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Home , Programmed cell death, Unicellular organism

Research 543 (2003) 235–249

Review The of programs as prerequisites of multicellularity Simone Huettenbrenner a,1, Susanne Maier a,1, Christina Leisser a, Doris Polgar a, Stephan Strasser a, Michael Grusch b, Georg Krupitza a,∗ a Institute of Clinical , University of Vienna, Währinger Gürtel 18-20, A-1090 Vienna, Austria b Institute of Research, University of Vienna, Borschkegasse 8a, A-1090 Vienna, Austria Received 4 September 2002; accepted in revised form 2 December 2002 In memoriam of my father, Hans Krupitza

Abstract One of the hallmarks of multicellularity is that the cellular fate is sacrificed for the benefit of a higher order of —the . The of cells in a results in swelling and membrane-rupture and inevitably spills cell contents into the surrounding tissue with deleterious effects for the organism. To avoid this form of necrotic death the cells of metazoans have developed complex self-destruction mechanisms, collectively called programmed , which see to an orderly removal of superfluous cells. Since evolution never invents new but plays variations on old themes by DNA , it is not surprising, that some of the genes involved in metazoan death pathways apparently have evolved from homologues in unicellular , where they originally had different functions. Interestingly some unicellular protozoans have developed a primitive form of non-necrotic cell death themselves, which could mean that the idea of an altruistic death for the benefit of genetically identical cells predated the invention of multicellularity. The cell death pathways of protozoans, however, show no to those in metazoans, where several death pathways seem to have evolved in parallel. Mitochondria stands at the beginning of several death pathways and also determines, whether a cell has sufficient energy to complete a death program. However, the endosymbiotic bacterial ancestors of mitochondria are unlikely to have contributed to the recent mitochondrial death machinery and therefore, these components may derive from mutated eukaryotic precursors and might have invaded the respective mitochondrial compartments. Although there is no direct evidence, it seems that the prokaryotic–eukaryotic created the space necessary for sophisticated death mechanisms on command, which in their distinct forms are major factors for the evolution of multicellular organisms. © 2003 Elsevier Science B.V. All rights reserved.

Keywords: ; ; ATP; Mitochondria; Evolution

Abbreviations: ADP, adenosine diphosphate; AIDS, acquired immune deficiency syndrome; AIF, apoptosis inducing factor; ANT, adenine nucleotide translocator; ATP, ; CAD, -activated DNAse; caspase, cysteine aspase; dATP, deoxy-adenosine triphosphate; DISC, death-inducing signaling complex; Ψ m, mitochondrial membrane potential; ER, endoplasmatic reticulum; Fas, Apo-1, or CD95; FasL, Fas-; FK506, immuno-suppressant isolated from Streptomyces sp.; IAP, ; mTOR, mammalian target of rapamycin; NAD, nicotinamide adenine dinucleotide; PT, pore transition; ROS, reactive oxygen ; TIR, toll-like interleukin- domain; TNF␣, ␣; TRAIL, TNF-related apoptosis-inducing ligand; z-VAD-fmk, benzyloxylcarbonyl Val-Ala-dl-Asp-fluoromethylketone, a specific inhibitor of ∗ Corresponding author. Tel.: +43-1-40-400-3487; fax: +43-1-405-34-02. E-mail address: [email protected] (G. Krupitza). 1 Contributed equally to first authorship.

