Apoptosis: lessons from in vitro systems

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Apoptosis research was born with the proposal by Kerr, Wyllie and Currie that there are two major types of cell deathi. The first of these, necrosis (passive or accidental cell death), follows from physical damage to cells (the cell membrane in particular), involves groups of cells, and has the deleterious end result of causing tissue inflammation. The second, which they termed apoptosis, is undergone by individual cells that are surrounded by healthy neighbours. Apoptosis is accomplished by a spectacular morphological transformation of the cell, which ultimately disassembles into membrane-enclosed vesicles (apoptotic bodies), and does not result in inflammatior?. Apoptosis requires ATP and, often, new protein synthesis. It therefore represents a form of active cellular suicide. This view of cellular mortality was seminal in defining active cell death as an interesting and important area for study. However, it is clear that some (perhaps many) forms of active cell death during development do not involve the hallmark morphogenetic events of classical apoptosis3. A particularly well-characterized example is provided by the death of the intersegmental muscles of the tobacco hawkmoth Munducca sexta4. Surprisingly, it also appears that some cell deaths that involve many of the classical features of apoptosis (cellular shrinkage, chromosome condensation, membrane blebbing, chromatin fragmentation) may actually follow different biochemical pathways in different cells5. Thus, when characterizing the molecular mechanisms of cell death, it will ultimately be important to move beyond the purely morphological definition of apoptosis to a biochemical definition of the pathways involved. What are the executive functions that trigger cellular suicide? Can this occur by more than one pathway? What are the functionaries that carry out the sentence and actually kill the cell? Why the apoptotic biochemists

pathway

is unkind

to

Why do we understand so little about the biochemical mechanisms involved in apoptosis? This is largely due to the nature of the pathway. Active cell death is a two-phase process. In the condemned phase, cells recognize a signal (external or internal) and become committed to death. This can also be referred to as a latent phase, as examination of these cells by microscopy reveals no obvious morphological changes. Condemned cells can even continue through the cell division cycle. For example, condemned chicken DU249 cells subsequently initiate and complete a normal mitotic division before undergoing apoptosis6. The second, or execution, phase is the stage at which all of the hallmark features of apoptosis occur. Apoptotic execution can be breathtakingly rapid. Video microscopy of cultures undergoing apoptotic death reveals that the entire execution phase, from its inception to the disassembly of the cell into apoptotic bodies, can be completed in as little as 15 minutes (G. Evan, pers. commun.). The major problem with biochemical studies of apoptosis is that death is cell-autonomous. TRENDS

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Apoptosis: lessons from in vitro systems Although apoptosis is a major factor in metazoan morphogenesis and tissue homeostasis, its underlying biochemical mechanisms are poorly understood. This is now beginning to change as cell-free systems for the study of apoptosis start to reveal some of the activities involved. As suggested by earlier genetic analyses, a proteinase with properties resembling. those of the interleukinl-/3-converting enzyme (ICE) has been shown to initiate the apoptotic cascade in vitro. Curiously, results obtained with the cell-free systems suggest that essential downstream effecters of the apoptotic response may be intrinsic components of healthy nuclei.

Condemned cells make the transition to apoptotic execution stochastically over a period of hours or days. Thus, the duration of the condemned phase is extraordinarily variable, even for synchronized clonal populations such as cell lines in vitro. As a result, any aliquot removed from experimental cultures contains cells in various stages of the process: condemned, execution and subsequent necrosis (apoptotic bodies eventually undergo necrotic degradation if not phagocytosed by neighbouring cells). Given the brevity of the execution phase compared with the condemnation phase, only -2O-40% of cells in in vitro cultures (and considerably fewer in tissues) are actually in the execution phase at the peak of apoptosis in the best experimental systems. This is a serious problem, particularly given the emerging realization that the execution process involves minute-by-minute changes in the spectrum of biochemical activities involved. New systems

for studying

apoptosis

One successful response to the synchrony problem has been to develop cell-free models for the study of apoptotic execution. Thus far, three rather different systems have emerged. They share several advantages: experiments are rapid; it is possible to add and remove proteins and other reagents that may not normally traverse the cell membrane; and it is possible to study apoptotic events without worrying about the effects of various treatments on other aspects of cellular physiology. However, these three systems also have significant differences. In the first, cytoplasm from apoptotic or control human promyelocytic HL-60 cells is mixed with isolated nuclei7. Cytoplasm from apoptotic cells 0 1995 Elsevier Science Ltd

William

C.

