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* From the Jewish Hospital Heart and Lung Institute, Louisville, KY Supported in part by a grant from the National Institutes of Health (HL02791), American Lung Association, Basic Grant FY-97-0590 from the March of Dimes Birth Defects Foundation, and Winthrop-University Hospital.
Correspondence to: Stuart Horowitz, PhD, Director, Research and Technology, Jewish Hospital Heart and Lung Institute, 217 E Chestnut St, Louisville, KY 40202; e-mail: stuart.horowitz{at}jhhs.org
A widely held opinion about cell death is that apoptosis follows a series of events that are genetically programmed and necrosis does not. This simple-minded notion is partially due to the problem that most thinking about cell death is dichotomous: cell death is either apoptosis or necrosis. Thus, necrosis is currently the term used to describe slow death associated with swelling from hyperoxia or low concentrations of oxidants,1 as well as very rapid death at extremely high oxidant doses,2 in addition to almost every other form of nonapoptotic, unscheduled death due to any number of catastrophic insults. Necrosis may be too vague a term to be of much use in this regard, and it is probably best to think of necrosis as a consequence, rather than a mode of cell death, because even apoptosis can eventually result in "secondary necrosis."3 However, it is conceivable that there are other modes of programmed cell death in addition to apoptosis, all of which currently fall under the heading of necrosis. So far, we are limited by the fact that the overwhelming majority of the tools available for the study of cell death have arisen from investigations of apoptosis. Recent progress in our laboratory indicates that hyperoxia-induced, nonapoptotic cell death is pathway driven, and that the pathways are at least partially divergent from those leading to apoptosis in the same cells. Herein we summarize these observations and discuss their implications.
Hyperoxia Does Not Cause Apoptosis
Our laboratory has a long-standing interest in lung injury resulting from ventilatory oxygen (O2) therapy. We focused on a lung epithelial cell model because the lung is a sensitive target of O2 toxicity. A549 cells are derived from human alveolar type II cells and have been extensively studied with respect to oxidant injuries.4 5 The results summarized in this section are reported in an article by Kazzaz et al.1 Cells were cultured either in 95% room air or 95% O2. The kinetics of hyperoxic A549 cell death are similar to those reported for other cell types,6 7 8 and several days pass before cell death becomes significant. Because physiologically and pathophysiologically relevant cell death from other oxidants is generally thought to occur via apoptosis2 9 10 11 and there is adequate time to trigger an apoptotic program, we tested whether hyperoxia also induces apoptosis.
The dye Hoecsht 33258 fluoresces brightly under ultraviolet excitation when bound to DNA. Apoptotic nuclei are much smaller and brighter than normal. However, the nuclei of cells exposed to hyperoxia were swollen and larger than control nuclei. In contrast, cells exposed to the oxidants hydrogen peroxide (H2O2) or paraquat (which generates intracellular superoxide) underwent apoptosis, as shown by their typically shrunken and brightly fluorescent nuclei. To independently assess apoptosis, we also utilized the in situ TUNEL (terminal deoxynucleotidyl transferase dUTP nick end labeling) assay. The TUNEL assay labels 3'-OH ends of DNA in chromatin, which result from endonuclease activation occurring during apoptosis.12 13 This approach showed that the vast majority of cells cultured in hyperoxia were TUNEL negative. In contrast, a large population of cells exposed to H2O2 or paraquat were clearly TUNEL positive. Another means of assessing whether cells are or are not apoptotic is to study their morphology by electron microscopy. When cells were exposed to 95% O2 for 6 days, they became enlarged, with swollen nuclei and mitochondria. By contrast, cells exposed to paraquat or H2O2 had condensed chromatin, a hallmark of apoptosis. These data indicate that hyperoxia did not result in apoptosis in these epithelial cells.
If the kinetics of cell death correlate with the extent of apoptosis throughout exposure, then apoptosis is indeed the operative mode of death. Conversely, if there is no correlation, cells would not have died via apoptosis. Using computer-aided image analysis of Hoechst-fluorescent cells, we established objective criteria for distinguishing apoptotic nuclei from normal ones. After quantifying the extent of apoptosis, we determined that there was no correlation between cell death and the extent of apoptosis at any time during hyperoxic exposure, as determined either by Hoecsht fluorescence or by counting TUNEL-positive nuclei. However, cell death and apoptosis were tightly correlated in cultures exposed either to H2O2 or paraquat.
