro- sis or apoptosis, depending on the severity of the dam- age to macromolecules (16, 17). Second, antioxidants and free radical scavengers have been reported to in- hibit apoptosis (18, 19). Finally, the addition of ROS themselves has been shown to induce apoptosis (20). However, the mechanism by which ROS, including 1 To whom correspondence should be addressed. Biochemical and Biophysical Research Communications 255, 477– 482 (1999) Article ID bbrc.1999.0208, available online at http://www.idealibrary.com on 477 0006-291X/99 $30.00 Copyright © 1999 by Academic Press All rights of reproduction in any form reserved.