Calpain I induces cleavage and release of apoptosis-inducing factor from isolated mitochondria

The translocation of apoptosis-inducing factor (AIF) from mitochondria to the nucleus has been implicated in the mechanism of glutamate excitotoxicity in cortical neurons and has been observed in vivo following acute rodent brain injuries. However, the mechanism and time course of AIF redistribution to the nucleus is highly controversial. Because elevated intracellular calcium is one of the most ubiquitous features of neuronal cell death, this study tested the hypothesis that cleavage of AIF by the calcium-activated protease calpain mediates its release from mitochondria. Both precursor and mature forms of recombinant AIF were cleaved near the amino terminus by calpain I in vitro. Mitochondrial outer membrane permeabilization by truncated Bid induced cytochrome c release from isolated liver or brain mitochondria but only induced AIF release in the presence of active calpain. Enzymatic inhibition of calpain by calpeptin precluded AIF release, demonstrating that proteolytic activity was required for release. Calpeptin and the mitochondrial permeability transition pore antagonist cyclosporin A also inhibited calcium-induced AIF release from mouse liver mitochondria, implicating the involvement of an endogenous mitochondrial calpain in release of AIF during permeability transition. Cleavage of AIF directly decreased its association with pure lipid vesicles of mitochondrial inner membrane composition. Taken together, these results define a novel mechanism of AIF release involving calpain processing and identify a potential molecular checkpoint for cytoprotective interventions.     

Calpain activation is not required for AIF translocation in PARP-1-dependent cell death (parthanatos)

Apoptosis-inducing factor (AIF) is critical for poly(ADP-ribose) polymerase-1 (PARP-1)-dependent cell death (parthanatos). The molecular mechanism of mitochondrial AIF release to the nucleus remains obscure, although a possible role of calpain I has been suggested. Here we show that calpain is not required for mitochondrial AIF release in parthanatos. Although calpain I cleaved recombinant AIF in a cell-free system in intact cells under conditions where endogenous calpain was activated by either NMDA or N -methyl- N '-nitro- N -nitrosoguanidine (MNNG) administration, AIF was not cleaved, and it was released from mitochondria to the nucleus in its 62-kDa uncleaved form. Moreover, NMDA administration under conditions that failed to activate calpain still robustly induced AIF nuclear translocation. Inhibition of calpain with calpastatin or genetic knockout of the regulatory subunit of calpain failed to prevent NMDA- or MNNG-induced AIF nuclear translocation and subsequent cell death, respectively, which was markedly prevented by the PARP-1 inhibitor, 3,4-dihydro-5-[4-(1-piperidinyl)butoxyl]-1(2H)-iso-quinolinone. Our study clearly shows that calpain activation is not required for AIF release during parthanatos, suggesting that other mechanisms rather than calpain are involved in mitochondrial AIF release in parthanatos.     

Bcl-2-family proteins and the role of mitochondria in apoptosis

Mitochondria are central to many forms of cell death, usually via the release of pro-apoptotic proteins from the mitochondrial intermembrane space. Some intermembrane space proteins, including cytochrome c, Smac/DIABLO, and Omi/Htra2, can induce or enhance caspase activation, whereas others, such as AIF and endonuclease G, might act in a caspase-independent manner. Intermembrane space protein release is often regulated by Bcl-2-family proteins. Recent evidence suggests that pro-apoptotic members of this family, by themselves, can permeabilize the outer mitochondrial membrane without otherwise damaging mitochondria. Mitochondria can contribute to cell death in other ways. For example, they can respond to calcium release from the endoplasmic reticulum by undergoing the mitochondrial permeability transition, which in turn causes outer membrane rupture and the release of intermembrane space proteins. Bcl-2-family proteins can influence the levels of releasable Ca(2+) in the endoplasmic reticulum, and thus determine whether the released Ca(2+) is sufficient to overload mitochondria and induce cell death.     