1383-5742/03/$ – see front matter © 2003 Elsevier Science B.V. All rights reserved. doi:10.1016/S1383-5742(02)00110-2 236 S. Huettenbrenner et al. / Mutation Research 543 (2003) 235–249

1. Introduction; life and the meaning of death existed in unicellular , albeit in different contexts. But even cell death mechanisms might have The term “life” describes a combination of pheno- existed in ancestral protozoans, because in a few con- types such as metabolic activity, its restriction to com- temporary unicellular parasites and in plex structures, growth, and the potential to identically discoideum we find cell death reminiscent self-reproduce. When we limit our observations to of apoptosis [2]. These mechanisms not only allow “death”, of course, does not occur in this differentiation into or cysts but enable the sur- description. In these primitive organisms death only vival of a in adverse conditions and hence it seems to be a consequence of environmental condi- seems that these mechanisms were mandatory to find tions that are not compatible with the biochemistry niches for multicellularity [2]. In this respect it is of and that accompanies “life”. In eukaryotes note that eukaryotic AIF could be tracked down to this accidental cell death constitutes the some of the diverse group of the , which are the called necrosis. In contrast, multicellular organisms assumed ancestors of today’s eukaryotes [13]. Also developed complex cell suicide mechanisms to cir- paracaspases which are found in Dictyostelium and cumvent necrosis and also in unicellular eukaryotes metazoans, and which are found in pro- non-necrotic cell death was described [1,2]. Prokary- tozoans, fungi and , indicate a common ancestry otes seem to lack those homologous components [12]. However, in unicellular eukaryotes these cysteine required to die in orchestrated fashion although the seem to serve in and commitment to suicide was observed during fruitbody have acquired their new function in apoptosis later on formation of Myxobacteria [3]. A few unicellular [13]. eukaryotes possess a primitive apoptogenic outfit The death effectors that operate in protozoans [2,4–7] which is however, not regulated on program. are still enigmatic and furthermore there exist also In general, there is an apparent lack of homologies protozoans that cannot undergo an apoptosis-like (orthologues) between unicellular death modules and phenotype. It has to await clarification whether the those of recent metazoans, and therefore, a secondary death machinery was lost by reductive evolution, or death machinery might have been acquired entirely whether those species without apparent self destruc- independent from higher organisms. In this respect, tion mechanisms represent an early phylogenic life the term apoptosis just defines the mechanism(s) lead- form. Alternatively, the death components in par- ing to the phenotype of resorbtive self-destruction. asitic unicellular eukaryotes might have developed In extension, “” includes during the -defense evolution. Notably, even in the invention of intrinsic and extrinsic trigger fac- prokaryotes “apoptotic” features have been described tors, as part of an integrated genetically determined [3,14,15] and even examples for shared homologies process, that becomes activated when appropriate exist: the TIR [13,16] is homologous in , [8–11]. plants, and . Toll receptors play a role in Once single cells started to organize into multicel- the immune defense against parasites and can elicit lular forms it was apparently advantageous to invent a apoptosis. Furthermore, bacterial cell death program as a means to shape structures and HtrA shows homology with mammalian HtrA2 [17]. to balance this complexity. This implicates that the in- However, bacteria require HtrA to tolerate thermal, dividual cellular fate was sacrificed for the benefit of osmotic and oxidative [18] and the chaperone a higher order of life, and seems to evidence the exis- function of the bacterial homologue changed to an tence of a principal archaic hierarchy of being. How- IAP-inhibitor, which is a that counteracts cas- ever, this point of view neglects the fact that evolution, pase activity in eukaryotes [19,20] in analogy to Smac as we know since , does not “invent” [21–23]. new genes with specific functions but varies and As the driving force, increased or new environ- adapts the existing outfit by DNA-mutations and this mental pressure, might have urged for different forms genetic plasticity allows to develop new shapes and of cell death, which were accomplished over time. advanced functionality. Thus, some components that We cannot track back the succession of mutations gradually gained functions for self destruction already that were necessary to evolve cell death programs S. Huettenbrenner et al. / Mutation Research 543 (2003) 235–249 237 mandatory for the evolution of higher order life forms. of these apoptotic defense systems, however, did not However, we know from such as cancer arise in the cells of the , but earlier in or Alzheimer’s , that loss- or gain-of-function evolution in less specialized cells favoring innocuous mutations of apoptosis genes destroy this complexity death as a prerequisite for the development and main- [24]. tenance of multicellular aggregations. Therefore, an- cient