Eamshaw is at the Dept of Cell Biology and Anatomy, Johns Hopkins University School of Medicine, 725 North Wolfe Street, Baltimore, MD 21205, USA.

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hand, S/M extracts provide a powerful system for studying the minute-byminute sequence of nuclear events during apoptotic execution. New facts about apoptosis

the biochemistry

of

Initial studies with the cell-free extracts have yielded several notable conclusions: l Essential factors triggering the onset of apoptotic execution are present in the cytoplasm, and not in the nucleus or other major organelles6. This is consistent with the observation that cytoplasts can undergo apoptosis in the absence of resident nuclei11,12. l While S/M extracts appear to require FIGURE 1 no cellular organelles, the Xenopus Left Isolated HeLa cell nuclei before exposure to apoptotic S phase/M phase (S/M) extract. extracts show a requirement for mitoRight: HeLa nucleus following incubation in S/M extract. Highly condensed chromatin domains chondria. Since Bcl-Z-mediated reprieve bleb outwards through the highly vesiculated nuclear membrane. Nuclear pores remain is possible in Xenopus, but not in S/M morphologically recognizable and apparently become concentrated in regions of the nuclear extracts, this suggests that mitochondria envelope between condensed chromatin domains2. Note that lamins A, B and C (not shown in are involved in the apoptotic pathway figure) are fully cleaved in these nuclei, and are no longer apparent in the nuclear envelope by before the execution phase begins. indirect immunofluorescence3’. The cluster of osmophilic particles at the centre of the nucleus l The earliest apoptotic event detected resembles nucleolar remnants seen in many apoptotic cells2. in these extracts is cleavage of PARP by prICE, a proteinase that resembles interleukin-l-B-converting enzyme (ICE) both with respect induces digestion of the nuclear chromatin within 15 minutes. This system is now being used to examto its cleavage site specificity and to its sensitivity to ine the role of protein kinases in apoptosis, and it has highly specific chloromethylketone inhibitors. prICE thus follows the Caenorhabditis elegans Ced3 protein been shown that the broad-spectrum protein kinase as the second ICE-like proteinase shown to function inhibitor staurosporine can activate cytoplasmic in an endogenous cell death pathway1°,13J4. PARP extracts from non-apoptotic cells thereby triggering was the first substrate for an ICE-like proteinase to be chromatin cleavage in added nucleis. identified in apoptosis. To date, genetic analysis in In the second in vitro system, extracts made from C. elegans has yielded no targets of the ted-3 gene Xenopus eggs laid more than 14 days after priming of product. We suspect that PARP is only one of a the frogs with hormone induce apoptotic execution number of cellular proteins cleaved by prICE during in demembranated sperm-nuclei after an initial, and apoptosis. Among these substrates are likely to be somewhat variable, lag phase9. These extracts are downstream regulators that go on to propagate responsive to factors known to regulate the apoptotic the cellular apoptotic response. The role of PARP response in vivo: addition of Bcl-2 can protect against cleavage in apoptosis is discussed below. induction of apoptosis in added nuclei. In addition, l prICE is not ICE. prICE cannot cleave recombinant the Xenopus extracts require the presence of mitopro-IL-l-B and canonical ICE cannot cleave PARPlO. chondria to exert their apoptotic effect on added nuclei. Thus, these extracts may yield powerful At least five human ICE-like proteinases either have been or will be described by the end of 1995 (Refs insights into the mechanisms of apoptotic regulation, including Bcl-2-mediated protection against 1.5-17). Of these, cpp32 (Ref. 17) appears to be the best candidate for prICE. Thus ICE, like the cyclinapoptosis. dependent kinase (cdk) p34cdcz,may turn out to be In the third system, extracts made from chicken the archetype of an extended family. It remains to be DU249 cells that become condemned following an determined which one of the ICE-like proteinases S phase/M phase synchronization regime (S/M extracts, see Fig. 1) rapidly cause added nuclei to cleaves PARP.Whether the various ICE-like proteinundergo apoptotic execution through to the ultimate ases will turn out to be functionally diverse, like the cdks, should make for very interesting reading in stage of disassembly into.membrane-enclosed apoptotic bodie@. The process begins extremely rapidly: forthcoming years. l prICE is essential for the initiation of apoptotic proteolytic cleavage of the nuclear DNA repair events in S/M extracts. If the extracts are incubated enzyme poly(ADP-ribose) polymerase (PARP)is comwith a highly specific ICE inhibitor (YVADplete within three minutes of addition of the nuclei chloromethylketonels) prior to the addition of (at 37”C)lO. This is rapid indeed considering that the nuclei, the nuclei undergo none of the characterized relevant proteinase must enter the nuclei before biochemical events of apoptosis. This provides a cleavage begins. It is thus not particularly surprising striking parallel to the position,of the ted-3 in the that these extracts do not seem to be especially useC. elegans cell-death pathway. In ted-3 mutants, none ful for the study of apoptotic regulation. On the other 218