Oxidative modification sensitizes mitochondrial apoptosis-inducing factor to calpain-mediated processing

Although processing of mitochondrial apoptosis-inducing factor (AIF) is essential for its function during apoptosis in most cell types, the detailed mechanisms of AIF cleavage remain elusive. Recent findings indicate that the proteolytic process is Ca²⁺-dependent and that it is mediated by a calpain located in the mitochondrial intermembrane space. We can now report that, in addition to a sustained intracellular Ca²⁺ elevation, enhanced formation of reactive oxygen species (ROS) is a prerequisite step for AIF to be cleaved and released from mitochondria in staurosporine-treated cells. These events occurred independent of the redox state of the mitochondria and were not influenced by binding of pyridine nucleotides to AIF. Chelation of cytosolic Ca²⁺ by BAPTA/AM suppressed the elevation of both Ca²⁺ and ROS, suggesting that the Ca²⁺ rise was the most upstream signal required for AIF processing. We could further show that the stimulated ROS production leads to oxidative modification (carbonylation) of AIF, which markedly increases its rate of cleavage by calpain. Accordingly, pretreatment of the cells with antioxidants blocked AIF carbonylation, as well as its subsequent cleavage and release from the mitochondria. Combined, our data provide evidence that ROS-mediated, posttranslational modification of AIF is critical for its cleavage by calpain and thus for AIF-mediated cell death.     

Calpain activation after mitochondrial permeability transition in microcystin-induced cell death in rat hepatocytes

Previous studies have shown that microcystin-LR (MLR), a specific hepatotoxin, induces onset of mitochondrial permeability transition (MPT) and apoptosis in cultured rat hepatocytes. Here we attempted to investigate the downstream events after the onset of MPT in MLR-treated hepatocytes. Various mitochondrial electron transport chain (ETC) inhibitors effectively prevented the onset of MPT, suggesting that the mitochondrial ETC plays an important role in MLR-induced MPT. MLR also induced mitochondrial cytochrome c release, which can be prevented by a specific MPT inhibitor (cyclosporin A, CsA), and by various ETC inhibitors. Interestingly, the release of cytochrome c did not activate caspase-9 and -3, the main caspases involved in apoptosis. Instead, MLR activated calpain in rat hepatocytes, probably through the increase of intracellular Ca(2+) released from mitochondria. Both ALLN and ALLM, two calpain inhibitors, significantly blocked MLR-induced calpain activation and subsequent cell death. CsA also prevented MLR-induced calpain activation and cell death, suggesting that the activation of calpain may be a post-mitochondrial event. These data demonstrate for the first time that calpain rather than caspases plays an important role in MLR-induced apoptosis.     

Bid-induced release of AIF from mitochondria causes immediate neuronal cell death

Mitochondrial dysfunction and release of pro-apoptotic factors such as cytochrome c or apoptosis-inducing factor (AIF) from mitochondria are key features of neuronal cell death. The precise mechanisms of how these proteins are released from mitochondria and their particular role in neuronal cell death signaling are however largely unknown. Here, we demonstrate by fluorescence video microscopy that 8-10 h after induction of glutamate toxicity, AIF rapidly translocates from mitochondria to the nucleus and induces nuclear fragmentation and cell death within only a few minutes. This markedly fast translocation of AIF to the nucleus is preceded by increasing translocation of the pro-apoptotic bcl-2 family member Bid (BH3-interacting domain death agonist) to mitochondria, perinuclear accumulation of Bid-loaded mitochondria, and loss of mitochondrial membrane integrity. A small molecule Bid inhibitor preserved mitochondrial membrane potential, prevented nuclear translocation of AIF, and abrogated glutamate-induced neuronal cell death, as shown by experiments using Bid small interfering RNA (siRNA). Cell death induced by truncated Bid was inhibited by AIF siRNA, indicating that caspase-independent AIF signaling is the main pathway through which Bid mediates cell death. This was further supported by experiments showing that although caspase-3 was activated, specific caspase-3 inhibition did not protect neuronal cells against glutamate toxicity. In conclusion, Bid-mediated mitochondrial release of AIF followed by rapid nuclear translocation is a major mechanism of glutamate-induced neuronal death.     

