The role of glycine in regulated cell death.

The cytoprotective effects of glycine against cell death have been recognized for over 28 years. They are expressed in multiple cell types and injury settings that lead to necrosis, but are still not widely appreciated or considered in the conceptualization of cell death pathways. In this paper, we review the available data on the expression of this phenomenon, its relationship to major pathophysiologic pathways that lead to cell death and immunomodulatory effects, the hypothesis that it involves suppression by glycine of the development of a hydrophilic death channel of molecular dimensions in the plasma membrane, and evidence for its impact on disease processes in vivo.

Manumycin A inhibits triple-negative breast cancer growth through LC3-mediated cytoplasmic vacuolation death.

Therapy resistance can be attributed to acquisition of anti-apoptotic mechanisms by the cancer cells. Therefore, developing approaches that trigger non-apoptotic cell death in cancer cells to compensate for apoptosis resistance will help to treat cancer effectively. Triple-negative breast cancers (TNBC) are among the most aggressive and therapy resistant to breast tumors. Here we report that manumycin A (Man A), an inhibitor of farnesyl protein transferase, reduces cancer cell viability through induction of non-apoptotic, non-autophagic cytoplasmic vacuolation death in TNBC cells. Man A persistently induced cytoplasmic vacuolation and cell death through the expression of microtubule-associated protein 1 light chain 3 (LC3) and p62 proteins along with endoplasmic reticulum (ER) stress markers, Bip and CHOP, and accumulation of ubiquitinated proteins. As inhibitors of apoptosis and autophagy failed to block cytoplasmic vacuolation and its associated protein expression or cell death, it appears that these processes are not involved in the death induced by Man A. Ability of thiol antioxidant, NAC in blocking Man A-induced vacuolation, death and its related protein expression suggests that sulfhydryl homeostasis may be the target of Man A. Surprisingly, normal human mammary epithelial cells failed to undergo cytoplasmic vacuolation and cell death, and grew normally in presence of Man A. In conjunction with its in vitro effects, Man A also reduced tumor burden in vivo in xenograft models that showed extensive cytoplasmic vacuoles and condensed nuclei with remarkable increase in the vacuolation-associated protein expression together with increase of p21, p27, PTEN and decrease of pAkt. Interestingly, Man A-mediated upregulation of p21, p27 and PTEN and downregulation of pAkt and tumor growth suppression were also mimicked by LC3 knockdown in MDA-MB-231 cells. Overall, these results suggest novel therapeutic actions by Man A through the induction of non-apoptotic and non-autophagic cytoplasmic vacuolation death by probably affecting ER stress, LC3 and p62 pathways in TNBC but not in normal mammary epithelial cells.

PI3K regulation of the SKP-2/p27 axis through mTORC2.

The cyclin-dependent kinase inhibitor p27 is a key regulator of cell-cycle progression. Its expression and localization are altered in several types of malignancies, which has prognostic significance in cancers such as renal cell carcinoma (RCC). S-phase kinase-associated protein 2 (SKP-2) is an F-box protein that is part of the SKP-1/Cul1/F-box ubiquitin ligase complex that targets nuclear p27 among many other cell-cycle proteins for proteosomal degradation. Its overexpression has been observed in several tumor types. Signaling by phosphoinositide 3-kinase (PI3K)/protein kinase B (AKT) has previously been shown to regulate the SKP-2/p27 axis. Recent evidence suggests that PI3K signaling may activate mammalian target of rapamycin complex 2 (mTORC2) activity. As PI3K signaling is known to regulate SKP-2 and p27, we sought to determine whether these effects were mediated by mTORC2. Here we provide additional genetic evidence that PI3K signaling activates mTORC2 kinase activity. We also demonstrate a novel role for mTORC2 in the modulation of nuclear p27 levels. In particular, mTORC2 signaling promotes the reduction of nuclear p27 protein levels through the increased protein expression of SKP-2. These are the first data to demonstrate a role for mTOR in the regulation of SKP-2. In concordance with these findings, mTORC2 activity promotes cell proliferation of RCC cells at the G1-S interphase of the cell cycle. Collectively, these data implicate mTORC2 signaling in the regulation of the SKP-2/p27 axis, a signaling node commonly altered in cancer.

Regulation of the mitochondrial permeability transition in kidney proximal tubules and its alteration during hypoxia-reoxygenation.

