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