We have made virtually identical observations on other lung epithelial cell lines, including mouse lung epithelial (MLE-12) cells and rat epithelial SV-40T2 cells, both of which are derived from type II cells. Likewise, we find that HeLa cells (epithelial cells derived from cervical carcinoma) do not undergo apoptosis in response to hyperoxia. One limitation of these experiments is that they all involve the use of continuously proliferating, transformed cells. Thus, their response might not reflect the biology of primary, diploid cells. To address this concern, we have recently exposed two different primary cell types of human lung to hyperoxia in culture: human vascular endothelial cells and normal human bronchial epithelial cells (NHBE). Importantly, neither of these cell lines underwent apoptosis in response to hyperoxia. Just as with the transformed cells, these primary cells swelled and died over a period of days of exposure to hyperoxia, although the endothelial cells were more resistant in culture than the NHBE cells. These experiments were performed at low seeding density (approximately 30% confluence), under conventional culture conditions, in which NHBE cells have a relatively nondifferentiated phenotype. It is also important to note that glucose consumption increases during hyperoxia, and it is essential to replenish culture media daily to avoid this potentially confounding variable. Taken together, these data indicate that many epithelial cell lines (as well as human microvascular endothelial cells of lung) do not die via apoptosis when cultured in hyperoxia. However, other cell types might be different. For example, Jyonouchi et al14 report that a few percent of Madin-Darby canine kidney (kidney epithelial) cells in culture die via apoptosis after 2 days in hyperoxia, but only when first grown to near-confluence.
Low-Dose Oxidants Do Not Kill by Apoptosis
Hyperoxic cell death is also different than death from other oxidants in terms of the kinetics of cell killing. At the oxidant doses typically studied10 15 16 17 18 and used herein, cells died in a matter of hours, while it took days for hyperoxia. To determine if oxidant-induced apoptosis occurs when the rate of cell death is substantially reduced, experiments were performed at much lower concentrations of H2O2 and paraquat. Importantly, virtually no apoptosis was observed at these lower oxidant concentrations,1 which is similar to cell death by hyperoxia. However, unlike hyperoxia, these low oxidant doses were not 100% lethal, and a subpopulation of cells began to adapt and divide. At intermediate oxidant doses, mixed modes of cell death occurred in the population, some dying by apoptosis, some by swelling. The significance of this observation for studies of oxidant-induced apoptosis cannot be overemphasized: to avoid the confounding variables that are likely to be associated with mixed-mode cell death in these kinds of experiments, relatively high levels of oxidants are required.
Inhibition of Apoptosis by Hyperoxia
It seems likely that at least some of the organellar and macromolecular sites of O2 damage are different from sites affected by other oxidants, since molecular O2 is not as reactive as oxygen-derived free radicals, diffuses throughout the cell, and can target virtually all organelles and cytosolic molecules. One possible explanation for the lack of apoptosis in hyperoxia is that one or more steps in the oxidative damage-induced pathway to apoptosis might be sensitive to direct oxidation by high levels of molecular O2. This would predict that hyperoxia can inhibit apoptosis in some cases. Consistent with this possibility is the observation that poly-adenosine diphosphate (ADP) ribosylation is defective in hyperoxia-injured cells.19 Poly(ADP)-ribose polymerase (PARP) must be cleaved for apoptosis to occur in some systems.20
To determine if hyperoxia can inhibit apoptosis, A549 and HeLa cells were grown in room air to subconfluence, and either exposed directly to concentrations of H2O2 that induce widespread (> 70%) apoptosis, or cultured in hyperoxia for 2 days and then exposed to H2O2. Cells were stained in Hoechst, analyzed, and the apoptotic index was determined. Preliminary results (unpublished) show that pretreatment with hyperoxia for 2 days (a time at which there was no cell death) reduced the level of HeLa cell apoptosis. These data indicate that hyperoxia can at least partially inhibit apoptosis in these cells, and nearly identical results were obtained with A549 cells. One possible explanation for the inhibition is that metabolic poisoning by hyperoxia is pleiotropic, adversely affecting many cellular processes including apoptosis. In other words, it might not be the hyperoxia itself that results in a specific failure to undergo apoptosis, rather, it could be a nonspecific consequence of overall cell poisoning. One way to test this notion is to repeat the experiment using cells that do not die because of hyperoxia. We accomplished this using mutant, hyperoxia-resistant HeLa-80 cells,21 22 which grow at normal rates in 80% O2, an otherwise lethal dose. HeLa-80 cells were grown in room air to subconfluence, and either exposed to H2O2 without preexposure, or first cultured in hyperoxia for 2 days and then exposed to H2O2. Similar to the parental HeLa-20 cells, culture in hyperoxia blunted subsequent H2O2-induced apoptosis in HeLa-80 cells. Because HeLa-80 cells do not actually suffer O2 toxicity under these conditions, these results suggest there may be a step in the pathway to apoptosis that is sensitive to levels of molecular O2. Alternatively, hyperoxia may activate a step that blocks a particular pathway to apoptosis. Although increased antioxidant enzymes can sometimes block apoptosis, 1 or 2 days of preexposure to hyperoxia does not induce increases in these enzymes in any of the cell lines we have tested.
