The genetic regulation of apoptosis

The genetic regulation Andrew The University

of apoptosis

H Wyllie

of Edinburgh

Medical

School,

Edinburgh,

UK

Dramatic advances, most of them within the past two years, have provided a picture of the genetic regulation of apoptosis in mammalian cells. Although much detail remains to be filled in, the general structure-concordant with programmed death in invertebrates-includes signalling systems, genetic determination of susceptibility, critical proteins capable of reversing or re-affirming the death sentence, and a common effector pathway driven by specific proteases. Current

Opinion

in Genetics

and Development

1995, 5:97-l

04

Introduction In this review, I seek to summarize some of the new information on the nature of the initial signals for apoptosis, the genetic regulation of cellular susceptibility to such signals, the multiple mechanisms that forestall or affirm enactment of a commitment to death, and the common effector pathway whereby death is finally expressed. Outstanding advances have been made in all these areas in the past year, allowing for the first time a general concept of the organization of apoptosis at the genetic level in mammalian cells (Fig. 1). Literature coverage here is necessarily selective (several recent reviews give a more historic perspective, or a broader picture [l-3]).

1 Susceptibility

ICH/ced

Other

1

3 protease

proteases

and nucleases

~.

0 1995 Current Opinion in Ceneticr ami Dewlqnnh

Initiating

stimuli

Fig. 1. Summary scheme of major events in apoptosis. Although this outline is liable to be proved an oversimplification, it seeks to emphasize a number of points: the existence of a state of susceptibility to apoptosis in which both physiological and injury-associated stimuli may trigger death; a complex interplay of positive and negative modulating factors influencing the final outcome of potentially lethal stimuli; and a common protease-activated effector pathway.

A recent attractive theory proposes that death is the inevitable default condition adopted by all isolated cells (with the exception of blastomeres) and is activated automatically in the absence of incoming survival signals from the environment [4]. It is also true, however, that death can be signalled directly, either as a result of cell injury or through cytokine-mediated pathways. Thus, engagement of the 55 kDa tumour necrosis factor (TNF)a receptor signals death in many cell types [5]. The death-determining domain is the more distal of two functional domains in the cytosolic portion, the terminal 20 amino acids being critical. At least one mechanism whereby ligation of the 55 kDa TNFa receptor is coupled to apoptosis is through activation of sphingomyelinase, releasing ceramide and phosphocholine firorn membrane sphingomyelin [6**,7]. Ceramide is itself a signalling molecule and can induce apoptosis after a lag of some hours, apparently by means of a proline-directed

serine/threonine kinase and (nerhaps) mitogen-activated protein kinase and NFK-B [8]. This pathway is independent of DNA damage, as it can be activated directly in isolated membrane preparations. It can be inhibited by protein kinase C and diacyl glycerol [9]. Ceramide levels also rise in cells doomed to die following exposure to ionizing radiation and hypoxia. Under these conditions, it appears improbable that the TNFa receptor is involved, although direct evidence to exclude this is not yet available. Fas/APO-1 is a second receptor that mediates apoptosis in some cell types [lo]. It is a 45 kDa transmembrane protein with some homology to the ‘death domain’

Abbreviations CTL-cytotoxic

T-lymphocyte; Rb (rb)-retinoblastoma

0 Current

ICE-interleukin-1 protein (gene);

Biology

B-converting TNF-tumour

Ltd ISSN 0959-437X

enzyme; necrosis

IL-interleukin; factor.

98

Oncogenes

.

and cell proliferation

of the TNFa 55kDa receptor in its single cytosolit domain. Despite this molecular similarity, signalling through Fas/APO-1 and the TNFa receptor appear to be by different routes, as cells engineered to bear both types of receptor die by apoptosis on engagement of F&s, but by necrosis on engagement of the TNFa receptor [ll’]. Moreover, pharmacological blockade of phospholipases, poly(ADP-ribose) polymerase and calcium metabolism inhibit TNFa-mediated death, but not apoptosis, and NFK-B is not activated following stimulation of apoptosis through the Fas/APO-1 receptor. The physiological Fas ligand has been identified recently [12”,13]. It is a 40 kDa transmembrane protein, a member of the TNF family, found on the surface of cytotoxic T-lymphocytes (CTLs), where it is inducible by T-cell receptor activation. A series of murine mutations in f&APO- 7 (e.g. Ipr and l@?) or in its ligand (g/d) have provided a powerful means of demonstrating the involvement of this system in various apoptosis-activating pathways [lo], but the intracellular signalling mechanisms remain obscure. The importance of the @S system in normal regulation of the immune system is demonstrated by the phenotype shared by animals homozygous for any of these mutations, which is an auto-immune disorder similar to human systemic lupus erythematosus, in which auto-reactive T cells are inappropriately conserved. Interestingly, although human patients with this disorder do not appear to have &s or&s ligand mutations, they do sometimes have a circulating soluble form of Fas that may block the activity of CTLs on their physiological targets [14*]. In addition to CTLs, hepatocytes and enterocytes express Fas/APO-1 and die in response to the Fas l&and, so it is possible that this system is a physiological regulator of apoptosis in cell lineages outwith the lymphoid system also. Nur77 is another newly identified receptor, signalling death in thymocytes stimulated via their CD3/T-cell receptor complex [15,16]. It is an ‘orphan’ zinc finger containing steroid receptor, the role of which in thymocyte apoptosis was discovered by subtractive hybridization methods, and it is of particular interest, as it demonstrates the selectivity of the pathways initiating death in thymocytes. Although necessary for apoptosis triggered by T-cell receptor occupancy, it is not involved in apoptosis following either dexamethasone treatment or withdrawal of interleukin (IL)-2.