Development of the mitochondrial permeability transition (MPT) can importantly contribute to lethal cell injury from both necrosis and apoptosis, but its role varies considerably with both the type of cell and type of injury, and it can be strongly opposed by the normally abundant endogenous metabolites ADP and Mg(2+). To better characterize the MPT in kidney proximal tubule cells and assess its contribution to injury to them, we have refined and validated approaches to follow the process in whole kidney proximal tubules and studied its regulation in normoxic tubules and after hypoxia-reoxygenation (H/R). Physiological levels of ADP and Mg(2+) greatly decreased sensitivity to the MPT. Inhibition of cyclophilin D by cyclosporine A (CsA) effectively opposed the MPT only in the presence of ADP and/or Mg(2+). Nonesterified fatty acids (NEFA) had a large role in the decreased resistance to the MPT seen after H/R irrespective of the available substrate or the presence of ADP, Mg(2+), or CsA, but removal of NEFA was less effective at restoring normal resistance to the MPT in the presence of electron transport complex I-dependent substrates than with succinate. The data indicate that the NEFA accumulation that occurs during both hypoxia in vitro and ischemic acute kidney injury in vivo is a critical sensitizing factor for the MPT that overcomes the antagonistic effect of endogenous metabolites and cyclophilin D inhibition, particularly in the presence of complex I-dependent substrates, which predominate in vivo.

A novel role for MAP1 LC3 in nonautophagic cytoplasmic vacuolation death of cancer cells.

Thiol reactive cyclopentenone prostaglandin, 15-deoxy-Delta(12,14)-prostaglandin J(2) (15d-PGJ2), induced a novel, nonapoptotic and microtubule-associated protein 1 light chain 3 (MAP1 LC3) dependent but nonautophagic form of cell death in colon, breast and prostate cancer cell lines, characterized by extensive cytoplasmic vacuolation with dilatation of endoplasmic reticulum (ER). Disruption of sulfhydryl homeostasis, which resulted in ER stress, accumulation of ubiquitinated proteins and subsequent ER dilation, contributed to peroxisome proliferator-activated receptor gamma (PPARgamma)-independent cell death by 15d-PGJ2. Absence of intracellular organelles in these vacuoles, shown by electron microscopy and unique fragmentation of lamin B, suggested this form of cell death to be different from autophagy and apoptosis. Cell death induced by 15d-PGJ2 is prevented by cycloheximide and actinomycin D, suggesting a requirement of new protein synthesis for death with cytoplasmic vacuolation. Here, we report for the first time that upregulation and processing of autophagy marker LC3 is an important event in nonautophagic cytoplasmic vacuolation and cell death. Notably, knockdown of LC3 conferred significant protection against 15d-PGJ2-induced cytoplasmic vacuolation and cell death, suggesting a novel role of LC3 in a death process other than autophagy.

cDNA cloning and promoter analysis of rat caspase-9.

Caspase-9 is the apex caspase of the mitochondrial pathway of apoptosis, which plays a critical role in apoptotic initiation and progression. However, gene regulation of caspase-9 is largely unknown. This is in part due to the lack of information on the gene promoter. Here we have cloned the full-length cDNA of rat caspase-9 and have isolated promoter regions of this gene. The rat caspase-9 cDNA of 2058 bp predicts a protein of 454 amino acids, which contains a caspase-recruitment domain ('CARD') at the N-terminus and enzymic domains at the C-terminus. The enzyme's active site, with a characteristic motif of QACGG, was also identified. Overall, rat and human caspase-9 have 71% identity. With the cDNA sequence, we subsequently isolated the proximal 5'-flanking regions of rat caspase-9 by the procedure of genomic walking. The 2270 bp genomic segment is 'TATA-less', but contains several GC boxes. Elements binding known transcription factors such as Sp-1, Pit-1, CCAAT-enhancer-binding protein (C/EBP), glucocorticoid receptor and hypoxia-inducible factor 1 (HIF-1) were also identified. When cloned into reporter gene vectors, the genomic segment showed significant promoter activity, indicating that the 5'-flanking regions isolated by genomic walking contain the gene promoter of rat caspase-9. Of significance is that the cloned promoter segments were activated by severe hypoxia, conditions inducing caspase-9 transcription. Thus, the genomic sequences reported here contain not only the basal promoter of rat caspase-9 but also regulatory elements responsive to pathophysiological stimuli including hypoxia.

Energetic determinants of tyrosine phosphorylation of focal adhesion proteins during hypoxia/reoxygenation of kidney proximal tubules.

