Báo cáo khoa học: Programmed cell death Apoptosis and alternative deathstyles pot

Eur J Biochem 271, 1638–1650 (2004) Ó FEBS 2004 doi:10.1111/j.1432-1033.2004.04084.x REVIEW ARTICLE Programmed cell death Apoptosis and alternative deathstyles ˜ Cinthya Assuncao Guimaraes* and Rafael Linden ¸˜ Instituto de Biofı´sica da UFRJ, Rio de Janeiro, Brazil Programmed cell death is a major component of both normal development and disease The roles of cell death during either embryogenesis or pathogenesis, the signals that modulate this event, and the mechanisms of cell demise are the major subjects that drive research in this field Increasing evidence obtained both in vitro and in vivo supports the hypothesis that a variety of cell death programs may be triggered in distinct circumstances Contrary to the view that caspase-mediated apoptosis represents the standard programmed cell death, recent studies indicate that an apoptotic morphology can be produced independent of caspases, that autophagic exe- cution pathways of cell death may be engaged without either the involvement of caspases or morphological signs of apoptosis, and that even the necrotic morphology of cell death may be consistently produced in some cases, including certain plants Alternative cell death programs may imply novel therapeutic targets, with important consequences for attempts to treat diseases associated with disregulated programmed cell death Introduction a violent and quick form of degeneration affecting extensive cell populations, characterized by cytoplasm swelling, destruction of organelles and disruption of the plasma membrane, leading to the release of intracellular contents and inflammation A remarkably distinct type of cell death was called apoptosis, identified in single cells usually surrounded by healthy-looking neighbors, and characterized by cell shrinkage, blebbing of the plasma membrane, maintenance of organelle integrity, and condensation and fragmentation of DNA, followed by ordered removal through phagocytosis [18,19] During the last 30 years, cell death has usually been classified within this dichotomy The work of Kerr and collaborators stirred interest in programmed cell death, both because it provided a visible object (the apoptotic profile) to be consistently approached in experimental studies prior to the disappearance of the dead cells, as well as due to the evidence provided for controlled events that justify the operational definition of ÔprogrammedÕ [20] At a first approximation, necrosis was attributed to accidental, uncontrolled degeneration, whereas apoptosis presented the defining characteristics of a cell death program Indeed, many research groups began to consider apoptosis and programmed cell death as a single entity, despite the knowledgeable criticism of pioneers in the field [21] Nonetheless, this simplified and generalized scheme neglects the exceptions; for example, morphologies of cell death that not fit in the original classification (reviewed in [22]) On the other hand, evidence is now available for multiple alternative cell death pathways, as well as for cross-talk of intracellular mechanisms involved in distinct aspects of cell degeneration This review will focus on the growing evidence that, besides apoptosis, autophagic and necrotic forms of cell degeneration may be programmed, and underlie cell death either in isolation or combined with mechanisms of apoptosis Programmed cell death is a major component of both normal development and disease [1–11] The roles of cell death during either embryogenesis or pathogenesis, the signals that induce or regulate this event, and the mechanisms of cell demise are common subjects that drive research in this field [12–17] The purpose of this article is to review major morphological, biochemical and molecular hallmarks of distinct forms of programmed cell death, and to examine the limits of some prevailing views of cell death modes and mechanisms The classical ultrastructural studies of Kerr and coworkers [18] provided evidence that cells may undergo at least two distinct types of cell death: The first type is known as necrosis, Correspondence to R Linden, Instituto de Biofı´ sica da UFRJ, ´ Centro de Ciencias da Saude, bloco G, Cidade Universitaria, ˆ ´ 21949–900, Rio de Janeiro, Brazil Tel.: + 55 21 25626553, Fax: + 55 21 22808193, E-mail: rlinden@biof.ufrj.br Abbreviations: AIF, apoptosis inducing factor; DAPk, death-associated protein kinase; DRP, DAPk-related protein kinase; FADD, Fas-associated protein with death domain; IAP, inhibitor of apoptosis; LEI, leucocyte elastase inhibitor; L-DNase II, LEI-DNase II; MPT, mitochondrial permeability transition; PCD, programmed cell death; PI3K, phosphatidylinositol-3-kinase; TNFa, tumor necrosis factor-alpha; TUNEL, TdT-mediated biotin-dUDP nick-end labeling Note: a website is available at http://www.biof.ufrj.br/neurogen *Present address: The Hebrew University of Jerusalem, Department of Biological Chemistry, Institute of Life Sciences, The Edmond J Safra Campus, Givat Ram, Jerusalem 91904 Israel (Received 12 January 2004, revised 17 February 2004, accepted 10 March 2004) Keywords: programmed cell death; apoptosis; autophagy; necrosis; neurodegenerative diseases Ó FEBS 2004 Alternative pathways of programmed cell death (Eur J Biochem 271) 1639 Defining features of programmed cell death Multiple mechanisms of apoptosis Despite the tremendous impact of research in apoptosis upon the understanding both of cellular and molecular mechanisms of cell demise, as well as of mechanisms of degenerative diseases, the confusion between apoptosis and programmed cell death has somewhat obscured the field Regardless of whether this paradox is attributable to either disconnection of modern science from its philosophical foundations [23] or to a more trivial neglect of classical papers (reviewed