Cellular

susceptibility

to apoptosis

Apoptosis can be induced in vitro by expression of c-myc in growth factor deprived cells [17]. All of the features of c-Myc that are considered important in its role in supporting cell proliferation appear to be equally essential for this induction of apoptosis - the Myc protein must dimerize with its partner Max [18*], and both the trans-

activation and DNA-binding domains are obligatory. It is now clear that c-myc induced apoptosis is part of a wider mechanism in which apoptosis-susceptible cells are generated by activators of cell cycle entry. Thus, deficiency of the retinoblastoma gene rb-7 [19’,20] or expression of E2F-1 [21*] can have the same effect, as can expression of human papilloma virus 16 E7 [22] or adenovirus ElA [23,24] (each of which inactivates d-1). In all these circumstances, the changes are incompetent to increase apoptosis without an additional factor, such as withdrawal of growth factors horn the medium, DNA damage horn ionizing radiation, or exposure to genotoxic drugs. Convincing evidence exists that the apoptosis in response to DNA injury (but seldom that caused by withdrawal of growth factors) is dependent on induction of wild-type cellular p53 protein [22-27.28*,29*]. There was a temptation to regard this apoptosis as the result of non-physiological conditions - a conflict set up artificially by the experimenter, with little relevance to the regulation of apoptosis in authentic tissue situations. However, myc-driven apoptosis can be aborted by specific growth factors, some of which are not (or are only weakly) mitogenic [30’]. An alternative hypothesis suggests dual control, whereby c-myc induces a state in which both DNA synthesis and apoptosis are permitted, the final choice depending on the availability or otherwise of a rescuing factor such as insulin-like growth factor 1, insulin, or epidermal growth factor. The essential difference between these two hypotheses is conceptual, rather than mechanistic. It is quite conceivable, for example, that the composition of the replication complex alters on encountering damaged DNA and that this alteration can act as a signal for apoptosis. A candidate for such a function-switching role in the replication complex might be ~21. This p53-dependent inhibitor of cyclin kinases binds to proliferating cell nuclear antigen in the replication complex, where its presence has an inhibitory effect on replication, but permits repair [31]. Attributing to ~21 all, or even any, of the coupling of p53 to apoptosis is a problem, however, as good evidence from some cell systems suggests that p53 induces apoptosis even in the presence of actinomycin D and cycloheximide in concentrations sufficient to completely inhibit expression of p53-induced molecules [32*]. Moreover, in several situations, ~21 production is independent of p53 and induces differentiation rather than apoptosis [33,34], suggesting that the coupling of p53 to apoptosis does not involve ~21. The situation is far from clear, however, and there are cell systems in which p53 and the resulting ~21 induction seem to be inseparable f?om induction of apoptosis [35]. These conflicts may be resolved when more is understood of the role of additional factors in the cellular background that condition susceptibility to apoptosis. Apoptosis is associated with c-myc activation or rb- 1 deprivation in many cell lineages, but there are also circumstances in which it occurs after the down-regulation of c-myc, in cells that remain in Go/G1 until the

The genetic

point of death. In this category come dexamethasoneinduced apoptosis of lymphoid cells [36], and apoptosis in prostate epithelium on androgen withdrawal [37*]. In neither of these situations does p53 play any role, as the apoptosis occurs with identical kinetics and quantity in p53-null animals. Erythroid progenitors deprived of er-ythropoitin [38] and myeloid cells deprived of IL-3 [39*] also enter apoptosis after down-regulation of c-myc. There may, therefore, be a separate class of susceptibility to apoptosis that requires the target cell to leave the cycle. The current seemingly incompatible data on the involvement or otherwise of cell cycle kinases in apoptosis.[40-42] might be reconciled were the existence of such different classesto be confirmed.