death-components are highly homologous from the primitive worm Caenorhabditis ele- 2. Necrosis—a threat to the higher order gans to the to , and the components increased in number and complexity Necrotic (accidental) cell death results from a vari- throughout phylogenic evolution [25]. ety of stresses, such as extreme physicochemical injury Below certain threshold limits of noxious insults (radicals, radiation, temperature, toxic trauma, etc.), cells vanish through apoptosis or may survive due osmotic imbalance, abrupt anoxia, energy deprivation to cellular de-toxification, DNA-repair, or emergency (sudden shortage of such as glucose), but mechanisms. However, upon severe and immediate also when apoptotic execution pathways are blocked impact the damage is either too massive to be re- after physiological apoptosis-induction. Hence, necro- paired or time is too short to allow for an apoptotic sis is always the outcome of severe acute insults that response. In either case, cells burst by a phenotype cause almost instantaneous membrane depolarization called primary necrosis. Depending on the extent of and disruption. the damage caused by stress factors, and whether or The destruction of permeability barriers (Ψ collapse) not energy supply is sufficient for the completion of upon stress and the resulting collapse of sepa- physiological emergency programs cells may still be- rated biochemical regulatory cycles that maintain come necrotic due to energy depletion, although the life-processes, is an inadequate mechanism for meta- apoptotic machinery might have been already sparked zoan cells to evade life, because of the threat to [26]. This constitutes the phenotype called secondary the superior hierarchy. Necrotic spillage of cellular necrosis. Therefore, cells that were exposed to necrotic components, which are otherwise stored away in stresses, may also exhibit apoptotic early stage phe- subcellular compartments, would induce inflamma- notypes [27–29]. tory reactions that affect neighboring cells which in Interestingly, a component of a DNA-proof-reading consequence become necrotic themselves, resulting and repair mechanism, poly(ADP-ribose) polymerase in a constant spreading of necrotic somatic areas. (PARP) [30,31], is the to date only known element that In general, inflammatory responses are physiological is sensitizing a cell for necrosis [32,33] but does not of the immune system in pathogen de- modulate apoptosis [34–36]. PARP utilizes NAD as a fense, but cell fluids that are normally not exposed substrate to synthesize and transfer poly(ADP-ribose) also elicit this response. Thus, necrosis is an inade- and upon prolonged PARP-activation the NAD-pool quate means to maintain homeostasis, because it can and the ATP-level drop dramatically and cells die lead to auto-immune reactions. To allow for proper, [37–40]. PARP−/− cells, that do not consume NAD in (stress-triggered) escape from life without damaging response to DNA-strand breaks, and therefore exhibit considerable tissue areas, it requires a mechanism improved ATP maintenance, are less sensitive towards which is non-toxic to the cell-neighborhood. The necrotic stimuli, and PARP−/− mice do not suffer immune system developed highly sophisticated apop- from cerebral [33]. Hence, ATP is consid- totic host-defense mechanisms against cells which ered to be a switch between apoptosis and necrosis are genetically altered- or “non-self”, thereby defend- [41–43]. However, recent discussion considers that ing off potentially dangerous cells and contributing ischemic brain damage is not only based on necrotic to homeostasis. Moreover, “activation induced cell but also on apoptotic cell death of the adjacent tissue death” evolved to down-modulate the immune re- [44], and furthermore, that ischemic cell death is a sponse, which is regulated by death ligand–death re- separate type of apoptosis [45]. Therefore, the role ceptor interactions, and to limit the expansion of cells of PARP in necrosis (but not in apoptosis) needs fur- responding to a particular antigen. The fundamentals ther clarification. Nevertheless, based on compelling 238 S. Huettenbrenner et al. / Mutation Research 543 (2003) 235–249 evidence, the current understanding is, that those com- compartment is a well buffered niche and perfect ponents that have evolved in multicellular organisms to safely store away life-threatening molecules. Two to allow for a death on program depend on energy arguments are supporting this hypothesis: (ATP), whereas necrosis is independent of ATP— (1) Death effectors (i.e. caspases 2, 9, and 3) are or more accurately—that energy depletion leads to only active at acidic pH but not in the mito- necrosis. chondrial, well buffered, inter-membrane com- partment. Thus, apoptogenic components would 3. The evolution of programmed cell death have developed by eukaryotes after the prokary- otic mitochondrial ancestor got manifested as . Death programs are tightly linked to eukaryotic– (2) and other unicellulars, which also contain bacterial endosymbiont co-evolution. Two distinct mitochondria lack the apoptogenic death compo- by bacterial endosymbiosis gave rise to nents known from metazoans. Thus, mitochondria eukaryotic realms. Firstly, ␣-proteobacteria develop- are not the