TiPS -June
2992 [Vol. 131
(1988) Pesticide Biochem. Physiol. 32,217-223 34 Pauron, D. et al. (1989) Biochemistry 28, 1673-1677 35 Liu, M-Y. and Plapp, F. W., Jr fl991) Pesticide Biochem. Physiol. 41, l-7
241 36 Klis, S. F. L., Vijverberg, H. P. M. and van den Bercken, J. (1991) Pesticide Biochem. Physiol. 39,210-218 37 Deecher, D. C. and Soderhmd. D. M. (1991) Pesticide Biochem. Physiol. 39, 130-137
The dioxin and peroxisome proliferator-activated receptors: nuclear receptors in search of endogenous ligands Lorecz Poellinger, Martin Gijttlicher and Jan-Ake Gustafsson Dioxins and peroxisome proliferators represent two diverse classes of xenobiotic compounds that induce transcription of specific genes encoding cytochrome P-450 drug-metabolizing enzymes. Signal transduction by these chemicals is mediated by two distinct nuclear receptors, one of which has recently been demonstrated to be a member of the steroid hormone receptor superfamily of ligand-activated transcription factors. However, no endogenous ligand has so far been identified for either of these nuclear receptors. Lorenz Poellinger, Martin Gattlicher and Jan-&e Gustafsson review properties of both these xenobiotic receptor systems and discuss how the molecular details in the receptor activation pathways compare with those of nuclear hormone receptors. ’ Nuclear receptors mediate signal transduction by steroid hormones, thyroid hormones and retinoic acid. In addition, xenobiotic compounds such as dioxin and clofibrate (a peroxisome proliferator) bind to or activate specific nuclear receptors. These two xenobiotic receptor systems share many mechanistic similarities with the nuclear hormone receptors. However, no endogenous ligands have so far been detected for the two xenobiotic receptors. The dioxin receptor Dioxin (used here to refer specifically to 2,3,7,%tetrachlorodibenzop-dioxin) and related halogenated aromatic hydrocarbons are a class of environmental pollutants that give rise to a plethora of biochemical and toxic responses, including the induction of transcription of genes encoding drugmetabolizing enzymes such as L. Poellinger is Associate Professor, M. Giittlicher is Research Associate and 1-A. Gustafsson is Professor and Chairman at the Department of Medical Nutrition, Karolinska Institutet, Huddinge University Hospital F-60, Novum, S-141 86 Huddinge, Sweden.
cytochrome P450IAl (reviewed in Refs 1, 2). Signal transduction by dioxin is mediated by the intracellular dioxin receptor protein. The dioxin receptor was deusing tected, ligand-binding assays, as an intracellular protein that could bind dioxin with high affinity and specificity and is found in virtually all rodent tissues examined (reviewed in Ref. 3). Noncovalent and covalent labelling studies4s5 as well as im-
-1022
*
GGCTCTTCTCACG&ACTCCGG
-1097
Cccncc~nGcGTG~~c'~'~
38 Salgado, V. L. (1990) Pesticide Sci. 26, 389-411 RHX?k methyl I-(IV-&a,&-hifluoro-ptolyl)carbamoyl-3-(4chlorophenyl)-4 methyl-2-pyrazolin-4-yl)carboxylate
munochemical experiments6 have revealed a molecular mass of about 100 kDa for the receptor monomer, which is present in very small amounts and has thus far confounded conventional cloning attempts. In the absence of extracellular stimulus or ligand, the dioxin receptor appears to be present in the cytoplasmic compartment of target cells in a latent, inactive (i.e. in which it doesn’t bind DNA) configuration (reviewed in Refs 3, 7). The process of ligand binding, however, induces an apparent translocation of the receptor to the cell nucleus and initiates the activation of the receptor to a DNAbinding form. Activation of the receptor in vitro’ is stimulated by ligands in a manner that reflects their relative binding affinities for the receptor protein in vitro and their relative potencies to induce cytochrome P45OIAl transcription in vivo. Several studies have established that the ligandactivated dioxin receptor exhibits sequence-specific DNA-binding to the motif TCACGC (Refs 3-12). This motif is conserved in dioxininducible, positive transcriptional control elements (Fig. I), which confer dioxin responsiveness to target promoters in vivo’3,14. The above model implicates the dioxin receptor directly in signal transduction. Thus, the dioxin
-1001
ITg. 1. DNA-regulatory sequences recognized by the ligand-activated dioxin receptor. Sequences are from dioxin response elements identitied in the rat cytochrome P45OlAl gene”(top
and middle)
and rat glutathione-Stransferase Ya gene’= (bottom).
Large
letters
and arrows indicate the position and oriintation, respective/~ of a conserved sequence motif. Small letters indicate deviating bases. Numbers indicate distance in base pairs relativeto tie start site of tmnsmiption.