in [20]), it is likely that progress in the identification and understanding of nonapoptotic forms of programmed cell death may have been unnecessarily delayed Indeed, well before the upsurge in the understanding of mechanisms of apoptosis, a clear warning had been issued to avoid confusion between the form of cell death called apoptosis, and the concept of programmed cell death as a sequence of events, but not necessarily those that led to the morphology of apoptosis [21] Although the original work that led to the concept of programmed cell death was carried out in developing organisms [24–29], there is nothing intrinsically developmental in the concept The apoptotic form has been long identified in adult tissues [18], and there is no evidence that any particular form of cell death, much less the operational concept of programmed cell death, can be attributed exclusively to either developing or mature cells Conversely, it has been argued that apoptotic forms of cell death induced by cytotoxic drugs or physical stimuli could not be taken as programmed cell death because the latter represents normal degeneration that is part of the life of an organism [30] However, those instances of induced degeneration reflected no less an orderly sequence of cellular events than naturally occurring cell death in the form of apoptosis found in developing organisms Thus, a simple and noncommittal definition of programmed cell death as Ôa sequence of events based on cellular metabolism that lead to cell destructionÕ is likely both to preserve the concept as originally defined, as well as to discard decorative qualifications based on particular experimental findings A disturbing example of the latter is the requirement for protein synthesis [31,32], that had a great impact in the acceptance of cell death as controlled by gene expression (and thus Ôgenetically programmedÕ) Not only many cells die under inhibition of either transcription or translation in a controlled way indistinguishable from that underlying cell death dependent on protein synthesis [33–35], but the rapid progress in the understanding of post-translational mechanisms of cell death has largely overshadowed transcriptional control and even the classical requirement for protein synthesis [36,37] The caveat that the sequence of events in programmed cell death must be based on cell metabolism allows for the irony that even the time between hitting a cell with a hammer and the death of the former is finite, notwithstanding that the intervening events are not resolvable with current techniques Acceptance of the minimalist concept should help attribute appropriate weight to alternative forms of programmed cell death, despite the overwhelming dominance of apoptosis in the literature Cell death with apoptotic morphology can be triggered by several stimuli, including intracellular stress and receptor-mediated signaling These signals feed into an evolutionarily conserved intracellular machinery of execution [36,38], the mechanisms of which have mainly been traced to the activity of the caspase family of cysteineproteases [39–41] Caspase-mediated apoptotic cell death has been extensively reviewed, e.g [16,36,38,42–44] Briefly, the caspases are synthesized as zymogens and upstream signals convert these precursors into mature proteases Initiator caspases (caspase-1, -2, -4, -5, -8, -9, -10 and -14] are activated via oligomerization-induced autoprocessing [45–50], while effector caspases [caspase-3, -6 and -7] are activated by other proteases, including initiator caspases and granzyme B Proteolytic cleavage of cellular substrates by effector caspases largely determines the features of apoptotic cell death ([51–53]; reviewed in [54–56]) Three major pathways have been identified according to their initiator caspase: the death receptor pathway involving caspase-8 [57], the endoplasmic reticulum stress pathway attributed to activation of caspase-12 [58], and the mitochondrial pathway, in which various signals can trigger the release of harmful proteins by mitochondria into the cytoplasm, leading to activation of caspase-9 and downstream cleavage of caspase-3, -7 or -6 [46,59–62] Although caspase-3 is widely involved in the execution of apoptosis [63], its effector functions may be dispensable for apoptotic-like cell death [64,65] The use of either pharmacological inhibitors or knockout animals further showed that cells can trigger alternative mechanisms of cell demise For example, sympathetic and dorsal root ganglion neurons deprived of nerve growth factor (NGF) die in a caspase-2dependent manner, but the same neurons derived from caspase-2 knockout mice still die following nerve growth factor deprivation, this time depending on activation of caspase-9, which does not occur in wild-type mice [66] Thus, rather than a single linear mechanism, alternative caspase-mediated pathways may be activated for apoptotic cell death, depending on whether a preferential caspase is blocked It is likely that the network of intrinsic regulatory pathways that impinge upon the activity of caspases, such as the inhibitors of apoptosis (IAPs) and IAP-binding proteins [67], may regulate the choice between alternative pathways in normal cells, depending on metabolic state, stage of differentiation and other conditions In addition, caspase inhibition fails to block programmed cell death with apoptotic morphology in several experimental models [68–72] For example, the ultrastructural features of apoptosis inducing factor (AIF)-induced cell death represent an example of a slight variation from the standard pattern of apoptotic morphology, which appears to be independent of caspase activation ([73]; see also [74]) Cell death pathways independent of caspase activation have been described, for example, even in some forms of cell death induced either by the Bcl-family