source of death components. How- ing to the mitochondria of all eukaryotic cells and ever, it was shown that Saccaromyces cervesiae secondly, that changed to [58–60], Schizosaccaromyces pombe [61], try- in evolution. Although we do not have evidence panosomes [1,6], [62] andafew today that more and diverse bacterial endosymbiosis other unicellular eukaryotes [63,64], can undergo had evolved, we can still take it as almost certain that apoptosis-like cell death upon exposure to ROS this had happened but was eradicated by competition [65,66]. or catastrophe. The Palomyxa palustris hosts endosymbiotic aerobic bacteria which metabolically Another hypothesis proposes that the evolution of substitute for the lacking mitochondria [2]. This ev- mechanisms, which provoke apoptosis-like pheno- idences that convergent endosymbiont evolution is types in a number of unicellular eukaryotes, have still going on. developed by the selective pressure of limited nutri- Many regulators of apoptosis are located in the mi- tional supply. This would eradicate affected genomes tochondrion or in the mitochondrial inter-membrane (turmoiled by genotoxic stress) in favor of intact ones space (caspases 2, 3, and 9 [46], cytochrome c [47], without poisoning limited nutritional resources by Bcl-2 [48], Bcl-XL [49,50], Bad [51], Bax [52], Bak necrotic spillage [26,58,59,61,65]. This hypothesis [53], Bim [54], Smac [55], HtrA2 [22], AIF [56,57]). suggests that unicellular eukaryotes, single However, we do not find these or homologous com- and virtually un-connected to each other, may have in- ponents (except HtrA) in contemporary bacteria. The vented a strategy for the benefit of the whole species, question arises: Did the aerobic bacterial predecessors that is reminiscent of the complex cellular interrela- of mitochondria possess apoptotic regulators that grad- tions and adaptations of metazoan tissue development. ually vanished in modern bacteria and other prokary- In retrospect, this makes the hypothetical species look otes? Usually systems increase in complexity during “altruistic”. In fact, slime molds, in which AIF acts as phylogenesis and, once mechanisms or structures got apoptogenic factor, can do both: live as single amoe- lost by reductive evolution, it has not been observed so boid individuals but can also aggregate and behave like far in biological systems that identical parts or pieces a multicellular organism in the search for nutrients [4]. were re-introduced later on by homologous structures Another “altruistic” hypothesis was put forward or components, but only by analogous ones due to [16], which speculates that parasitic , that secondary (i.e. fish fins versus would have been lethal for protozoans, might stand dolphin fins). at the very beginning of the development of apopto- A few hypotheses were brought up, to explain how genic mechanisms. In this hypothesis, the altruistic the apoptotic outfit might have evolved in metazoans. suicide of the infected individual (the individual cell One hypothesis speculates [46] that the metazoan would die anyway) would also destroy the parasite, apoptotic machinery “invaded” the mitochondrial which could otherwise move on to the next, presum- membrane interspace during evolution, because this ably closely related protozoan. Thereby it preserves S. Huettenbrenner et al. / Mutation Research 543 (2003) 235–249 239 the rest of the population and this “altruism” exerts a 4.1.2. Caspase-independent apoptosis selective advantage for the species. Currently we know that the execution of apoptotic These hypotheses have in common, that a death degradation can be achieved not only by machinery gradually developed from already exist- executioner caspases 3, 6, and 7 and their effector ing polypeptides by successive mutational events. The CAD ([70,71], which generates 180 bp DNA frag- new functions benefited an assumed ancestral ments and multiples thereof, but also by AIF in a population of unicellular individuals, which included caspase-independent fashion. AIF activates a nuclear the option to get on the evolutionary way to invent DNAse [72] which cuts genomic DNA into 50 kb frag- multicellular complexity in the future. This may exem- ments giving rise to a distinct nucleo-morphological plify, that a primitive suicide machinery was the pre- phenotype of chromatin condensation, which is called requisite to start metazoan evolution, which is widely stage I [73]. Whereas CAD generates the phenotype accepted, yet has to be proven. of stage II chromatin condensation (see Fig. 1). The Regardless whether the basic apoptotic machin- two mechanisms of apoptosis execution exist to- ery emerged in the one way or the other, within the gether and may cooperate. It needs to be determined framework of the ancestral metazoan self-destruction whether either of the pathways can be triggered sep- “” provided by the endosymbiont, distinct arately depending on the cell type, the context, or the multi-component death mechanisms emerged. stimulus. The question why two apoptosis pathways evolved in parallel remains open. We find numerous examples showing convergent evolutions of organs 4. Distinct genetic programs to terminate life and structures, which in contrast to homologies (or- thologues), are termed analogies (paralogues). Thus, Several morphologically and biochemically dis- might as well have invented self-destructing cernible forms of programmed cell death have been mechanisms a second or third time simultaneously described. or in parallel, utilizing the same frame structures such as the mitochondrial inter-membrane space (because of advantage), but developing distinct func- 4.1. Type I cell death tional components (because no homologies of apop- totic molecules between yeast and metazoans were 4.1.1. Caspase-dependent apoptosis found to date). Throughout cell death evolution the This is a physiological process of cell suicide elim- proapoptotic mammalian factor Smac [55] and the inating superfluous or unwanted cells. Apoptotic cell fruitfly factors Reaper, Grim and Hid [74] function death involves the orchestrated action of catabolic in a similar way by inhibiting IAPs, but they are not enzymes (proteases, nucleases) within the limits of homologous. intact plasma membranes and is accompanied by a characteristic change of nuclear morphology (see 4.2. Type II cell death Fig. 1) and chromatin biochemistry (stepwise DNA degradation; [67]). Specific cysteine proteases (cas- Cell death by , or type II cell death, pases) cleave their targets after aspartic acid residues involves . Upon induction, cytoplasmic and catalyze a highly selective pattern of protein material and relevant subcellular such as degradation. Moreover, cellular organelles remain ER and/or Golgi become sequestered and parts of morphologically intact (biochemically only subtle their membranes assemble to . These changes of organelles become manifest in dying cells further integrate with lysosomes and build up the au- such as membrane permeabilization and partial pro- tophagic , which can be visualized by stain- tein degradation), whereas cells shrink and reduce ing with mono-dansylcadaverine [75] (see Fig. 1). intracellular potassium level [68,69]. In the aftermath Even mitochondria become degraded despite a few of apoptosis, because the remaining material is in required for ATP-supply. Furthermore and in contrast relative small units, they are readily phagosytosed by to apoptosis, the cytoskeleton remains intact (see unaffected neighboring cells. Fig. 1). This is a fundamental difference to apoptosis Fig. 1. Doxorubicin simultaneously induces apoptosis, necrosis and formation in SKBR-3 breast cancer cells; SKBR-3 cells were triple-stained with Hoechst 33258 (2.5 ␮g/ml), propidium iodide (1 ␮g/ml) and mono-dansylcadaverine (1:3000 dilution of a saturated 50% ethanol solution) and photographed with a 400-fold magnification using a Zeiss Axiovert microscope connected to a UV-lamp and a Zeiss DAPI filter no.: 02 (left side panels; H-P-C). Right side panels show the identical microscopical frames photographed with phase contrast (P-C). Panels (A): Control cells were treated with solvent and exhibit normal nuclear Hoechst 33258 chromatin staining (dark blue). (a) Shows a cell undergoing spontaneous apoptosis at an early stage (bright blue nuclear staining due to chromatin condensation). Panels (B): Cells were treated with 5 ng/ml doxorubicin for 96 h. (a) Points at an early apoptotic (bright blue). Exposure to doxorubicin clearly increases the apoptosis rate. Panels (C): Cells were treated with 40 ng/ml doxorubicin. (a) Shows a growing number of late apoptotic cells, which are in the process of losing membrane integrity. Due to intrusion of propidium iodide, which causes the merging of the colors red and blue, the chromatin stains pink-white. Panels (D1): Arrow (n) points at a necrotic cell. Due to disrupted membranes, as a hallmark of necrosis, propidium iodide intrudes resulting in evenly pink-white nuclear staining. Condensed chromatin, which is typical for apoptosis does not occur in necrotic cells. Arrow (a) points at an apoptotic cell and arrow (au) at a cell which exhibits autophagosome formation detected by mono-dansylcadaverine (blue cytoplasmic spots). The nuclear morphology of this cell (au) is still intact and prevents intrusion of propidium iodide and hence, the chromatin is only stained by Hoechst 33258 (blue nucleus). Therefore, the cell is presumably alive, or considered as “not dead yet”, and might recover upon drug removal. However, the cell shown in panels (D2) by arrow (au), which also forms autophagosomes, but lacks nuclear staining, has reached an advanced stage of autophagy and is certainly not viable any more. The remaining cell structure (see phase contrast; D2) suggests that cytoplasmic structure- are still preserved as a hallmark of type II cell death. By which mechanism the chromatin vanishes remains enigmatic. In panels (D1) and (D2) cells were treated with 100 ng/ml doxorubicin and the images were additionally magnified two-fold and brightened by Adobe Photoshop program for improved demonstration. S. Huettenbrenner et al. / Mutation Research 543 (2003) 235–249 241