Characteristics of mitochondrial calpains

Calpains are considered to be cytoplasmic enzymes, although several studies have shown that calpain-like protease activities also exist in mitochondria. We partially purified mitochondrial calpain from swine liver mitochondria and characterized. Only one type of mitochondrial calpain was detected by the column chromatographies. The mitochondrial calpain was stained with anti-mu-calpain and calpain small subunit antibodies. The susceptibility of mitochondrial calpain to calpain inhibitors and the optimum pH differ from those of cytosolic mu- and m-calpains. The Ca(2+)-dependency of mitochondrial calpain was similar to that of cytosolic mu-calpain. Therefore, we named the protease mitochondrial mu-like calpain. In zymogram analysis, two types of caseinolytic enzymes existed in mitochondria and showed different mobilities from cytosolic mu- and m-calpains. The upper major band was stained with anti-mu-calpain and calpain small subunit antibodies (mitochondrial calpain I, mitochondrial mu-like calpain). The lower band was stained only with anti-calpain small subunit antibody (mitochondrial calpain II, unknown mitochondrial calpain). Calpastatin was not detected in mitochondrial compartments. The mitochondrial calpain processed apoptosis-inducing factor (AIF) to truncated AIF (tAIF), releasing tAIF into the intermembrane space. These results indicate that mitochondrial calpain, which differs from mu- and m-calpains, seems to be a ubiquitous calpain and may play a role in mitochondrial apoptotic signalling.     

Involvement of endoplasmic reticulum in paclitaxel-induced apoptosis

The participation of the mitochondrial pathway in paclitaxel-induced apoptosis has been well documented. After addition of paclitaxel to U937 cells, however, we observed an early expression of five endoplasmic reticulum (ER) stress response genes that preceded the release of cytochrome c from the mitochondria and the cleavage of the caspases. Involvement of the ER was supported by the following evidence. Paclitaxel treatment not only activated calpain and caspase-4, but also induced a gradual increase in the cytosolic Ca(2+) concentration at 3-6 h. Paclitaxel-induced apoptosis can be inhibited by the calpain inhibitor calpeptin and IP(3) receptor inhibitors. Either buffering of the cytosolic Ca(2+) or inhibition of mitochondrial calcium uptake reduced BiP expression. These inhibitors also reduced mitochondrial apoptotic signals, such as mitochondrion membrane potential disruption, cytochrome c release and eventually reduced the death of U937 cells. Paclitaxel-induced Bax/Bak translocation to the ER and Bax dimerization on the ER membrane occurred within 3 h, which led to a Ca(2+) efflux into cytosol. Moreover, we found that cytochrome c translocated to the ER after releasing from mitochondria and then interacted with the IP(3) receptor at 12-15 h. This phenomenon has been known to amplify apoptotic signaling. Taken together, ER would seem to contribute to paclitaxel-induced apoptosis via both the early release of Ca(2+) and the late amplification of mitochondria-mediated apoptotic signals.     

Calcium release from the rat liver mitochondria during collapse of the membrane potential

Ca(2+)-release from rat liver mitochondria after protonophore (carbonyl cyanide m-chlorophenylhydrazone, CCCP)-induced membrane depolarisation is studied. It is shown that the release of calcium is accompanied by an increase of the inner mitochondrial membrane permeability as the result of the opening of permeability transition pore (PTP). Calcium is released from mitochondria through the uniporter working in reverse mode and also by PTP mechanism which accounts for ruthenium red (RR)-insensitive component of total. Ca(2+)-release. Unlike Ca2+, the strontium release from the mitochondria is completely sensitive to RR, specific uniporter blocker, which shows the absence of rapid Sr(2+)-efflux mechanisms other than uniporter of bivalent cations. The data obtained also give an evidence that the lifetime of the open state of the pore is limited, and barrier properties of the mitochondrial membrane are restored after the closure of the pore.     

Mitochondrial regulation of cell death: Processing of apoptosis-inducing factor (AIF)

Apoptosis might proceed through the activation of both caspase-dependent and -independent pathways. Apoptosis-inducing factor (AIF) was discovered as the first protein that mediated caspase-independent cell death. Initially, it was regarded as a soluble protein residing in the intermembrane space of mitochondria, from where it could be exported to the nucleus to participate in large-scale DNA fragmentation and chromatin condensation. However, later it was demonstrated that AIF is N-terminally anchored to the inner mitochondrial membrane. Hence, AIF must be liberated from its membrane anchor prior to being released into the cytosol. The current knowledge about the molecular mechanisms regulating the processing and release of AIF from the mitochondria will be summarized and discussed in this review.     