Death-modulating

factors

The preceding section has outlined circumstances in which cells become susceptible to apoptosis. Near-total ignorance reigns at present on the molecular basis of this susceptibility, although the suggestion has been made that susceptible cells accumulate more of the apoptosis effector proteins in a pre-activated state [43]. This was suggested on the basis of a relatively crude assay of endogenous endonuclease activity, and it will be interesting to see whether it is confirmed when more specific assays of these proteins become available. Commitment to death in susceptible cells can be modified by a set of proteins that can re-affirm or forestall the death sentence. A small protein encoded by the Drosophila reaper gene reaffirms death [44”]. The reaper gene is expressed in doomed cells 1-2 hours before the first morphological changes of apoptosis. Deletion mutations inhibit all the programmed deaths of normal development, the excess deaths that result from a variety of mutations in other developmental genes, and even those following ionizing radiation. Hence, the point of action of the wild-type reaper gene must lie after the convergence of these different death-specifying pathways. The gene product affirms death, rather than altering differentiation, as the result of reaper deletion is accumulation of supernumerary, but normally differentiated, cells. Moreover, some of the normal deaths can be restored in reaper-deletion embryos by expression of a reaper transgene. Nonetheless, reaper is not itself an essential component of the apoptosis effector pathway, because even in redper-deficient embryos, apoptosis with normal morphological features can still be induced by sufficiently high doses of ionizing radiation. So far, no mammalian homologue of reaper has been identified. The DAD1 gene has been shown to forestall the death sentence [45]. It was discovered through study of a temperature-sensitive mutant BHK21 cell line and en-

regulation

of apoptosis

Wyllie

codes a 12.5 kDa protein with no substantial homology to other currently known genes. The most fully described set of death-modulating proteins is the Bcl-2 family [46]. Bcl-2 is a 25-26 kDa protein with a membrane anchor at its carboxyl terminus [47], by which it localizes to outer mitochondrial membranes, endoplasmic reticulum, and the nuclear membrane [48-501. Membrane localization does not appear to be essential for the death-forestalling effect of Bcl-2, however, as mutants that lack the anchor still confer some survival function [51]. Two highly conserved domains (BH-1 and BH-2) permit formation of dimers between Bcl-2 and its partner, Bax [52’], and between Bax and other members of the Bcl-2 family (e.g. Bcl-XL and Bclxs). Strikingly, Bcl-2 shows overall sequence homology (and near identity in the dimerization domains) with the Caenorhabditis elegans death-inhibitory protein CED-9 [53**]. Coprecipitation experiments in mammalian cells expressing epitope-tagged Bcl-2 and Bax demonstrate that the death-inhibitory activity resides in Bcl-2-Bax heterodimers, whereas Bax-Bax homodimers facilitate death [54*]. Bcl-XL and Bcl-XS, alternative splice variants from a single transcription unit, appear to have similar complementary functions [55]. The role of Bcl-2-Bcl-2 homodimers is not yet fully determined. No evidence exists for Bcl-2 expression on its own ever causing an increase in cell proliferation. Rather, it acts as a survival factor, forestalling death in the cells in which it is expressed, whether or not they are in cycle [56*]. Deaths initiated by many different stimuli are blocked by Bcl-2 expression, including those dependent upon p53 [57], ionizing radiation, and (in lymphocytes) dexamethasone, ionophore treatment, and cross-linking of the T-cell receptor (reviewed in [2]). Some evidence exists for hypoxic cell death in neurones also being aborted by Bcl-2 expression [58’]. These observations suggest that Bcl-2 acts late in the pathway between the initial stimulus and the first irreversible effector event. It is not a universal passport to survival, however, for it appears to play no part in negative selection of intrathymic lymphocytes. Thus, although Bcl-2 null animals show catastrophic involution of thymus and spleen some weeks after birth [59*], this does not coincide with negative selection, nor is a substantial defect in negative selection seen in animals expressing transgenic Bcl-2 in intrathymic CD3-positive cells [60]. The regulation of Bcl-2, although imperfectly understood, is clearly of great complexity. In addition to the switch f?om death-inhibitory to death-affirming effects, according to the relative intracellular concentrations of Bcl-2, Bax, and the other interacting members of the family, Bcl-2 binds to a series of (as yet) incompletely characterized molecules designated Nipl-3 (to which ElB also binds) [61*], the Raf kinase [62], and perhaps R-Ras [63]. Further, the Bax : Bcl-2 ratio rises (favouring formation of death-affirming Bax-Bax homodimers) in response to p53 expression [64,65’].