Apoptotic Pathways in Lung Epithelial Cells
We addressed the question of whether any apoptosis-related genes are regulated in lung epithelial cells that undergo either oxidant-induced apoptosis or necrosis. Nedd-2 is a member of the interleukin-1ß converting enzyme (ICE) family of proteases, and other members of the ICE family have been shown to be activated in apoptosis. Western blots show that Nedd-2 is cleaved when A549 cells are driven into widespread apoptosis by H2O2. Immunoflourescence assays indicate that not only Nedd-2, but also ICE itself, was activated by H2O2. We have also observed that the enzyme PARP is proteolytically cleaved, and is then translocated to the nucleus in apoptotic A549 cells. In contrast, we have found that that neither PARP nor ICE are induced during cell death from hyperoxia, suggesting that some of the molecular pathways to oxidant-induced cell death are distinct, depending on the oxidant, dose, and mode of death.
Transcription Factors and Hyperoxic Signaling
Apoptotic cell death can be prevented by the activation of nuclear
factor-kappa B (NF-
B),23
24
25
26
a multisubunit
transcription factor that regulates expression of genes involved in
inflammation, infection, and stress.27
The induction of
NF-B may be part of a survival mechanism used to escape cell
death.28
We examined NF-B in cells exposed to lethal
concentrations of H2O2 (which causes apoptosis)
or hyperoxia. Despite the apparent activation and induction of NF-B
by molecular O2, we observed that the cells do not escape
death.29
These observations are summarized below.
Following release from inhibitory binding protein IB, NF-B
translocates from cytosol to the nucleus, where it regulates
transcription. NF-B activation was studied during hyperoxia by
immunofluorescence of the p65 subunit of NF-B. In control A549 cells
grown in room air, immunofluorescence was weak, and evident primarily
in the cytoplasm, although there was limited fluorescence in the nuclei
of some cells. Nuclear fluorescence was more prominent by 30 min of
hyperoxia, and it increased over the course of 1 day. The cells showed
some signs of swelling by 24 h, and fluorescence became more
intense not only in the nuclei, but also in the cytoplasm of many
cells. In contrast to hyperoxia, H2O2-induced
apoptosis was associated with the nuclear evacuation of NF-B. In the
few cells that had not undergone apoptosis, nuclear fluorescence for
NF-B was observed. The translocation of p65 to the nucleus is
consistent with NF-B activation. Increased immunofluorescence not
only suggested activation, but also an increased level of p65 protein.
Western blots show that NF-B levels were increased after 30 min of
exposure to 95% O2, and peak levels were achieved by
24 h. In contrast, H2O2-induced apoptosis
caused no increased NF-B protein, and there was even a slight
decrease after 2 h.
To determine if increased levels of p65 protein were correlated with
increased messenger RNA (mRNA) abundance, Northern blot analyses were
performed. By 30 min after O2 exposure, there was a slight
increase in steady-state levels of p65 mRNA. This increased over the
course of 1 day and remained elevated for 2 days. In contrast,
H2O2-induced apoptosis was not associated with
increased NF-B expression.