protein Bax [75], as well as in cell death involving the activation of other proteases, such as calpain [76], proteasome [77] and serine proteases The latter enzymes have an important role in early chromatin cleavage [78], and are activated in the classical 1640 C Assuncao Guimaraes and R Linden (Eur J Biochem 271) ¸ ˜ ˜ model of apoptosis of thymocytes induced by glucocorticoids [79] Serine proteases participate in a cell death pathway that involves the activation of the endonuclease leucocyte elastase inhibitor (LEI)-DNase II (L-DNase II), and is not inhibited in HeLa cells by pancaspase inhibitors [80] Activation of L-DNase II was first described in lens cell differentiation, which is related to apoptosis [81] The activation of this enzyme also occurs under other physiological conditions, such as the death of retinal cells during development [82] The key molecule of this pathway is LEI, which is a member of the superfamily of protease inhibitors called serpins (serine protease inhibitors) In its active form, LEI inhibits elastase, cathepsin G and probably other proteases [83] LEI can undergo post-translational modifications either under acidic pH or by the action of proteases, including elastase Once LEI is exposed to these conditions, a decrease in the molecular mass is observed simultaneously with the appearance of endonuclease activity [84] The DNase generated by the action of the serine protease elastase was named L-DNase II, as it shows dependence on the same ions and pH required by DNase II Recent reports shows that the serine protease Omi/HtrA2 is a mitochondrial direct X-chromosome-linked inhibitor of apoptosis protein (XIAP)-binding protein, which is released from mitochondria upon induction of apoptosis together with cytochrome c and Smac/Diablo ([85,86]; reviewed in [87]), and its release can be inhibited by Bcl-2 [88] These data suggest that in some cases there may be a cooperative action between serine proteases and caspases in the execution of cell death The data show that the classically defined apoptotic morphology can be achieved either by activation of caspases, or through the mediation of other families of proteases [Fig 1], although the exact cytological features of cell demise may vary slightly among these various forms of apoptosis Ó FEBS 2004 Autophagy and autophagic cell death As part of normal development, cells depend on a strictly regulated balance of protein synthesis and degradation, as well as organelle biogenesis and dismantlement While proteasome-mediated degradation is responsible for most of the protein recycling, the turnover of organelles is mainly attributed to autophagy [89] Autophagy occurs in many eukaryotic cell types, where organelles and other cell components are sequestered into lysosomes and degraded The lysosome is a cellular compartment enriched in hydrolases able to cleave proteins, lipids, nucleic acids and carbohydrates that may lead to organelle degradation through macroautophagy [90] Autophagy has been described both as a means to resist starvation, and as part of cellular remodeling during differentiation, metamorphosis, aging, cell transformation, physiological whole-organ changes such as growth of the uterus during pregnancy and its atrophy after childbirth, as well as in the removal of anomalous cellular components that accumulate following toxic insults or during cell death [91] In the nervous system, for example, morphological signs of autophagy are observed in physiological processes, such as the removal of outer segments of retinal photoreceptors by the pigment epithelium [92], which is not associated with cell death Notwithstanding, autophagic profiles identified by ultrastructural features have been associated with cell death in certain circumstances [22] Cells in the early stages of autophagy contain several autophagic vacuoli, and both the nucleoplasm and the cytoplasm appear slightly darkened, although nuclear structure still appears normal Mitochondria and the endoplasmic reticulum are sometimes dilated, and the Golgi apparatus is often enlarged The plasma membrane loses specializations such as microvilli and junctional complexes, and blebbing can occur In several cases, an intense endocytosis is observed, and this probably Fig Multiple pathways to apoptosis, both dependent and independent of the activation of caspases The diagram summarizes the major components of the pathways reported to underlie cell death in various types of cells and tissues Ó FEBS 2004 Alternative pathways of programmed cell death (Eur J Biochem 271) 1641 leads to a reduction in the area of the plasma membrane During late stages, both the number and size of vacuoli increase, and many of them contain myelin figures or are filled with lipids, which appear as pale gray inclusions in the cytoplasm [22] The nucleus of a cell undergoing autophagic cell death can become pyknotic and identifiable as such by light microscopy, either in early or in late stages of the degenerative process Nevertheless, this nuclear condensation is neither as common nor as remarkable as that of apoptosis The late autophagic cell debris is frequently removed by heterophagy, but this tends to occur in very late stages, and seems to be less conspicuous than the clearance of apoptotic bodies [22] Autophagic cell death is not an exclusive feature of multicellular organisms In the protozoan pathogen Leishmania donovani, treatment with antimicrobial peptides induced cytoplasmic vacuolization and dismantling of the cellular organization without disruption of the plasma membrane, with no nuclear fragmentation or DNA laddering, and independent of caspase-like activity Instead, monodansylcadaverine, a biochemical marker of autophagy, specifically labeled the vacuoles induced by antimicrobial peptides [93] Endostatin, an inhibitor of angiogenesis, was shown to induce