99

100

Oncogenes

Effector

and cell proliferation

.

mechanisms

Analysis of the phenotype of C. eleganscell death mutants permits the effects of the death-determining genes to be ordered in a simple linear genetic pathway, in which ad-9 lies ‘upstream’ of ted-4 and ted-3. (This genetic order does not of course preclude the possibility that the product of ted-9 may inhibit the function of the ted3 product, and so act ‘downstream’ in the biochemical sense.) CED-4 has a zinc finger motif and shows homology to transcription factors, whereas CED-3 is a cysteine protease with both structural and functional homology to the mammalian interleukin-1 p-converting enzyme (ICE) family [66”]. Although mammalian ICE, expressed from a transgene, can partially restore the ‘death phenotype to ted-3 null nematode larvae, it shows substantial sequence divergence from CED3 in its amino-terminal region. Better overall structural homology is shared by the product of a second mammalian gene, Ich-1 [67”] (initially called Nedd-2 [68”]). CED3, human and murine ICE, and ICH-1 share a highly conserved pentapeptide proteolytic active site Gln-AlaCys-Arg-Gly (in the three-letter amino acid code), in which the central cysteine is critical. Reiterating a theme found elsewhere in the death pathway, two splice variants of ICH-1 exist, the larger with death-promoting and the smaller with death-inhibiting properties. Two recently described cell-&ee systems promise to shed further light on the effector events in apoptosis. In one, an apoptosis-promoting activity (assayed by its effects on the morphology and chromatin configuration of indicator HeLa cell nuclei) is recovered f?om extracts made from aphidicolin-synchronized mitotic DU249 chicken hepatoma cells [69’]. This activity is an ICE-like cysteine protease and cleaves poly(ADP-ribose) polymerase, in a manner closely similar to that observed in authentic apoptosis, at a Glu-Val-Asp/Gly sequence almost identical to the proteolytic cleavage site in IL-IS. Nonetheless, it is not ICE, as it fails to cleave IL-lp itself. It may prove to be a member of the rapidly expanding family of ICElike proteases such as ICH-1. The second cell-free system [7@] relies on the observation that Xenopur egg extracts, under appropriate Table

1. Viral

death-inhibitory

, Summary

Protein

of DNA

viral

gene

It is probable that the cysteine proteases lie at the apex of a cascade of proteolytic events in the common effector pathway of apoptosis. A variety of proteins in apoptotic cells undergo proteolytic digestion, and the process can be arrested by protease inhibitors of several specificities [72]. Moreover, the proteases of cytotoxic T cell granules (fragmentins) are not cysteine proteases, although in their target cells they appear to switch on effector events yet further downstream [73]. The cell-free assays also show that the nucleolytic events (historically of substantial significance in establishing the internally organized character of apoptosis) lie clearly downstream of the cysteine protease activation. They consist of progressive cleavage of DNA, first to very large fragments (30-50 kb) and only later (and in some cell types not at all) to the familiar nucleosomal ‘ladder’, by enzymes with different cation requirements [74-761.

Dysregulation

of apoptosis

and cancer

As many of the genes involved in the regulation of apoptosis are known to play a role in carcinogenesis, and cancer is, arguably, a condition characterized by the appearance of supernumerary abnormal cells, the issue arises as to whether cancer is the result of deficient regulation of apoptosis. The founder cells of cancers might be those in which, because of such deficiency,

proteins.

virus , Adenovirus Adenovirus , Human papilloma virus 16 Simian virus 40 , African swine fever virus Herpesvirus samiri Epstein-Barr virus Epstein-Barr virus 1Cowpox virus

conditions, cause a rapid apoptosis-like phenomenon in co-incubated hepatocyte nuclei. The active principle is inhibited by calpain, suggesting that it also includes a cysteine protease. It is not immediately present in fresh extracts, but requires a short incubation at (amphibian) body temperature. Addition of recombinant Bcl-2 over this time prevents appearance of the apoptosis-promoting activity and, like Bcl-2, the protease co-localizes with mitochondrial membranes. Reports’from other systems attribute the function of Bcl-2 to reduction in generation of reactive oxygen species [58], or to increased cellular resistance to their action [71]. In the Xenopus extracts, however, known inhibitors of oxidative damage have no effect on the generation of the protease.

ElB ElB

Homology

55 kDa 19kDa E6

T% LMWS-HL ORF 16 BHRFl LMP-1 Crm A products

with

Bcl-2 Bcl-2 Bcl-2 -

functions

inhibitory

Function

Functional

homologue

Protects

Inactivates p53 of Bcl-2; binds Bcl-2 binding Inactivates p53 Inactivates p53 and Rb-1

protein

from death; same cell localization as Bcl-2 [84,85] May induce cellular Bcl-2, but see 1861 Inhibitor of ICE-like proteases; protects from death [87]

to the cell death

pathway.