Fe(2+) induces a transient Ca(2+) release from rat liver mitochondria

Isolated mitochondria loaded with Ca(2+) and then exposed to Fe(2+) show a transient release of Ca(2+). The magnitude of this response depends on the Ca(2+) loading and the kinetics of the response depends on the concentration of added Fe(2+). We investigated the Fe(2+)-induced Ca(2+) release mechanism by measuring mitochondrial Ca(2+) uptake in the presence of Fe(2+). The presence of Fe(2+) inhibits Ca(2+) uptake two times. Since mitochondria can cycle Ca(2+) across their inner membrane, the suppression of Ca(2+) uptake, but not release, results in an elevation of the extramitochondrial Ca(2+), thereby varying the steady state. The transient release of Ca(2+) initially observed from mitochondria appears to occur via the electroneutral 2H(+)/Ca(2+)-exchange mechanism, since it can be markedly decreased by cyclosporin A and does not involve lipid peroxidation. When Fe(2+) accumulation is completed, reuptake of released Ca(2+) into mitochondria resumes. Finally, we propose that Fe(2+) either inhibits Ca(2+) entry at the uniporter or is transported by it into the matrix.     

Mitochondrial modulation of calcium signaling at the initiation of development

Fertilization triggers cytosolic Ca(2+) oscillations that activate mammalian eggs and initiate development. Extensive evidence demonstrates that Ca(2+) is released from endoplasmic reticulum stores; however, less is known about how the increased Ca(2+) is restored to its resting level, forming the Ca(2+) oscillations. We investigated whether mitochondria also play a role in activation-associated Ca(2+) signaling. Mitochondrial dysfunction induced by the mitochondrial uncoupler FCCP or antimycin A disrupted cytosolic Ca(2+) oscillations, resulting in sustained increase in cytosolic Ca(2+), followed by apoptotic cell death. This suggests that functional mitochondria may participate in sequestering the released Ca(2+), contributing to cytosolic Ca(2+) oscillations and preventing cell death. By centrifugation, mouse eggs were stratified and separated into fractions containing both endoplasmic reticulum and mitochondria and fractions containing endoplasmic reticulum with no mitochondria. The former showed Ca(2+) oscillations by activation, whereas the latter exhibited sustained elevation in cytosolic Ca(2+) but no Ca(2+) oscillations, suggesting that mitochondria take up released cytosolic Ca(2+). Further, using Rhod-2 for detection of mitochondrial Ca(2+), we found that mitochondria exhibited Ca(2+) oscillations, the frequency of which was not different from that of cytosolic Ca(2+) oscillations, indicating that mitochondria are involved in Ca(2+) signaling during egg activation. Therefore, we propose that mitochondria play a crucial role in Ca(2+) signaling that mediates egg activation and development, and apoptotic cell death.     

Outer mitochondrial membrane localization of apoptosis-inducing factor: mechanistic implications for release

Poly(ADP-ribose) polymerase-1-dependent cell death (known as parthanatos) plays a pivotal role in many clinically important events including ischaemia/reperfusion injury and glutamate excitotoxicity. A recent study by us has shown that uncleaved AIF (apoptosis-inducing factor), but not calpain-hydrolysed truncated-AIF, was rapidly released from the mitochondria during parthanatos, implicating a second pool of AIF that might be present in brain mitochondria contributing to the rapid release. In the present study, a novel AIF pool is revealed in brain mitochondria by multiple biochemical analyses. Approx. 30% of AIF loosely associates with the outer mitochondrial membrane on the cytosolic side, in addition to its main localization in the mitochondrial intermembrane space attached to the inner membrane. Immunogold electron microscopic analysis of mouse brain further supports AIF association with the outer, as well as the inner, mitochondrial membrane in vivo. In line with these observations, approx. 20% of uncleaved AIF rapidly translocates to the nucleus and functionally causes neuronal death upon NMDA (N-methyl-d-aspartate) treatment. In the present study we show for the first time a second pool of AIF in brain mitochondria and demonstrate that this pool does not require cleavage and that it contributes to the rapid release of AIF. Moreover, these results suggest that this outer mitochondrial pool of AIF is sufficient to cause cell death during parthanatos. Interfering with the release of this outer mitochondrial pool of AIF during cell injury paradigms that use parthanatos hold particular promise for novel therapies to treat neurological disorders.     