the formation of autophagic vacuoles in endothelial cells Cell death was not prevented by antioxidants or caspase inhibitors, but was reduced by 3-methyladenine, a specific inhibitor of autophagy, and serine and cysteine lysosomal protease inhibitors [94] Neuregulin (NRG; a ligand of ErbB), also activates ErbB-2/ErbB-3 heterodimers and induces cell death of prostate cancer LNCaP cells Neuregulin-induced cell death was not inhibited by broadspectrum caspases inhibitors, but was blocked by 3-methyladenine [95] Ionizing radiation induced a dose-dependent suppression of cell proliferation and autophagic cell changes in several glioblastoma multiform cell lines [96] Arsenic trioxide, an agent that causes remission in patients with acute promyelocytic leukemia and multiple myeloma without severe sideeffects, was shown to inhibit proliferation of glioma cell lines The G2/M arrest was accompanied by ultrastructural features of autophagy, and was inhibited by the autophagy inhibitor bafilomycin A1, whereas general caspase inhibitors did not block As2O3-induced cell death [97] In neuroblastoma cells, dopamine leads to autophagic changes characterized by the presence of numerous cytoplasmic vacuoles with inclusions, and accompanied by mitochondrial aggregation, activation of the stress-response kinases SAPK/JNK and p38, and increased a-synuclein expression Both cell viability and the increase in a-synuclein expression were prevented by antioxidants, by the specific inhibitors of p38 and SAPK/JNK, and by 3-methyladenine [98] Thus, various agents can lead to autophagic cell death in tumor cell lines Indeed, Bursch and collaborators [99] had long since shown that the MCF-7 breast carcinoma cell line, which does not express caspase-3 [100], undergoes autophagic cell death upon treatment with tamoxifen More recent work showed that in apoptotic cell death induced by tyrphostin A25 in the human colon cancer cell line HT29/HI1, early stages of the death process are associated with depolymer- ization of actin and degradation of intermediate filaments In contrast, during tamoxifen-induced autophagic cell death of MCF-7 cells, intermediate and microfilaments are redistributed, but largely preserved even beyond the stage of nuclear collapse [101] These data support the concept that autophagic cell death is a separate form of programmed cell death that is distinctly different from apoptosis In keeping with this interpretation, intensive irradiation led to up to 30% cell death in MCF-7 cells without any signs of apoptosis In this case, cell death was accompanied by the formation of acidic vesicular organelles and lamellar structures, which was prevented by 3-methyladenine However, following low-dose irradiation, the presence of acidic vesicular organelles correlated with an increased chance of survival, suggesting that moderate signs of autophagy may be associated with a defensive reaction of nonlethally damaged cells [102] The data are consistent with the view that nonlethal injury can trigger an autophagic defensive reaction, whereas harsh treatment of certain cells can lead to cell death largely dependent on autophagy itself The first step of autophagy is the formation of an autophagosome, which occurs when a portion of the cytoplasm is engulfed by a double membrane vacuole that does not contain either acid phosphatases or aryl-sulphatase activity The double membrane is derived from ribosomefree areas of rough endoplasmic reticulum [91] After a maturation period that includes the acidification of the vacuole, hydrolases are inserted into the autophagosome by fusion with pre-existing lysosomes or elements deriving from the Golgi complex This process appears to involve mannose-6-phosphate receptors located in the autophagosome membrane, resulting in the formation of a degradative vacuole limited by a single membrane named an autolysosome [103] Vacuole formation can be regulated by amino acids and hormones and by stress [104,105] Similarly to yeast [90], autophagy in mammalian cells is highly dependent on phosphorylation events In mammalian hepatocytes, the phosphorylation of the ribosomal protein S6 correlates strongly with inhibition of macroautophagy [106] The activity of the p70S6-kinase is regulated by the mTor kinase, and inhibition of S6 phosphorylation caused by inactivation of mTor with rapamycin induces autophagy even under nutrient-rich conditions In yeast, inhibition of the Tor2 kinase results in activation of protein phosphatase 2A and induction of autophagy [107] Also, in hepatocytes, the effect of the phosphatase inhibitor okadaic acid upon protein phosphatase 2A inhibits the autophagic process [108] Furthermore, various classes of phosphatidylinositol3-kinase (PI3K) control the autophagic pathway in distinct ways: class IA PI3K inhibits cytoplasm sequestration and degradation, while class III stimulates the sequestration of cytoplasm, implicating the PI3K family as key regulators of the autophagic pathway [109] In a recent study, Inbal and coworkers [110] showed that the expression of death-associated protein kinase (DAPk) and DAPk-related protein kinase (DRP)-1, members of a family of Ca2+/calmodulin-regulated Ser/Thr death kinases, triggered two major caspase-independent cytoplasmic events These were membrane blebbing, a feature common to various forms of cell death, and extensive autophagy, which is typical of autophagic cell death Furthermore, either the expression of the dominant negative mutant of Ó FEBS 2004 1642 C Assuncao Guimaraes and R Linden (Eur J Biochem 271) ¸ ˜ ˜ Fig A summary diagram of the major components identified in pathways leading to autophagy These may lead to either cell death or recovery from an insult such as aminoacid deprivation DRP-1 or DAPk antisense mRNA reduced autophagy induced by antiestrogens, amino acid starvation, or administration of