1611

The genetic regulation of apoptosis Wyllie

DNA damage inflicted by carcinogens had failed to trigger ‘apoptosis [77] (Fig. 2). Conversely, the defect might lie not in the apoptosis pathway, but in the regulation of DNA repair, so that cells that would have survived DNA damage with accurate repair do so with previously disallowed sequence inaccuracies or recombination events. It is difficult to distinguish between these possibilities, but I discuss the relevant evidence below.

Cent&k

injury

0 1995 Curt

Opinion in Genetics and Develqment

Fig. 2. Postulated origin of cancer cells through failure of apoptosis after genotoxic damage. The normal outcome of genotoxic damage is considered to be p53dependent apoptosis, but deficiency in p53 permits survival of cells with mutations.

First, some apoptosis is present in every tumour, hence if defects in apoptosis are intrinsic to tumorigenesis, they must be in its regulation rather than in the effector pathway Second, oncogenic DNA viruses (including those with the smallest genomes) incorporate mechanisms in their transforming strategy that seem to be designed to abort death (Table 1). Third, transformed cells that lack ~53, and cannot, therefore, couple DNA damage to apoptosis by this route, are resistant to many types of chemotherapeutic agent [29*]. This makes it feasable that tumours with the relatively common drugresistant phenotype have been selected from the outset on the basis of resistance to damage-induced apoptosis. Fourth, in three tissues of genetically modified animals (lens [78’], retina [79’], and choroid plexus [go’]), sequestration of Rb leads to a state of high incidence of apoptosis. whereas removal of p53 as well as Rb produces tumours, suggesting that these result from ‘rescue’ of cells previously doomed to apoptosi: by a p53-dependent pathway Finally, in experiments of similar design conducted on human diploid fibroblasts that were then exposed to a metabolic negative growth signal (the drug N-phosphonoacetyl-L-aspartate), cells lacking functional Rb die, or produce clonogenic survivors at the low firequency of around 5 x 10-6, whereas those lacking both Rb and p53 show lo-fold higher survival [Sl”]. Moreover, the survivors that lack p53 have unstable aneuploid genomes with intrachromosomal amplification involving the the multifunctional drug-resistance gene CAD (carbamyl phosphate synthase, aspartate transcarbamylase, dihydro-orotase), features consistent with those observed in many authentic neoplasms, whereas the survivors

lacking Rb alone are near-diploid amplification.

and without

101

gene

Against this admittedly circumstantial evidence, that neoplasms arise hrn cells normally committed to apoptosis, are experiments in which transgenic Bcl-2 was used to rescue such doomed potential tumour founder cells. It can be argued that, asBcl-2 forestalls the death of cells undergoing apoptosis by p53-dependent pathways [57,82], the spontaneous tumour incidence of Bcl-2 transgenic animals should be as great and include as aggressive a spectrum of tumours as that of p53-null animals. In fact this is far from the observed result, the Bcl-2 transgenic animals having both less aggressive tumom-s and a lower tumour incidence [83’]. There is no guarantee, however, that the bcI-2 transgene used would have been expressed in all cells, even of the restricted lineages under study, and, even if it were, that it would have effected rescue Iiom apoptosis in all cases (the levels of Bax being unknown). Moreover, as Bcl-2 rescue may also permit repair of cells with DNA damage, a cell with intact p53 that survives DNA damage because of Bcl-2 expression may be fundamentally different corn one that survives the same damage because of p53 deficiency.

Conclusions In this review, I have summarized recent findings that clarify the regulation of cell death. Some of its elements are highly conserved across the phyla. It is also clear that the death programme can be coupled in many ways to changes in local concentrations of cytokines or to injury. Many of the key molecules involved in cell death appear to have partners (such as Bcl-2 and Bax), or splice variants (such as Bcl-XL or ICH-1) with opposite action, or may themselves stimulate either apoptosis or proliferation (such as as c-Myc) depending on prevailing cytokine conditions. Cell death thus appears (not surprisingly) to be subject to many checks and balances. Although it is attractive to consider that more insight into the regulation of death might lead to new cancer therapies, much of the detail of the cell death pathways has still to be resolved.

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AH ment

Wyllie,

Cancer

of Pathology,

I
Teviot Place. Edinburgh

University

Campaign

Laboratories,

of Edinburgh

EH8 YAG, UK.

I)epart-

Medical School,