Anaerobic mitochondrial metabolism of alpha-ketoglutarate and aspartate or alpha-ketoglutarate and malate can prevent and reverse severe mitochondrial dysfunction during reoxygenation after 60 minutes of hypoxia in kidney proximal tubules.(34) The present studies demonstrate that, during hypoxia, paxillin, focal adhesion kinase, and p130(cas) migrated faster by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, their phosphotyrosine (pY) content decreased to approximately 5% of that in oxygenated tubules without changes in total protein, and the normally basal immunostaining of beta1 and alpha6 integrin subunits, pY, and paxillin was lost or markedly decreased. During reoxygenation without supplemental substrates, recovery of pY and basal localization of the focal adhesion proteins was poor. alpha-Ketoglutarate and aspartate, which maintained slightly higher levels of ATP during hypoxia, also maintained 2.5-fold higher levels of pY during this period, and promoted full recovery of pY content and basal localization of focal adhesion proteins during subsequent reoxygenation. Similarly complete recovery was made possible by provision of alpha-ketoglutarate and aspartate or alpha-ketoglutarate and malate only during reoxygenation. These data emphasize the importance of very low energy thresholds for maintaining the integrity of key structural and biochemical components required for cellular survival and reaffirm the value of approaches aimed at conserving or generating energy in cells injured by hypoxia or ischemia.

Protection of ATP-depleted cells by impermeant strychnine derivatives: implications for glycine cytoprotection.

Glycine and structurally related amino acids with activities at chloride channel receptors in the central nervous system also have robust protective effects against cell injury by ATP depletion. The glycine receptor antagonist strychnine shares this protective activity. An essential step toward identification of the molecular targets for these compounds is to determine whether they protect cells through interactions with intracellular targets or with molecules on the outer surface of plasma membranes. Here we report cytoprotection by a cell-impermeant derivative of strychnine. A strychnine-fluorescein conjugate (SF) was synthesized, and impermeability of plasma membranes to this compound was verified by fluorescence confocal microscopy. In an injury model of Madin-Darby canine kidney cells, ATP depletion led to lactate dehydrogenase release. SF prevented lactate dehydrogenase leakage without ameliorating ATP depletion. This was accompanied by preservation of cellular ultrastructure and exclusion of vital dyes. SF protection was also shown for ATP-depleted rat hepatocytes. On the other hand, when a key structural motif in the active site of strychnine was chemically blocked, the SF lost its protective effect, establishing strychnine-related specificity for SF protection. Cytoprotective effects of the cell-impermeant strychnine derivative provide compelling evidence suggesting that molecular targets on the outer surface of plasma membranes may mediate cytoprotection by strychnine and glycine.

Up-regulation of apoptosis inhibitory protein IAP-2 by hypoxia. Hif-1-independent mechanisms.

Hypoxia is a key determinant of tissue pathology during tumor development and organ ischemia. However, little is known regarding hypoxic regulation of genes that are directly involved in cell death or death resistance. Here we report the striking induction by severe hypoxia of the anti-apoptotic protein IAP-2. Hypoxic cells with IAP-2 up-regulation became resistant to apoptosis. IAP-2 was induced by hypoxia per se rather than by the secondary effects of hypoxia, including ATP depletion and cell injury. The inductive response did not relate to alterations of cellular redox status or arrest of mitochondrial respiration. On the other hand, IAP-2 induction was attenuated by actinomycin D, suggesting a role for gene transcription. In vitro nuclear run-on assays demonstrated specific increases in IAP-2 transcriptional activity after hypoxia exposure. HIF-1, the primary transcription factor that is responsible for multiple gene activation under hypoxia, does not have a role in IAP-2 expression. HIF-1 and IAP-2 were induced by different degrees of hypoxia; severe hypoxia or anoxia was required for IAP-2 induction. Moreover, cobalt chloride and desferrioxamine activated HIF-1 but not IAP-2. Finally, IAP-2 was induced by severe hypoxia in mouse embryonic stem cells that were deficient of HIF-1. Thus, this study not only provides the first demonstration of hypoxic regulation of an anti-apoptotic gene but also suggests the participation of novel hypoxia-responsive transcription mechanisms.

Bcl-2 prevents Bax oligomerization in the mitochondrial outer membrane.