interferon-c The finding of DRP-1 inside the autophagic vesicles suggests a direct involvement of this kinase in the process of autophagy Liang and collaborators [111] showed that beclin-1, a protein that interacts with bcl-2, promotes autophagy in both an autophagy-defective yeast cell line and in the MCF-7 cell line, which normally does not express detectable levels of beclin-1 It was also shown that beclin-1 expression is frequently low in epithelial breast carcinomas, but is widely expressed in normal tissue The authors suggested that beclin-1 may be a mammalian gene for autophagy, and that it inhibits tumorigenesis through activation of this cell death pathway [111] Kihara and coworkers [112] suggested that beclin-1 is a component of the PI3K complex, which is also required for autophagy, and that beclin-1 and PI3K control autophagy as a complex at the Trans Golgi network [112] However, in contrast with the poor autophagic response of MCF-7 cells to amino acid deprivation [111], strong autophagic responses were elicited in this cell line both by tamoxifen [101] and by irradiation [102] Thus, either beclin-1 can be replaced by other autophagy-inducing proteins, or alternative pathways of autophagy may operate under various stimuli The death of cerebellar Purkinje cells in lurcher animals is due to a mutation in GluRd2 that results in its constitutive activation (GluRd2-Lc) Yue and collaborators [113] showed that GluRd2, nPIST and beclin-1 interact, and that autophagy can be induced by nPIST and beclin-1 synergy and by the mutated GluRd2-Lc, but not by the wildtype GluRd2 Dying lurcher Purkinje cells displayed morpholo- gical features of autophagy in vivo, providing evidence both for a direct link between GluRd2-Lc receptor and the autophagic pathway in these cells [113], and that beclin-1 can exert its autophagic functions through interaction with multiple proteins in the cells [111–113] Current evidence therefore indicates that that in at least some instances, autophagy may lead to a caspase-independent program of cell demise that fits the concept of programmed cell death, subject to complex, multivariate control [Fig 2] The next section will examine its relationship with apoptosis Apoptosis vs autophagy Notwithstanding the abundant evidence for a role for both autophagy and apoptosis in various diseases, their interplay in those pathologies is not yet fully understood In Parkinson’s disease, the ultrastructural study of neurons of the substantia nigra of affected patients showed signs of autophagy, as well as apoptosis [114] However, it has been shown that expression of a-synuclein mutants, a condition frequently found among certain families with Parkinson’s disease, induces autophagic cell death with no caspase activation, due to alterations of the ubiquitin-dependent protein degradation system [115] This suggests that there may be no obligatory interdependence between apoptosis and autophagy in the pathology of this disease In Alzheimer’s disease, the endosomal–lysosomal system was found to be enlarged and highly activated, showing that endocytosis and/or autophagy are accelerated in the neurons of affected brains, even at early stages The early activation of this system could be related to major Ó FEBS 2004 Alternative pathways of programmed cell death (Eur J Biochem 271) 1643 etiological factors of the disease, such as defective membrane proteins, apolipoprotein E function, or altered processing of the amyloid precursor protein [116] In Huntington’s disease, it was suggested that the accumulation of a mutant isoform of the protein huntingtin in lysosomal compartments can activate cell death by autophagy [117,118] Finally, in experimental prion disease, bovine or mice brains inoculated with scrapie-infected brain tissue showed signs of autophagy, suggesting that the accumulation of the protease-resistant prion protein isoform can lead to sequestration and autophagy of portions of the cytoplasm and eventually to neuron loss [119] Furthermore, giant vacuoli were observed in experimental prion disease induced in hamsters The incidence of these vacuoli correlates with the inoculation pathway, the intensity of the process and the incubation period, and they are most numerous following intracerebral inoculation The emergence of vacuoli was chronologically related to the appearance of fibrils associated with prion disease, suggesting that this process may be related to disturbed protein turnover and processing of the prion protein [120] Despite the evidence for autophagy, there are several reports of apoptotic cells detected by in situ nick-end DNA labeling in human cases of all Alzheimer’s, Huntington’s and Creutzfeldt–Jakob diseases [121–123] The mode of cell death in neurodegenerative disorders remains a matter of controversy [124], and it is possible that both apoptotic and nonapoptotic cell death coexist in the brains of affected patients This problem is further complicated by claims that the TdT-mediated biotin-dUDP nick-end labeling (TUNEL) technique may stain not only apoptotic cells [125,126] For example, cell death of keratinocytes induced by 5-fluoruracil exhibits autophagic ultrastructural features, such as cytoplasm vacuolation and chromatin detachment from the nuclear membrane, alongside apoptotic features such as TUNEL staining and both a decrease in size and increase in granularity observed by cytometric analysis [127] The authors suggested that in this case the TUNEL-positive cells were dying by autophagy, and cast doubt on the identification of apototic cell death solely using nick-end labeling techniques, such as in some of the neuropathological surveys cited above The data show that the incidence of mixed cell death features may be more common among various cell types than predicted by previous studies The same stimulus can sometimes lead to the activation