Table 1 Distinct mechanisms causing cell demise Apoptosis Autophagy Necrosis Physiological/pathological features Induced by various physiological and noxious Induced by various physiological and Induced upon severe stress, toxic stimuli, and during development noxious stimuli, and during development exposure and/or ATP-depletion No inflammatory response No inflammatory response Inflammatory response Affection of individual cells Affection of individual cells Massive affection of tissue areas Active process Active process Passive process Morphological features Blebbing of intact outer membranes Sequestration of cytoplasmic material Swelling of nucleus, organelles and the entire cell Preserved organelles Chromatin condensation Degradation of ER, Golgi, and partly of Disruption of membranes mitochondria Apoptotic body formation Formation of autophagosomes Spilling of cell constituents Biochemical features Moderate PT PT? Severe PT Transient collapse of Ψ m Permanent collapse of Ψ m Requirement of ATP Requirement of ATP ATP-independent Mitochondrial efflux of Cycto-C Other mitochondrial contributions? De-polymerization and of cytoskeleton Redistribution preservation of the cytoskeleton Dependence on caspases and/or AIF Caspase-independent; role of AIF? Non-random DNA fragmentation DNA-fragmentation at random? Molecular features Promoted by Bad, Bim, Bid, AIF, and mitotic Promoted by Beclin-1 stimuli in absence of survival signals Regulated by p70-S-6 and mTOR Inhibited by Bcl-2, Bcl-XL Inhibited by abrogation of autophagosome Prevented by Bcl-2, and formation PARP-inhibition All three types of cell death exhibit distinct but also similar features at the molecular, biochemical and physiological level as far as known to date. To investigate the type of cell death induced by various triggers, the examination of the morphological features is still a reliable method for an initial discrimination. Many of the morphological features can be determined by middle or high resolution fluorescence phase contrast microscopy in combination with vital staining methods (e.g. Hoechst 33258, propidium iodide, mono-dansylcadaverine) using appropriate fluorescence filters. Some of the morphological criteria need to be examined by electron microscopy, which makes investigations more tedious. To speed up the discrimination analysis phase contrast fluorescence microscopy in combination with biochemical–analytical methods such as monitoring of the stability of cytoskeletal proteins and caspase-mediated cleavage of target proteins by Western blotting, can be used to determine the type of cell death. However, it is not unusual that a cell population undergoes all three death types upon the same stimulus. Even in the same cell the distinct death features may occur. Abbreviations: PT, mitochondrial pore transition; ATP, adenosine triphosphate; Cyto C, cytochrome c; AIF, apoptosis inducing factor; ER, endoplasmatic reticulum; mTOR, mammalian target of rapamycin; Ψ m, mitochondrial membrane potential.

(type I cell death; see Table 1), in which mitochondria [79]. Despite the autophagy-inducing gene beclin1 are the relevant organelles which remain preserved [80], a Bcl-2-interacting protein, which is homologous throughout the process, whereas , cytokeratins to yeast autophagy gene apg6, molecular components and become fragmented [76]. There are reports of type II cell death still await to be determined. demonstrating that autophagy proceeds independent apg genes interact with mTOR (mammalian target of caspases [77,78]. This implicates that several sui- of rapamycin) and thus might link mTOR, FK506, cide mechanisms evolved in parallel. Recent data, and S6-kinase to autophagic processes. Interestingly, however indicates that cell death, which was triggered type I and type II cell death can occur in mixed form by toxins directed against other organelles such as the within the same tissue or cell type (see Fig. 1) and ER, is also dependent on mitochondrial pore transition even within the same cell. Autophagy which is known 242 S. Huettenbrenner et al. / Mutation Research 543 (2003) 235–249 from yeast to the D. discoideum [81] to apoptosis but force or reverse autophagic for- mammalians [76,82] implicates that self-destructing mation will elucidate whether autophagic processes mechanisms already existed before the splitting of are sufficient for cell death induction. ancestral unicellulars into fungi and metazoans. The prime function of autophagy in yeast is the degrada- tion and the recycling of cytoplasmic constituents and 5. Cell-death programs as prerequisites of organelles in response to nutritional starvation. How- organized life ever, in higher eukaryotic cells its independence of or interdependence with apoptosis needs to be further During embryonic development of the nervous clarified [83,84]. Experiments that specifically inhibit system [85], the finger digits, or the ovary [86],but

