EDITORIAL FOCUS
Living and dying with reactive species Focus on "Peroxynitrite induces apoptosis of HL-60 cells by activation of a caspase-3 family protease"

Harry Ischiropoulos

Institute for Environmental Medicine and Biochemistry and Biophysics, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania 19140-6068

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TRADITIONALLY, PERTURBATION IN cellular redox capacity and metabolic status by reactive species-mediated oxidative stress has been associated with necrosis. Recent studies, however, have implicated reactive species as inducers of apoptosis (2, 4, 5, 8, 10). Apoptosis is an essential process of cell elimination critical for maintaining tissue homeostasis in health and disease. It is characterized by a number of well-defined morphological and structural changes. A number of physiological and chemical agents have been shown to trigger apoptosis. Agents that induce perturbations in membrane function, cytoskeleton, signal transduction, mitochondria, protein synthesis, and DNA integrity provoke apoptotic cell death (12).

Although much progress has been made in understanding oxidative stress-mediated death, little is known regarding the executioners of apoptosis provoked by reactive species. The article in focus, a study by Lin et al. (Ref. 7, see p. C855 in this issue), provides evidence that peroxynitrite-mediated apoptosis in HL-60 cells is executed by the induction of caspase-3. Peroxynitrite is the product of the reaction of superoxide (one electron reduced oxygen) with nitrogen monoxide. Caspases, the cysteine aspartases, are a class of programmed cell death genes that have been characterized as major executioners of apoptosis triggered by tumor necrosis factor receptor activation or Fas-APO1 activation (11). Data in this paper show that exposure of HL-60 cells to concentrations of peroxynitrite, which previously were found to induce apoptosis, induced proteolytic cleavage of procaspase-3 enzyme to caspase-3. Moreover, the activation of caspase-3 was demonstrated by the proteolytic cleavage of poly(ADP-ribose) polymerase, one of the natural substrate(s) of caspase-3. Specific inhibitors of the caspase-3 pathway were also shown to rescue HL-60 cells from peroxynitrite-induced apoptosis. Overall, this study represents one of the few examples that has delineated a specific pathway of apoptotic cell death mediated by reactive species. Although the data provide solid evidence for the role of caspase-3 in executing cell death in this model, the reason for the activation of caspase-3 by peroxynitrite remains to be defined. Activation of caspase-3 may result from mitochondrial release of cytochrome c or possibly by direct activation of the proteolytic conversion of procaspase-3 to active caspase-3. Mitochondria constitute a significant target for peroxynitrite. To date, however, there is no indication that peroxynitrite induces release of cytochrome c from mitochondria. Peroxynitrite has been shown to alter mitochondrial membrane potential, electron transport, and activity of manganese superoxide dismutase, all of which increase intracellular levels of both calcium ions and partially reduced oxygen species (1, 9). Intracellular increases in both calcium ions and partially reduced oxygen species have been linked to induction of apoptosis.

Inherent in their biochemical properties, reactive species react with a number of biological molecules. Thus the role of reactive species in initiating specific activation of the apoptotic execution machinery remains elusive. The concentration of a biological target, the reaction rate constants, and the proximity of reactive species generation with the putative biological target may provide the rationale to explain this apparent specificity. Superoxide, hydrogen peroxide, nitric oxide, and peroxynitrite, unlike strong oxidants such as hydroxyl radical, react with biological targets in a rate-controlled manner. Superoxide, nitric oxide, and peroxynitrite have been shown to selectively react with heme proteins, iron-sulfur, zinc-sulfur, protein cysteine, and tyrosine residues (1, 3, 9). Enzymes regulating bioenergetics, transcription factors, and the iron regulatory proteins contain iron-sulfur and/or zinc-sulfur centers. The activity of different kinases, transcription factors, and ion channels is redox sensitive and is found to be dependent on critical cysteine and tyrosine residue(s). Nitrosation or nitration of these critical cell targets by nitric oxide and peroxynitrite is expected to alter their activities. Moreover, since apoptosis is a gated checkpoint-controlled process, reactive species are not required to be the proximal effector molecules; perturbation of redox-regulated secondary pathways described above satisfy the requirement for selective signals triggering apoptosis.

It is also becoming apparent that reactive species have paradoxical and even diametrically opposite effects in different cell systems, because they can either induce or prevent apoptosis (2, 4-8, 10). Evidence exists to implicate nitric oxide as a mediator as well as an inhibitor of apoptosis. Moreover, peroxynitrite may also share similar properties. This is a reasonable supposition, because, as discussed above, apoptosis appears to be a cell-specific process, quite often regulated by a number of redox-sensitive checkpoints. It is not known if all cells express the same pathways for the execution of apoptosis. Furthermore, a number of other factors may determine the mode of cell death. These factors may include the magnitude and duration of exposure to reactive species, cellular antioxidant capacity, efficiency of repair of oxidant-modified biomolecules, ability to sustain metabolic requirements, and trophic support. Perhaps, more importantly, a number of unrecognized cell-specific responses that function as sensors for reactive species may also exist and regulate cell death and adaptive responses. Studies such as the one by Lin et al. (7) in specific cell systems will provide the knowledge required to piece together the puzzle of living and dying with reactive species.

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2.   Bonfoco, E., D. Krainc, M. Ankarcrona, P. Nicotera, and S. A. Lipton. Apoptosis and necrosis: two distinct events induced, respectively, by mild and intense insults with N-methyl-Daspartate or nitric oxide/superoxide in cortical cell cultures. Proc. Natl. Acad. Sci. USA 92: 7162-7166, 1995[Abstract].

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7.   Lin, K.-T., J.-Y. Xue, M. C. Lin, E. G. Spokas, F. F. Sun, and P. Y.-K. Wong. Peroxynitrite induces apoptosis of HL-60 cells by activation of a caspase-3 family protease. Am. J. Physiol. 274 (Cell Physiol. 43): C855-C860, 1998[Abstract/Free Full Text].

8.   Lin, K.-T., J.-Y. Xue, M. Nomen, B. Spur, and P. Y.-K. Wong. Peroxynitrite-induced apoptosis in HL-60 cells. J. Biol. Chem. 270: 16487-16490, 1995[Abstract/Free Full Text].

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11.   Villa, P., S. H. Kaufmann, and W. C. Earnshaw. Caspases and caspase inhibitors. Trends Biochem. Sci. 22: 388-393, 1997[Medline].

12.   Wertz, I., and M. R. Hanley. Diverse molecular provocation of programmed cell death. Trends Biochem. Sci. 21: 359-364, 1996[Medline].


AJP Cell Physiol 274(4):C853-C854
0363-6143/98 $5.00 Copyright © 1998 the American Physiological Society