of distinct and independent cell signaling pathways Tumor necrosis factor-alpha (TNFa)-induced cell death in an acute T-lymphoblastic leukaemic cell line developed with an apoptotic pattern that was preceded by autophagy The compound 3-methyladenine, which inhibits the formation of autophagosomes, also inhibited the cytolysis and DNA fragmentation induced by TNFa However, inhibition of the fusion of lysosomes with autophagosomes by asparagine did not block TNFa-induced apoptosis, and amino acid and protein deprivation enhanced TNFa-induced autophagy, but not apoptosis These data suggest that, at least in this case, early stages of autophagy are required for, but not necessarily result in, TNF-a induced apoptosis [128], and also that a cell can switch between apoptosis and autophagy as the dominant form of cell death However, there are clear examples of interdependence between apoptosis and autophagy Following damage to peripheral nerves in adult rats, oligodendrocytes undergo ultrastructural features of apoptotic cell death, but membrane-bound cytoplasmic organelles typical of autophagy were also noticed in the cytoplasm of these cells [129] In sympathetic neurons from the superior cervical ganglia, substantial autophagic activity was activated by pro-apoptotic factors, and treatment of these cells with 3-methyladenine decreased their rate of apoptosis [130] This was also observed in serum-deprived PC12 cells, and was accompanied by changes in the activity of lysosomal proteases, particularly cathepsins B and D [131] Alternative pathways of execution of cell death induced by blockade of protein synthesis were shown among a relatively homogenous population of postmitotic undifferentiated cells in the developing retinal tissue Inhibition of protein synthesis induced in the immature retina various post-translational, mitochondria-dependent pathways of cell death In one pathway autophagy precedes the sequential activation of caspases-9 and -3, and DNA fragmentation, while, in parallel, caspase-6-dependent mechanisms led to a TUNEL-negative form of cell death Evidence was also provided for additional cell death mechanisms dependent on caspase-9 activity, which may be engaged upon selective inhibition of execution caspases [132] These results support the hypothesis of interdependence of autophagy and caspase-dependent cell death pathways Camougrand and coworkers [133] showed in yeast that the expression of Bax induces cell death with characteristics of both apoptosis and autophagy, providing a newly identified function of Bax in autophagic cell death [133] Developmental cell death of motoneurons in the hawkmoth Manduca sexta depends on caspase activation and the loss of mitochondrial function, showing an accumulation of autophagic bodies and vacuoles Motoneurons displayed a normal nuclear ultrastructure, without chromatin condensation, although they were found to be TUNEL-positive, which is diagnostic of fragmented DNA These results indicate that the steroid-induced, caspase-dependent motoneuron cell death exhibits intermingled features of both autophagy and apoptosis [134] In the fruitfly Drosophila, autophagic cell death depends on steroid-regulated genes encoding transcription regulators, which appear to activate other genes involved in cell degeneration The latter include genes that function in apoptosis, such as caspases, showing that caspase function is required for autophagic cell death during Drosophila development (for a review of apoptosis and autophagy interplay during Drosophila development see [135,136]) The autophagy inhibitor 3-methyladenine also increased the sensitivity of HT-29 colon cancer cells to apoptosis induced by sulindac sulfide, an inhibitor of cyclo-oxygenase Mutants that have a low rate of autophagy were more sensitive to sulindac sulfide-induced apoptosis than parental HT-29 cells, and the rate of cytochrome c release was higher in mutant cells than in HT-29 cells, suggesting that autophagy could delay apoptosis by sequestering mitochondrial death-promoting factors such as cytochrome c [137] In this context, autophagy may represent an attempt of the cell to recover from a noxious stimulus, rather than a necessary stage of execution of cell death 1644 C Assuncao Guimaraes and R Linden (Eur J Biochem 271) ¸ ˜ ˜ The available data thus indicate that cells may die by a caspase-independent autophagic process either with or without showing signs of apoptosis In some cases, cells may choose between the autophagic and the apoptotic execution pathways Nevertheless, several experiments show that the limits between apoptosis and autophagy may be tenuous, and suggest either considerable overlap or interdependence of both programs of cell demise Mitochondria and cell deaths Mitochondria have a central role both in cellular homeostasis and pathological conditions, as not only they serve as the major energy factory of living cells, but they can also either trigger or amplify the signals that lead to cell death [46,59–62] Permeabilization of the mitochondrial membrane has been associated with cell death by apoptosis [59,138], by mechanisms that are not yet completely elucidated (reviewed in [139,140]) Induction of permeability transition in the inner mitochondrial membrane may be accompanied by release of cytochrome c, Smac/Diablo, AIF and endonuclease G, all of which lead to mitochondria-dependent apoptotic forms of cell death [141–146] Notwithstanding, the collapse of the mitochondrial transmembrane potential as a consequence of permeability transition is not a universal early feature of apoptosis [147–149] Although mitochondrial involvement in apoptosis is broadly documented, permeabilization of mitochondria seems to be an event also shared by autophagy and necrosis Mitochondria from hepatocytes spontaneously depolarize after nutrient deprivation before