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of the genetically programmed deaths occur. Instead, cells that are normally deleted remain alive and functionallg, l Apoptosis involves the action of multiple proteinases. The dissolution of the lamina in apoptosis is accompanied by proteolytic degradation of the lamin subunits20J1. The lamin proteinase, which we term Lamp, has an inhibitor profile and kinetics distinct from prICE. Nonetheless, inactivation of prICE inactivates LamP (Y. Lazebnik, A. Takahashi et al., unpublished). This suggests that LamP may either be downstream of prICE in a proteinase cascade, or it may be a distinct ICE-like activity functioning in parallel with prICE. 0 Other factors, in addition to prICE and Lamp, are required for apoptosis in vitro. The effects of phosphotyrosiney and staurosporines suggest that specific kinases and phosphatases may be involved. Other activities include a Zn2+-sensitive factor (not the endonuclease)6,9, and factors sensitive to GTPyS and ionomyciny. The identification of these factors will be of considerable interest. Why degrade

PARP during

apoptosis?

At present, the role of PARPin apoptosis is unclear (see Ref. 22 and references therein), and it is possible that cleavage of PARPby prICE may have nothing to do with the initiation of apoptosis. However, PARP cleavage might be essential for the completion of apoptosis. PARP has two zinc fingers near its N-terminus, defining a domain that binds to DNA breaks with nanomolar affinity 23. Binding to DNA breaks activates the C-terminal catalytic domain, stimulating it to catabolize NAD and polymerize ADP-ribose onto the central automodification domain. PARP has a very high activity and, if massively activated by the chromatin fragmentation that frequently occurs in cells undergoing apoptosis, it could deplete cellular ATP storesz4. This would endanger the apoptotic pathway, since the formation of apoptotic bodies requires ATP25. One purpose of PARP cleavage may be to protect the apoptotic pathway and avoid inflammation. Cleavage of PARP by prICE protects the apoptotic cell from PARP activation in three ways. First, the cleavage separates the DNA-binding and catalytic domains. This renders the catalytic domain insensitive to DNA cleavagez6. Second, the N-terminal DNAbinding fragment can act as a dominant negative inhibitor of intact PARPby competing for free DNA endsz7. Third, the cleavage occurs in the middle of the PARPbipartite nuclear localization signa128.Thus, any of the 85 kDa C-terminal fragment present in the cytoplasm will be unable to enter the nucleus. This fragment retains enzymatic activity and may stimulate apoptosis by modifying target cytoplasmic polypeptides. This rationale for PARP cleavage is readily testable. Mutation of D214 to A should render the enzyme immune to cleavage by prICE. For example, this mutation rendered a decapeptide inactive as a competitive inhibitor of prICE in titrolo. In order to determine the role of PARP cleavage (if any) in apoptosis, TRENDS