Proton transport is necessary for divalent metal cations release from deenergized mitochondria

The release of divalent cations (Ca2+ and Sr2+) from rat liver mitochondria after membrane depolarization with protonophore (carbonyl cyanide m-chlorophenyl hydrazone, CCCP), sodium azide and K(+)-ionophore (valinomycin) was studied. It is stated that membrane depolarization itself is not sufficient for cations release from mitochondrial matrix (provided that mitochondrial permeability transition pore is blocked by cyclosporin A). Complete delivering of divalent cations is observed only after protonophore (CCCP) addition to suspension of deenergized mitochondria. The data show that membrane permeabilisation to hydrogen ions (H+) is necessary for complete cation release from the mitochondrial matrix. The enhancement in K(+)-conductivity of mitochondrial membrane (by valinomycin), on the contrary, is not able to provide complete delivering of cations from mitochondria. It is shown that quantity of divalent metal cation released from mitochondria (depolarized and permeabilized for K+ as well) is proportional to the concentration of protonophore (but not K(+)-ionophore) introduced in the incubation medium. The data obtained lead to the conclusion that H(+)-permeabilization of the mitochondrial membrane is necessary for the complete release of Ca2+ and Sr2+ from mitochondria after membrane depolarization. The possible mechanism of divalent metal cations release from deenergized mitochondria is discussed.     

Ca2+-induced oxidative stress in brain mitochondria treated with the respiratory chain inhibitor rotenone

In this study we show that micromolar Ca(2+) concentrations (>10 microM) strongly stimulate the release of reactive oxygen species (ROS) in rotenone-treated isolated rat forebrain mitochondria. Ca(2+)-stimulated mitochondrial ROS release was associated with membrane lipid peroxidation and was directly correlated with the degree of complex I inhibition by rotenone. On the other hand, Ca(2+) did not increase mitochondrial ROS release in the presence of the complex I inhibitor 1-methyl-4-phenylpyridinium. Cyclosporin A had no effect on Ca(2+)-stimulated mitochondrial ROS release in the presence of rotenone, indicating that mitochondrial permeability transition is not involved in this process. We hypothesized that Ca(2+)-induced mitochondrial oxidative stress associated with partial inhibition of complex I may be an important factor in neuronal cell death observed in the neurodegenerative disorder Parkinson's disease.     

High- and low-calcium-dependent mechanisms of mitochondrial calcium signalling

The Ca(2+) coupling between endoplasmic reticulum (ER) and mitochondria is central to multiple cell survival and cell death mechanisms. Cytoplasmic [Ca(2+)] ([Ca(2+)](c)) spikes and oscillations produced by ER Ca(2+) release are effectively delivered to the mitochondria. Propagation of [Ca(2+)](c) signals to the mitochondria requires the passage of Ca(2+) across three membranes, namely the ER membrane, the outer mitochondrial membrane (OMM) and the inner mitochondrial membrane (IMM). Strategic positioning of the mitochondria by cytoskeletal transport and interorganellar tethers provides a means to promote the local transfer of Ca(2+) between the ER membrane and OMM. In this setting, even >100 microM [Ca(2+)] may be attained to activate the low affinity mitochondrial Ca(2+) uptake. However, a mitochondrial [Ca(2+)] rise has also been documented during submicromolar [Ca(2+)](c) elevations. Evidence has been emerging that Ca(2+) exerts allosteric control on the Ca(2+) transport sites at each membrane, providing mechanisms that may facilitate the Ca(2+) delivery to the mitochondria. Here we discuss the fundamental mechanisms of ER and mitochondrial Ca(2+) transport, particularly the control of their activity by Ca(2+) and evaluate both high- and low-[Ca(2+)]-activated mitochondrial calcium signals in the context of cell physiology.     

Endoplasmic reticulum Ca(2+) signaling and calpains mediate renal cell death

The goal of the current study was to determine the roles of ATP content, endoplasmic reticulum (ER) Ca(2+) stores, cytosolic free Ca(2+) (Ca(2+)(f)) and calpain activity in the signaling of rabbit renal proximal tubular (RPT) cell death (oncosis). Increasing concentrations (0.3-10 microM) of the mitochondrial inhibitor antimycin A produced rapid ATP depletion that correlated to a rapid and sustained increase in Ca(2+)(f), but not phospholipase C activation. The ER Ca(2+)-ATPase inhibitors thapsigargin (5 microM) or cyclopiazonic acid (100 microM) alone produced similar but transient increases in Ca(2+)(f). Pretreatment with thapsigargin prevented antimycin A-induced increases in Ca(2+)(f) and antimycin A pretreatment prevented thapsigargin-induced increases in Ca(2+)(f). Calpain activity increased in conjunction with ER Ca(2+) release. Pretreatment, but not post-treatment, with thapsigargin or cyclopiazonic acid prevented antimycin A-induced cell death. These data demonstrate that extensive ATP depletion signals oncosis through ER Ca(2+) release, a sustained increase in Ca(2+)(f) and calpain activation. Depletion of ER Ca(2+) stores prior to toxicant exposure prevents increases in Ca(2+)(f) and oncosis.     