The differential activation and expression of NF-B during different
modes of cell death imply the existence of different signaling
pathways. We therefore undertook a study aimed at deciphering the
transcriptional regulatory events that occur in the early phases of
oxidant-induced death. A typical early response to stress involves the
transient expression of the c-Fos and c-Jun protooncogenes. Moreover,
the transcription complex known as activator protein-1 (AP-1) includes
the Fos-Jun dimer and not only is reported to be redox sensitive, but
is also activated in lungs of hyperoxic rats.30
To examine
AP-1 regulation during hyperoxia, mouse lung epithelial (MLE-12) cells
were exposed to 95% O2 for up to 24 h. At various
time points, cells were harvested and RNA assayed by Northern blots for
Fos and Jun expression. Relative to control cells (that were plated at
the same time as hyperoxic cells, and also transferred to fresh
medium), both c-Fos and c-Jun transcript levels were elevated
transiently (at 30 min) and then returned rapidly to baseline. However,
both mRNAs were again increased in abundance at 16 to 24 h
(unpublished observations).
The c-Fos promoter can be regulated by the p42 and p44 mitogen-activated protein (MAP) kinase or MAP kinase cascade in some cases.31 To investigate whether p42 and p44 MAP kinases are activated by hyperoxia, we used antibodies raised against the phosphorylated or activated form of the proteins. No changes were detected in the levels of phosphorylated p42 or p44 at any time during exposure to hyperoxia. In contrast, within 10 min of incubation in an apoptosis-inducing concentration of H2O2, there was a significant increase in phosphorylated p42 and p44. These data indicate that signaling events are different in these two modes of oxidant-induced lung epithelial cell death.
Increased abundance of c-Fos and c-Jun transcripts are often associated with activation of the corresponding proteins. Jun phosphorylation, a step in its activation, was therefore assayed. Western blots were incubated with an antibody specific for ser-63-phosphorylated c-Jun. A notable rise in the level of Jun-phosphate was observed within 0.5 h. The appearance of a second, higher-migrating band suggested additional phosphorylation at ser-73. Like c-Jun mRNA, Jun-phosphate levels decreased after this brief rise, but increased again at 16 to 24 h. Jun phosphorylation can occur by Jun kinase (JNK) or by p38. As a first step toward examining a possible role for JNK, we performed a JNK "pull-down" experiment. In this assay, cell lysates were incubated with affinity beads bound to a glutathione-S-transferase-Jun fusion protein, used to "pull down" JNK. Unlike the biphasic increase in Jun-phosphate and Jun mRNA, an increase in JNK was only evident at the late phase, at 16 to 24 h. This observation suggests that early and late signaling of Jun activation may be different.
These preliminary data on hyperoxia signaling indicate that NF-B
translocation (and presumptive activation) is not a result of the
p42/p44 MAP kinase pathway, but probably a downstream consequence of
activation of the JNK pathway. Moreover, c-Jun and its phosphorylated
products are increased in a two-phase fashion, the first occurring
within 30 min, and the second after 16 h. Based on these
observations, and the fact that the Fos-Jun transcription complex AP-1
has been shown to have a role not only in the immediate-early response
to stress,32
but also in cell death,33
we
hypothesize a multiple signal transduction pathway in hyperoxia-induced
cell death. Specific elements of this working hypothesis are being
tested in our laboratory.
Acknowledgements
ACKNOWLEDGMENT: The experiments described were performed at and by members of the CardioPulmonary Research Institute, Mineola, NY.
References
B in preventing TNF-alpha-induced cell death. Science 274,782-784B. Science 274,787-789B activation prevents cell death. Cell 87,565-576[CrossRef][ISI][Medline]
B is activated by hyperoxia but does not protect from cell death. J Biol Chem 272,20646-20649This article has been cited by other articles:
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  D S Urquhart, H Montgomery, and A Jaffe Assessment of hypoxia in children with cystic fibrosis Arch. Dis. Child., November 1, 2005; 90(11): 1138 - 1143. [Abstract] [Full Text] [PDF]
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 S. V. Truong, M. M. Monick, T. O. Yarovinsky, L. S. Powers, T. Nyunoya, and G. W. Hunninghake Extracellular Signal-Regulated Kinase Activation Delays Hyperoxia-Induced Epithelial Cell Death in Conditions of Akt Downregulation Am. J. Respir. Cell Mol. Biol., December 1, 2004; 31(6): 611 - 618. [Abstract] [Full Text] [PDF]
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