Fig. 2. Similar, distinct, and overlapping triggers of different cell death mechanisms; type I apoptosis is elicited by intrinsic signals such as ROS production, over-expression, DNA damage, physical stress factors and cytotoxic chemicals. Exposure to these factors leads to mitochondrial pore transition (m-PT) which is controlled by some of the Bcl-2 family of proteins. Anti-apoptotic Bcl-2 members prevent pore transition, whereas pro-apoptotic Bad may promote this process. Cytochrome c (Cyto-C) and/or apoptosis inducing factor (AIF) are released through this pore and initiate assembly followed by caspase 3 activation and nuclear stage II chromatin condensation, and/or nuclear stage I chromatin condensation, respectively. Therefore, both mechanisms, which are physiological processes, can cooperate in chromatin degradation. In contrast, intense noxious stimuli can override subtly regulated membrane alterations causing long lasting membrane ruptures and the ultimate collapse of the mitochondrial membrane potential (Ψ m) which results in the over-production of reactive oxygen species (ROS) and in the depletion of ATP otherwise required for the orchestrated apoptotic program. The outcome is necrotic cell demise. This cascade leading to ATP depletion is triggered very rapidly in response to a severe damage of the DNA. Upon less severe stresses, necrosis can still occur later on, after the apoptotic program has been already sparked, and when ATP is gradually running short. Hence, both apoptotic and necrotic features may be observed in a cell population or even within the same cell. Type II apoptosis is elicited by ligand-mediated activation of death receptors such as Fas, TNF-receptor I, TRAIL-receptor, to which the death-inducing signaling complex (DISC), which includes , becomes assembled. DISC-recruited caspase 8 is processed and activated by the proximity to other caspase 8 molecules and now activates downstream (executioner) caspases 3, 6, and 7. Caspase 8 can also amplify the receptor-triggered death signal by processing Bid. The carboxy-terminus of Bid translocates to the mitochondria which causes the release of cytochrome c and the unleashing of the intrinsic apoptosis pathway. S. Huettenbrenner et al. / Mutation Research 543 (2003) 235–249 243 also in adult organisms [11], minutely regulated daily 6. Apoptosis–necrosis: similar start–different death processes maintain health and integrity. Ev- finish ery second, several millions of cells of the body undergo apoptosis, i.e. in conditions of home- There is convincing evidence that mitochondria are ostasis each is compensated by one event central regulators of apoptosis [101,102]. As one of of apoptosis. Such as to select B cell in the ger- the very first events in response to multiple stresses minal centers of lymph nodes by follicular T cells or physiological induction, mitochondrial PT causes [87], or to remove aged phosphatidylserine-exposing ion efflux [103] thereby disturbing the mitochondrial erythrocytes by macrophages—otherwise blood ves- membrane potential Ψ m [26]. sels would plug [88,89]. Hence, in the blood sys- One consequence of Ψ m disruption is interference tem, cell death on program has to be as abundant with mitochondrial oxidative phosphorylation result- as cell replication. Once this fine tuned balance is ing in the decrease of ATP [104,105]. Another con- disturbed (even when occurring at sub-detectable ex- sequence is the uncoupling of the respiratory chain tent), sooner or later life-threatening such leading to the hyperproduction of ROS, affection of as and leukemia will become mani- the plasma redox potential, and subsequent irreversible fest. Lymphocyte selection in germinal centers is oxidation of thiols and membrane , as well as based on ligand-receptor mediated cell death [90,91] the depletion of glutathione [106]. Further, the influx 2+ which is an additional specialization and acquisited of H2O and Ca ions, will cause swelling of the cy- phylogenically later to the regulatory repertoire of toplasm and organelles due to osmotic pressure, and apoptosis. This type I apoptosis, which is exerted finally the cell bursts and spills the fluids into the peri- across caspase 8 (extrinsic pathway triggered by, cellular space. This phenomenon describes a classic e.g. FasL, TNF␣, TRAIL, see Fig. 2), is assumed necrotic phenotype (see Fig. 2). Cytochrome c, which to be independent of mitochondria [92], and is set together with Apaf 1 induces caspase 9 in higher eu- in contrast to type II apoptosis (intrinsic pathway karyotes, and AIF, which directly translocates into the triggered by, e.g. toxic compounds, stress, but also nucleus to induce caspase-independent stage I apop- during development; see Fig. 2), which is exerted tosis, are also released through the activated PT–pore by caspase 9 [93–97]. Interestingly, inhibition of the complex [16,107] (see Fig. 2). Very likely AIF, which mitochondrial pathway by Bcl-XL-overexpression mediates degradation of genomic DNA into 50 kb frag- prevents Fas-triggered apoptosis [98]. This may be ments, exerts its activity during apoptosis and necro- explained by the observation that DISC-activated sis [108]. Therefore, the extent of Ψ m disturbance caspase 8 can cross-talk to and activate the mitochon- may determine between life and death as a point of no drial caspase 9 by the Bid-bypass [99,100], thereby return. connecting the extrinsic to the intrinsic pathway The PT–pore complex is controlled by the anti- and amplifying receptor-mediated death signals (see apoptotic molecules Bcl-2 and Bcl-XL [56,109,110] Fig. 2). and upon ectopic overexpression, Bcl-2 inhibits PT Since apoptosis is required for develop- [56,111] and prevents not only apoptosis [112,113] ment and tissue homeostasis the dis-regulation of but also necrosis [114–119]. Thus, early apoptotic cell death programs results in various pathologies. onset and necrosis start with similar events such as An abnormal resistance to apoptosis induction causes PT [120–125]. ANT, a component of the PT–pore