The peripheral-type benzodiazepine receptor is involved in control of Ca2+-induced permeability transition pore opening in rat brain mitochondria

The peripheral-type benzodiazepine receptor (PBR) is an 18 kDa mitochondrial membrane protein with still elusive function in cell death. Here, we studied whether PBR is involved in Ca2+-induced permeability transition pore (PTP) opening in isolated rat brain mitochondria (RBM). PTP opening is important in mitochondrial events leading to programmed cell death. Immunoblots revealed a single 18 kDa anti-PBR antibody-immunoreactive band in purified RBM. Adenine nucleotide transporter, a key PTP component, was found in the PBR-immunoprecipitate. In isolated intact RBM, addition of a specific anti-PBR antibody [H. Li, Z. Yao, B. Degenhardt, G. Teper, V. Papadopoulos, Cholesterol binding at the cholesterol recognition/interaction amino acid consensus (CRAC) of the peripheral-type benzodiazepine receptor and inhibition of steroidogenesis by an HIV TAT-CRAC peptide, Proc. Natl. Acad. Sci. U.S.A. 98 (2001) 1267-1272] delayed Ca2+-induced dissipation of membrane potential (psi(m)) and diminished cyclosporine A-sensitive Ca2+ efflux, which are both indicative for the suppression of PTP opening. Moreover, anti-PBR antibody caused partial retention of Ca2+ in the mitochondrial matrix in spite of psi(m) dissipation, and reduced activation of respiratory rate at Ca2+-induced PTP opening. A release of pro-apoptotic factors, AIF and cytochrome c, from RBM was shown at threshold Ca2+ load. Anti-PBR antibody blocked the release of AIF but did not affect the cytochrome c release. Addition of ATP was able to initiate PTP closing, associated with psi(m) restoration and Ca2+ re-accumulation. At the same time mitochondrial protein phosphorylation (incorporation of 32P from [gamma-32P]ATP) occurred and anti-PBR antibody was able to inhibit phosphorylation of these proteins. The endogenous PBR ligand, protoporphyrin IX, facilitated PTP opening and phosphorylation of the mitochondrial proteins, thus, inducing effects opposite to anti-PBR antibody. This study provides evidence for PBR involvement in PTP opening, controlling the Ca2+-induced Ca2+ efflux, and AIF release from mitochondria, important stages of initiation of programmed cell death.     

Calcium influx through receptor-operated channel induces mitochondria-triggered paraptotic cell death

We address the specific role of cytoplasmic Ca(2+) overload as a cell death trigger by expressing a receptor-operated specific Ca(2+) channel, vanilloid receptor subtype 1 (VR1), in Jurkat cells. Ca(2+) uptake through the VR1 channel, but not capacitative Ca(2+) influx stimulated by the muscarinic type 1 receptor, induced sustained intracellular [Ca(2+)] rises, exposure of phosphatidylserine, and cell death. Ca(2+) influx was necessary and sufficient to induce mitochondrial damage, as assessed by opening of the permeability transition pore and collapse of the mitochondrial membrane potential. Ca(2+)-induced cell death was inhibited by ruthenium red, protonophore carbonyl cyanide m-chlorophenylhydrazone, or cyclosporin A treatment, as well as by Bcl-2 expression, indicating that this process requires mitochondrial calcium uptake and permeability transition pore opening. Cell death occurred without caspase activation, oligonucleosomal/50-kilobase pair DNA cleavage, or release of cytochrome c or apoptosis inducer factor from mitochondria, but it required oxidative/nitrative stress. Thus, Ca(2+) influx triggers a distinct program of mitochondrial dysfunction leading to paraptotic cell death, which does not fulfill the criteria for either apoptosis or necrosis.