ATP depletion results in Bax translocation from cytosol to mitochondria and release of cytochrome c from mitochondria into cytosol in cultured kidney cells. Overexpression of Bcl-2 prevents cytochrome c release, without ameliorating ATP depletion or Bax translocation, with little or no association between Bcl-2 and Bax as demonstrated by immunoprecipitation (Saikumar, P., Dong, Z., Patel, Y., Hall, K., Hopfer, U., Weinberg, J. M., and Venkatachalam, M. A. (1998) Oncogene 17, 3401-3415). Now we show that translocated Bax forms homo-oligomeric structures, stabilized as chemical adducts by bifunctional cross-linkers in ATP-depleted wild type cells, but remains monomeric in Bcl-2-overexpressing cells. The protective effects of Bcl-2 did not require Bcl-2/Bax association, at least to a degree of proximity or affinity that was stable to conditions of immunoprecipitation or adduct formation by eight cross-linkers of diverse spacer lengths and chemical reactivities. On the other hand, nonionic detergents readily induced homodimers and heterodimers of Bax and Bcl-2. Moreover, associations between translocated Bax and the voltage-dependent anion channel protein or the adenine nucleotide translocator protein could not be demonstrated by immunoprecipitation of Bax, or by using bifunctional cross-linkers. Our data suggest that the in vivo actions of Bax are at least in part dependent on the formation of homo-oligomers without requiring associations with other molecules and that Bcl-2 cytoprotection involves mechanisms that prevent Bax oligomerization.

Anaerobic and aerobic pathways for salvage of proximal tubules from hypoxia-induced mitochondrial injury.

We have further examined the mechanisms for a severe mitochondrial energetic deficit, deenergization, and impaired respiration in complex I that develop in kidney proximal tubules during hypoxia-reoxygenation, and their prevention and reversal by supplementation with alpha-ketoglutarate (alpha-KG) + aspartate. The abnormalities preceded the mitochondrial permeability transition and cytochrome c loss. Anaerobic metabolism of alpha-KG + aspartate generated ATP and maintained mitochondrial membrane potential. Other citric-acid cycle intermediates that can promote anaerobic metabolism (malate and fumarate) were also effective singly or in combination with alpha-KG. Succinate, the end product of these anaerobic pathways that can bypass complex I, was not protective when provided only during hypoxia. However, during reoxygenation, succinate also rescued the tubules, and its benefit, like that of alpha-KG + malate, persisted after the extra substrate was withdrawn. Thus proximal tubules can be salvaged from hypoxia-reoxygenation mitochondrial injury by both anaerobic metabolism of citric-acid cycle intermediates and aerobic metabolism of succinate. These results bear on the understanding of a fundamental mode of mitochondrial dysfunction during tubule injury and on strategies to prevent and reverse it.

Serine protease inhibitors suppress cytochrome c-mediatedcaspase-9 activation and apoptosis during hypoxia-reoxygenation.

We have shown that reoxygenation of hypoxic rat kidney proximaltubule cells leads to apoptosis. This is mediated by translocation ofBax from the cytosol to mitochondria, accompanied by release ofmitochondrial cytochrome c (cyt.c). The present studyhas examined the proteolytic mechanisms responsible for apoptosisduring hypoxia-reoxygenation. Caspases were activated duringhypoxia, as shown by cleavage of fluorogenic peptide substrates. By5 h caspase-3-like activity to cleave carbobenzoxy-Asp-Glu-Val-Asp-7-amino-4-trifluoromethyl coumarin was increased approx. 30-fold. Thiswas accompanied by specific processing of pro-caspase-3, -8 and -9 intoactive forms. Caspase activation during hypoxia was blocked bycarbobenzoxy-Val-Ala-Asp-fluoromethyl ketone and overexpression of Bcl-2. Of particular interest, caspase activation was also suppressed bythe chymotryptic inhibitors N-tosyl-L-phenylalaninechloromethyl ketone (TPCK) and Ala-Pro-Phe chloromethyl ketone (APF),and the general serine protease inhibitor 4-(2-aminoethyl)benzenesulphonyl fluoride. Inhibition of caspase activationby these compounds resulted in arrest of apoptosis. On the other hand,the serine protease inhibitors did not prevent release of mitochondrialcyt.c during hypoxia, suggesting that these compounds blockeda critical step in post-mitochondrial caspase activation. Furtherstudies using an in vitro reconstitution model showedthat cyt. c/dATP stimulated caspase-9 processing and downstreamcaspase activation were significantly suppressed in the presence ofTPCK and APF. Based on these results, we speculate that serineproteases may be involved in post-mitochondrial apoptotic events thatlead to activation of the initiator, caspase-9.

Mitochondrial dysfunction during hypoxia/reoxygenation and its correction by anaerobic metabolism of citric acid cycle intermediates.