malformations, cancer, or autoimmune diseases due complex [126], which is also regulated by Bcl-2 to the persistence of superfluous, mutated cells, or [127] exchanges mitochondrial matrix ATP for cellu- cell-specific immunocytes, respectively. Conversely, lar ADP [121,128]. ATP:ADP ratios are supposed to acute apoptosis upon by toxin-producing mi- determine whether a cell will vanish by necrosis or croorganisms, during ischemia–reperfusion damage, orchestrate an apoptotic [129]. The intensity or infarction will result in enhanced decay of cells, of noxious stimuli (stress, toxic compounds, or even whereas chronically increased apoptosis induces dis- physiological death factors) correlates with the extent eases such as neurodegenerative and neuromuscular of ATP depletion and therefore, with the type of cell disorders and AIDS. death. Exhausting ATP in consequence of excessive 244 S. Huettenbrenner et al. / Mutation Research 543 (2003) 235–249 trauma blocks the apoptotic execution machinery (4) ATP might just be needed to maintain intact mem- downstream of PT and necrosis prevails [103]. Also branes, which are a major feature of apoptosis. specific inhibition of caspases by z-VAD-fmk ren- ders cells to exit by a necrotic phenotype although Upon DNA-damage by genotoxic stresses (e.g. ra- identical stimuli induce apoptosis when z-VAD-fmk diation, radicals, or alkylating agents), the activation is omitted [52,103,130]. This demonstrates that of DNA-repair enzymes and PARP causes the con- necrosis–apoptosis decisions are not only taken by sumption of ATP. ATP. Hypothetically the physiological inhibitors of Therefore, in cases of severe DNA-damage the en- caspases, the IAP family members, might also deter- ergy level will decrease below certain threshold levels mine by which mode cells will demise. This has not and either energy and/or time are insufficient to un- been investigated so far. Interestingly, it was shown dergo the entire repair or apoptosis program and the that caspases can cause PT, thus their activation occurs demise is “necrotic”. not only downstream but also upstream of a mito- In presence of pro-apoptotic stimuli artificial de- chondrial death decision [131,132]. Nevertheless, the pletion of ATP by inhibitors of the respiratory chain status of Ψ m which depends on the stress type and (e.g. rotenone, [138]), or by interference with glycol- impact, not only switches between life and death, but ysis (deoxyglucose, or S-nitrosoglutathione; [139]), gives also major directives for death modes such as the balance is shifted towards necrosis [117,134].In necrosis, type II apoptosis, and caspase-independent contrast, restoration of ATP by enhanced glycolysis apoptosis by AIF (see Fig. 2). The current observa- (fructose, glucose) restores the apoptotic phenotype tions support the notion that a substantial number of [29,43]. This is in agreement with the observation effectors of the apoptotic machinery developed within that damages which otherwise would cause necrosis (or from?) the mitochondrial PT–pore complex, (due to high dosage) result in apoptotic execution which is the primordial necrosis effector, unless bar- when energy-levels are artificially maintained, which riers that separate and regulate biochemical/metabolic has impact for chemotherapy, because unwanted processes (inevitable to maintain “life”) are well side-effects due to necrotic damage can be avoided preserved. [43]. However, acute toxicity might result in acciden- tal cell death so quickly (e.g. primary necrosis) that artificial energy restoration alone would not suffice to 7. Prevention of necrosis in favor of apoptosis proceed with an intrinsic death program. It is evident that distinct self destruction programs, As demonstrated by various experiments, depletion which stand in apparent contrast to necrosis, co-exist or replenishment of ATP favors necrosis or apoptosis, within the same cells and can get started depending respectively [36,43,133,134]. Nevertheless, it is not on the context and on the stimuli. The fact, that such completely understood, by which ATP-dependent step death-programs evolved despite the high price of en- the apoptosis–necrosis-decision is taken: ergy consumption—necrotic cell death would be “cost free”, evidences that apoptosis and other forms of ac- (1) ATP is required to keep survival pathways such tive cell death are major factors in the evolution of as the AKT pathway active. This however should multicellular organisms. be expected to decide only between life and death but not apoptosis and necrosis. (2) Although the components for apoptosis are Acknowledgements (partly) prefabricated in the cell it has been shown that de novo transcription and translation may be We want to thank Prof. W. Bursch for helpful dis- required in some cases. Both processes demand cussions and the donation of mono-dansylcadaverine. ATP. This work was supported by the Hezfeldersche (3) dATP (and therefore ATP), together with cy- Familienstiftung, the Fund of the Austrian National tochrome c and Apaf 1, is needed for caspase 9 Bank No. 9298 and the Unruhe Privatstiftung to assembly into the active apoptosome [135–137]. G.K. S. Huettenbrenner et al. / Mutation Research 543 (2003) 235–249 245

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