their capture by an acid lysosomal compartment, suggesting that a permeability transition occurs before the normal autophagic process [150] The authors proposed that cells respond to mitochondrial permeability transition (MPT) in a graded manner When MPT occurs only in a few mitochondria, autophagy is activated, leading to lysosomal degradation of the affected organelles and cessation of the signals that stimulate autophagy When a larger number of mitochondria are permeabilized apoptosis occurs, probably due to the higher concentration of molecules such as cytochrome c and AIF in the cytoplasm And finally, when virtually all mitochondria in the cell are affected, MPT promotes necrosis, attributed to the uncoupling of oxidative phosphorylation and accelerated ATP hydrolysis by mitochondrial ATPase [151] This interpretation is consistent with the hypothesis that autophagy, apoptosis and necrosis are part of a continuum of degenerative events [152,153] Programmed necrosis The death and elimination of cells by apoptosis remains unnoticed by the body’s immune system, while the release of the intracellular content of necrotic cells into the extracellular space induces an inflammatory response, constituted by the activation of resident phagocytes and attraction of leukocytes into the necrotic area [18] Until recently, necrosis has often been viewed as an accidental and uncontrolled cell death process Nevertheless, there is growing evidence that necrotic and apoptotic forms of cell Ó FEBS 2004 death may share more similarities than originally thought (reviewed in [16,154–156]) Necrosis has indeed been found to be a potential substitute for apoptosis during development Development-related loss of spinal cord and brainstem neurons was not impaired upon genetic deletion of both caspase-3 and caspase-9, although the morphology of the forebrain suffered a marked perturbation [157] Furthermore, the loss of interdigital cells in the mouse embryo, a prototype of programmed cell death, still occurs by necrosis either upon caspase inhibition by drugs, or in mice bearing a mutation in the apoptosis protease-activating factor (APAF-1) gene Such necrotic cell death was also observed in normal wild-type mice [158] Several apoptosis-related molecules have been implicated in necrosis-like forms of cell death The antiapoptotic proteins Bcl-2 and Bcl-xL as well as caspase inhibitors delay necrotic cell death induced by cyanide, rotenone or antimycin A [159] Bcl-2 can also protect neural cells from necrotic cell death induced by the depletion of glutathione [160] These results indicate that Bcl-2/Bcl-xL and caspases modulate not only apoptotic but also some forms of necrotic cell death In a Jurkat-derived cell line (JB-6) that is deficient in caspase-8, the forced multimerization of Fas-associated protein with death domain (FADD) induced caspaseindependent cell death No DNA fragmentation was observed and dying cells showed neither condensation nor fragmentation of cells and nuclei, but the cells and nuclei swelled in a manner similar to that seen in necrosis [161] In other studies, caspases-3 and -7 were activated in cell death of murine L929 fibrosarcoma cells transfected with human Fas receptor, but peptide inhibitors of caspase-1 and -3 failed to block cell degeneration, and microscopical analysis showed features of necrosis after a delay of h [162] These results suggest the existence of two distinct pathways of cell death triggered by the engagement of Fas receptors, which may lead either to classical caspase-dependent apoptosis or to necrosis Chi and coworkers [163] showed that the expression of oncogenically mutated ras gene in human glioma and gastric cancer cell lines causes a necrotic-like cell death characterized by cytoplasmic vacuoles derived from lysosomes Dying cells had relatively well-preserved nuclei that were negative for TUNEL staining This oncogenic Rasinduced cell death occurred in the absence of caspase activation and was not inhibited by the overexpression of Bcl-2 [163] These morphological descriptions of cell death recall the recently suggested concept of necrosis-like programmed cell death (PCD), where this term was used to Ô… define PCD in the absence of chromatin condensation, or at best with chromatin clustering to specklesÕ [16] It seems that the cell death often classified as aborted apoptosis, where PCD is initiated in the presence of caspase inhibitors, also meets this requirement because cells end up dying by alternative routes that are independent of caspase activation (reviewed in [164]) Biochemical mechanisms of the execution of necrosis are beginning to be identified in plants Recessive genetic mutations in the Rn locus in soybean lead to progressive browning of the root, accompanied by incidence of necrotic Ó FEBS 2004 Alternative pathways of programmed cell death (Eur J Biochem 271) 1645 as well as apoptotic cell death within the same tissue, but in distinct cells [165] The appearance of necrotic cells in the root preceded visible browning and lesion formation at the macroscopic level, exhibiting a flocculent nucleoplasm, increased vacuolation, condensed cytoplasm and presence of swollen malformed organelles [165] Rcr3 is a plant disease-resistance protein that recognizes pathogens and activates plant defenses This protein is a secreted papain-like cysteine endopeptidase and is specifically required for Cf-2 function, e.g resistance to pathogens in tomato, a hypersensitive response that results in cell death Genetic analysis showed that Rcr3 is allelic to the Ne (Necrosis) gene, which suppresses the Cf-2-dependent autonecrosis induced by plant pathogens [166] These data may represent, at least in plants, the first examples of genes related to the induction of necrotic-like PCD It is not known whether any of the necrosis-related genes of plants have homologs