Kidney proximal tubule cells developed severe energy deficits during hypoxia/reoxygenation not attributable to cellular disruption, lack of purine precursors, the mitochondrial permeability transition, or loss of cytochrome c. Reoxygenated cells showed decreased respiration with complex I substrates, but minimal or no impairment with electron donors at complexes II and IV. This was accompanied by diminished mitochondrial membrane potential (DeltaPsi(m)). The energy deficit, respiratory inhibition, and loss of DeltaPsi(m) were strongly ameliorated by provision of alpha-ketoglutarate plus aspartate (alphaKG/ASP) supplements during either hypoxia or only during reoxygenation. Measurements of (13)C-labeled metabolites in [3-(13)C]aspartate-treated cells indicated the operation of anaerobic pathways of alphaKG/ASP metabolism to generate ATP, yielding succinate as end product. Anaerobic metabolism of alphaKG/ASP also mitigated the loss of DeltaPsi(m) that occurred during hypoxia before reoxygenation. Rotenone, but not antimycin or oligomycin, prevented this effect, indicating that electron transport in complex I, rather than F(1)F(0)-ATPase activity, had been responsible for maintenance of DeltaPsi(m) by the substrates. Thus, tubule cells subjected to hypoxia/reoxygenation can have persistent energy deficits associated with complex I dysfunction for substantial periods of time before onset of the mitochondrial permeability transition and/or loss of cytochrome c. The lesion can be prevented or reversed by citric acid cycle metabolites that anaerobically generate ATP by intramitochondrial substrate-level phosphorylation and maintain DeltaPsi(m) via electron transport in complex I. Utilization of these anaerobic pathways of mitochondrial energy metabolism known to be present in other mammalian tissues may provide strategies to limit mitochondrial dysfunction and allow cellular repair before the onset of irreversible injury by ischemia or hypoxia.

Development of porous defects in plasma membranes of adenosine triphosphate-depleted Madin-Darby canine kidney cells and its inhibition by glycine.

Studies during the past decade have led to the recognition of a fundamental, widely expressed mechanism of structural damage in energy-deprived cells, which is suppressed by physiologic levels of glycine and is independent of Ca2+ availability or alterations of cytosolic free Ca2+. To gain insight into this process, Madin-Darby canine kidney (MDCK) cells were depleted of adenosine triphosphate (ATP) by a mitochondrial uncoupler in glucose-free medium, and intracellular free Ca2+ was clamped at 100 nM to avoid calcium cytotoxicity. Although the ATP-depleted cells swelled and blebbed and their plasma membranes appeared to be under tension, they nevertheless became permeable to macromolecules. The plasma membranes of these cells retained structural continuity, as determined by morphologic observations, and confocal microscopy of a plasma membrane protein label (Biotin: Ultra Avidin-Texas Red) and a lipid label (NBD-sphingomyelin). Using fluoresceinated dextrans of graded molecular size, membrane permselectivity was examined noninvasively by confocal microscopy. Measured as inside/outside ratios of fluorescence intensity, the permeability indices showed progressively greater restriction to diffusion of increasingly larger dextran molecules across plasma membranes, with sharp break-points between 70,000 and 145,000 daltons (d). The results indicated that the membranes behaved as if they were perforated by water-filled channels or "pores," with size-exclusion limits of molecular dimensions. The membrane defects evolved from small pores permeable only to propidium iodide (668 d) and the smallest dextran (4,000 d), before enlarging with time to become permeable to larger dextrans. Inclusion of glycine during ATP depletion did not affect cell swelling or blebbing but completely prevented the development of permeability defects. Treatment of cells before ATP depletion with a membrane-impermeant homobifunctional "nearest neighbor" cross-linking agent, 3,3' dithiobis(sulfosuccinimidylpropionate), suppressed the development of permeability defects, even in the absence of glycine. These observations suggest that the cellular abnormality that is suppressed by glycine involves rearrangement of plasma membrane proteins to form water-filled pores large enough to leak macromolecules.

Intracellular Ca2+ thresholds that determine survival or death of energy-deprived cells.