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it will be interesting to construct cell lines expressing this mutant form of PARP and ask whether they can successfully undergo apoptosis. Apoptosis:

a ‘cyanide-tooth’

model

Experiments with cell-free extracts also suggest some surprising conclusions concerning the nature of the downstream effecters of prICE. This follows from the observation that inhibition of prICE by YVAD-chloromethylketone blocks not only PARP cleavage, but also chromatin fragmentation, lamin cleavage and morphological changeslO. This can be explained either if the downstream effecters are present (soluble) in latent form in S/M extracts and are activated by addition of nuclei (it is not clear how this would work biochemically), or if the essential downstream effector of prICE are present in the added nuclei and are activated by prICE. The latter is potentially significant, since the nuclei studied in the S/M-extract experiments are not from apoptotic cells. In fact, nuclei from a wide range of cultured cells, as well as from chick embryos, undergo apoptotic changes in the extracts. Thus, the downstream effectors of apoptosis may be ubiquitous in the nuclei of growing cells. This is consistent with the view of Rafp9 that the ‘default state’ of all cells is to die unless they are kept alive by specific signals. It also fits with the observation that some cell types will undergo apoptosis even if protein synthesis is blocked by cycloheximide, and may explain the provocative finding that nuclei in heterokaryons can respond independently to apoptotic stimuli”O. If it is true that nuclei carry within them the seeds of their own destruction, then this could have profound implications for the biochemical analysis of apoptosis. For example, if the lamin proteinase turns out to be an integral component of the lamina, this enzyme may prove to be extremely difficult to study: it may not function in solution. Similar considerations may apply to the nuclease(s) that fragment chromatin. Thus, detailed biochemical reconstruction of the apoptotic mechanisms may proceed hand in hand with advances in our understanding of nuclear structure. References 1 KERR, J. F. R., WYLLIE, A. M. and CURRIE, A. R. (1972) Br. 1. Concer24,239-275 2 WYLLIE, A. H., KERR, 1, F. R. and CURRIE, A. R. (1980) ht. Rev. Cytol. 68,251-305 3 CLARKE, P. C. H. (1990) Amt. hnbryol. 181, 195-213 4 SCHWARTZ, L. M., SMITH, S. W., JONES, M. E. E. and OSBORNE, B. A. (1993) Proc. Nat/ Acad. SC;. USA PO, 980-984 5 GJERTSEN, B. T., CRESSEY, L. I., RUCHAUD, S., HO&E, G., IANOTTE, M. and D0SKELAND, S. 0. (1994) j. Cell Sci. 107, 3363-3377 6 LAZEBNIK, Y. A., COLE, S., COOKE, C. A., NELSON, W. G. and EARNSHAW, W. C. (1993) /. Cell Biol. 123, 7-22 7 SOLARY, E., BERTRAND, R., KOHN, K. W. and POMMIER, Y. (1993) Blood 81, 1359-l 368 8 BERTRAND, R., SOLARY, E., O’CONNER, P., KOHN, K. W. and POMMIER, Y. (1994) Exp. Cell Res. 211, 314-321 9

NEWMEYER, D. D., FARSCHON, Cell 79. 353-364

D. M. and REED, 1. C. (1994)

Acknowledgements I thank Eddie Wood for development of the S/M extracts; Yuri Lazebnik for recognizing, over my objections, that they were apoptotic and, together with Atsushi Takahashi, for many fine experiments and ideas; Bill Nelson, Scott Kaufmann and Guy Poirier for many stimulating conversations in two enjoyable collaborations; Gerard Evan and Wei Wu He for helpful discussions; and Scott Kaufmann, Chuck Yang, llya Goldberg and Miguel Martins for comments on the manuscript. Experiments from our laboratory were funded by a grant from the Human Frontier Science Program.

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