in animal cells However, components of a necrotic program are beginning to emerge Integration of the BH3 protein BNIP3 to mitochondria triggers a necrotic-like form of PCD [167] The necrotic-like cell death triggered by engagement of the Fas/FADD signal transduction system in mammalian cells was reported to depend on the kinase RIP as an effector molecule [168] These data support the hypothesis that, besides uncontrollable necrosis that may occur following either massive mechanical aggression or harsh chemical treatment of tissues, a program of cell demise with morphological characteristics of necrosis may be found with components largely distinct from either apoptosis or autophagy Conclusion and perspectives Increasing evidence obtained in many model systems both in vitro and in vivo supports the hypothesis that a variety of cell death programs may be triggered in distinct circumstances [Fig 3] Contrary to the widely held view that Fig A summary diagram of the various forms of degeneration that may follow cell stress or damage Distinct forms of programmed cell death are indicated in italics within the boldly outlined boxes Cell demise is the usual outcome of either the somewhat variable forms of apoptosis mediated by multiple, alternative pathways, or of programmed necrosis Autophagy may lead to cell death either directly or through apoptosis, as well as mediate cellular recovery caspase-mediated apoptosis represents the standard PCD, recent studies indicate that an apoptotic morphology can be produced without the involvement of caspases, that autophagic execution pathways of cell death may be engaged without either the involvement of caspases or morphological signs of apoptosis, and that even the necrotic morphology of cell death may be consistently produced in some cases, including the natural history of at least some plants Particularly in the case of autophagic cell death, but to a lesser extent also in the case of controlled necrosis, components have been identified at the molecular level that justify the assumption of an intracellular program mediating either form of cell death under upstream command Distinct from the idea that a requirement for protein synthesis defines genetically programmed cell death, the ÔprogramsÕ appear in general to be ready for action Together with abundant evidence that the apoptosis execution pathways are essentially independent of protein synthesis, both autophagy and programmed necrosis have so far been traced to post-translational signal transducers, such as protein kinases and phosphatases In all of these cases, it would appear that only the simplest definition of PCD, much like the original concept of Ôa sequence of events … that lead to death of the cellÕ [20] would resist close scrutiny There is much to be said in favor of undecorated concepts, which more often than not represent the essential features upon which myriad variations can be identified, quite typical of biological systems Despite the long-standing evidence for alternative cell death strategies, it is still often assumed that caspasemediated apoptosis is either the major, or the most frequent, form of PCD Although statistics may eventually prove this point, this is by no means warranted Indeed, the dominance of apoptosis among the published examples of PCD may be due to the fact that apoptosis is the only form of cell death that has long been categorized not only on the basis of its defining ultrastructural changes, but also on the basis of 1646 C Assuncao Guimaraes and R Linden (Eur J Biochem 271) ¸ ˜ ˜ cytological features recognizable by either conventional light microscopy or by relatively simple histochemical or immunohistochemical techniques Reviewers frequently demand TUNEL or caspase assays as tests of the nature of programmed cell death, and are usually satisfied with a simple positive, unqualified response, even though this positive identification is often achieved for only some of the dying cells in either naturally occurring or experimentally induced cell death More thorough examination of the forms of cell death in each circumstance is needed to assess, for example, the view that morphologically distinct pathways of cell death may be part of a continuum of degenerative events Only recently, techniques such as the monodansylcadaverine assay have become available to test for autophagic cell death in cell populations [169] and no simple assay is as yet available for the positive identification of necrosis Thus, it remains to be evaluated how frequently the alternative cell death forms also occur where apoptosis has been positively identified, whether it is caspase-mediated or not A further and pressing issue is how the autophagic and apoptotic pathways interact Evidence is available both for independent as well as for tandem programs for each of these forms of cell dismantlement The issue is complicated by the fact that the presence of autophagic vacuoles per se is not enough evidence that the cell is committed to degeneration, because autophagy may be part of a cell defense mechanism Admittedly, the rather unpopular view that apoptosis, or at least caspase-mediated apoptosis, may well have been grossly overrated has a significant chance to underlie a minority of cases, after all But it has already been worthwhile in the form of increasing awareness concerning alternative cell death styles These must be critically evaluated, in lieu of the prevailing assumption that the overwhelming and excellent science behind apoptosis may have definitively set the scene for all the hot stories related to programmed cell death Alternative cell death programs most probably imply novel therapeutic targets, and have important consequences for attempts to treat diseases associated with disregulated programmed cell death 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