Increase of intracellular ionized or free Ca2+ is thought to play a central role in cell death due to ATP depletion. However, concurrently operative mechanisms of injury that do not require intracellular Ca2+ increases have made it difficult to test this hypothesis or to determine the concentrations at which intracellular Ca2+ becomes lethal. The predominant Ca2+-independent mechanism of injury during ATP depletion involves the loss of cellular glycine. This type of damage can be fully inhibited by adding the amino acid exogenously. Using glycine to suppress Ca2+-independent plasma membrane damage, we have examined the effect of intracellular Ca2+ elevations on cell viability during ATP depletion. Madin-Darby canine kidney (MDCK) cells were depleted of ATP by incubation with a mitochondrial uncoupler in glucose-free medium. Free Ca2+ concentration in the medium was varied between 26 nmol/L and 1.25 mmol/L in the presence of a Ca2+ ionophore. Measurements with the Ca2+ probes fura-2, furaptra, and fura-2FF showed that intracellular Ca2+ was clamped at extracellular levels under these conditions. Cell survival during ATP depletion was indicated by viable cells recovered 24 hours later. The results show that ATP-depleted cells can sustain high levels of intracellular Ca2+ (100 micromol/L) for prolonged periods and remain viable if plasma membrane damage is prevented by glycine. Cell death was observed only when intracellular free Ca2+ was allowed to increase beyond 100 micromol/L, and this was associated with dramatic nuclear alterations: chromatin condensation, loss of nuclear lamins, and breakdown of DNA into large 50- to 150-kb fragments. Our studies demonstrate unexpectedly high resistance of cells to calcium cytotoxicity if glycine that is lost during ATP depletion is restored. In addition, they provide insights into novel mechanisms of nuclear disintegration and DNA damage that are triggered when the high thresholds of intracellular Ca2+ required for cell death are exceeded.

Role of hypoxia-induced Bax translocation and cytochrome c release in reoxygenation injury.

We investigated mechanisms of cell death during hypoxia/reoxygenation of cultured kidney cells. During glucose-free hypoxia, cell ATP levels declined steeply resulting in the translocation of Bax from cytosol to mitochondria. Concurrently, there was cytochrome c release and caspase activation. Cells that leaked cytochrome c underwent apoptosis after reoxygenation. ATP depletion induced by a mitochondrial uncoupler resulted in similar alterations even in the presence of oxygen. Moreover, inclusion of glucose during hypoxia prevented protein translocations and reoxygenation injury by maintaining intracellular ATP. Thus, ATP depletion, rather than hypoxia per se, was the cause of protein translocations. Overexpression of Bcl-2 prevented cytochrome c release and reoxygenation injury without ameliorating ATP depletion or Bax translocation. On the other hand, caspase inhibitors did not prevent protein translocations, but inhibited apoptosis during reoxygenation. Nevertheless, they could not confer long-term viability, since mitochondria had been damaged. Omission of glucose during reoxygenation resulted in continued failure of ATP production, and cell death with necrotic morphology. In contrast, cells expressing Bcl-2 had functional mitochondria and remained viable during reoxygenation even without glucose. Therefore, Bax translocation during hypoxia is a molecular trigger for cell death during reoxygenation. If ATP is available during reoxygenation, apoptosis develops; otherwise, death occurs by necrosis. By preserving mitochondrial integrity, BCL-2 prevents both forms of cell death and ensures cell viability.

Mechanisms of cell death in hypoxia/reoxygenation injury.

Investigation of death pathways during cell injury in vivo caused by ischemia and reperfusion is of clinical importance, but technically difficult. Heterogeneity of cell types, differences between organ systems, diversity of death paradigms and exacerbation of tissue damage caused by inflammation are only some of the variables that need to be taken into account. With respect to the identification of necrosis and apoptosis in affected organs, technical issues related to preparation artifacts, occurrence of internucleosomal DNA cleavage in necrotic as well as apoptotic cells and other overlaps in death pathways bear consideration. In that caspase independent as well as caspase dependent processes cause cell death and that caspase inhibitors can act as anti-inflammatory agents, evaluation of ischemic death mechanisms in parenchymal cells needs to be performed with caution. When the effects of inflammation are removed by appropriate in vitro studies using purified or cultured cells, several mitochondrial factors that lead to cell death can be studied. Substantial evidence exists for the participation of electron transport defects, mitochondrial permeability transitions (MPT) and release of cytochrome c from mitochondria, effected by pro-apoptotic proteins such as Bax. The anti-apoptotic protein Bcl-2 exerts an overriding protective role in this type of injury by preserving mitochondrial structure and function. In contrast, caspase inhibitors cannot offer long-term protection to ischemically injured parenchymal cells regardless of how effectively they can inhibit apoptotic events, if the cells have suffered permanent mitochondrial damage impairing respiration.