TiPS -June 1992 Wok 231
242
active receptor 200 kDa DNA-binding activity
@and-binding activity no DNA-bindingactivity
i!i~*~ 8 3 r
8 zf =
b z 8 !?! .E :: 5
release of hsp90; homo- or heterodimerization
phosphorylation?
r
0
P
dioxin
TCACGC target DNA activation of the dioxin receptor showing proposed critical steps during the activation process. For ancephral si@city hsp90 is represented as a dimer. The active 200 kDa dioxin receptor form represents either a homodimer of the . _ lcgandbrndbrg 100kDa mceptor or, aftematively, a hetemdimeric complex of the receptor with a distinct, non-ligand-bindiing protein @ethaps Amt? See textj (hatched box). Finally. the receptor appears to require phosphorylatton for DNA-binding activity.
FQ.2 heel for&u&dependent
receptor, like steroid hormone receptors (reviewed in Ref. IS), the NF-KB transcription factor (reviewed in Refs 16, 17) or yinterferon-activated transcription factors18, acts as a messenger to transmit the gene induction signal from the cytoplasm to the nucleus and its target transcription unit. Moreover, the dioxin receptor together with most, if not all, members of the steroid receptor superfamily functionally represent gene regulatory proteins that require ligand-dependent conversion from a covert precursor to an active form. Ligand-dependent activation of the dioxin receptor At present the mechanism of activation of the dioxin receptor to a functional species is poorly understood. A non-dioxin-binding factor, AmPg, which bears similarity to the broad class of helix-loop-helix regulatory factors (reviewed in Ref. 20) appears to be required for nuclear translocation of the ligand-activated form of receptor. In addition, preliminary evidence suggests that the DNAbinding activity of the dioxin receptor is regulated by phosphorylation”. Moreover, it has previously been shown that the inactive form of dioxin receptor forms a stable heteromeric complex with the 90 kDa heat shock protein hsp90, which does not
take part in ligand binding (Refs 22, 23). In this context hsp90 appears to function as an inhibitory protein, preventing the receptor from binding to DNArecognition elementsz4. Thus, dioxin-induced activation of the receptor requires release of hsp90. A model that summarizes our current understanding of the ligand-dependent activation of the dioxin receptor is illustrated in Fig. 2. Interestingly, the specific DNA-binding activity of a number of steroid receptors, most notably that of the glucocorticoid receptor, has also beel. shown to be repressed by association with hsp90 (Ref. 25). In addition, hsp90 appears to modulate the steroidbinding activity in vitro26,27and, possibly as a consequence thereof, hormone responsiveness in vivoz8 of the glucocorticoid receptor. In agreement with these observations, when the dioxin receptor is not complexed with hsp90 it appears to form a less stable complex with dioxin”. Therefore hsp90 may serve as a cellular chaperon molecule, which, in addition to repressing receptor activity, directly determines the signal-responsiveness of a distinct subclass of nuclear receptors (including the dioxin and glucocorticoid receptors) by stabilizing and maintaining a ligand-binding conformation of these proteins. The architecture of the activated
form of the dioxin receptor is still an unresolved issue. The in vivo activated form of receptor detected in nuclear extracts of dioxin-treated cells is a 200 kDa protein’g*24,30. Moreover, DNA crosslinking studies have indicated an association between the 100 kDa dioxin receptor and a protein of very similar molecular mass to form a complex of about 200 kDa (Ref. 31). Taken together, these data suggest that the active form of receptor is either a homodimer of the 100 kDa dioxinbinding subunit or that it is a heteromeric complex of the ligand-binding subunit and an unrelated protein of similar molecular mass. The latter possibility is favoured by genetic data from dioxin-resistant, mutant hepatoma cells, showing that three distinct gene products affect functioning of the dioxin receptors’. In this context it is interesting to note that the putative helix-loophelix motif of the Amt factor facilitating nuclear translocation of the dioxin receptor has been implicated in mediating formation of homomeric- or heteromerit complexes and in regulation of the DNA-binding specificity of a broad class of transcription factors (reviewed in Refs 20,33). It is not yet known, however, if the Amt factor modulates dioxin receptor function by directly associating with the receptor to
TiPS -June
1992 Wol. 131
form a heteromeric complex. Moreover, it cannot yet be conclusively ruled out that the activated 200 kDa complex simply represents a homodimeric form of the receptor. In the case of several steroid hormone receptors, homodimerization has been shown to be critical for DNA-binding activity34.35. In contrast, multiple and distinct cell type-specific proteins appear to modulate the functional activity of the crretinoic acid receptor (another member of the steroid receptor superfamily) by the formation of heteromeric protein complexes36. Physiological ligand for the dioxin receptor? Since the known ligands of the dioxin receptor are mainly environmental contaminants of industrial origin (reviewed in Ref. 2), they are most probably not the natural ligands for this receptor. In view of the great number of mechanistic similarities between the dioxin receptor and steroid hormone receptor superfamily systems, it is conceivable that the physiological ligand for the dioxin receptor is an as yet unidentified hormone. By analogy to retinoic acid, dioxin is able to modulate differentiation processes (reviewed in Refs 1,2). Thus, it is also possible that the endogenous dioxin receptor ligand may represent an unknown morphogen. The dioxin receptor binds neither steroid hormones1 nor retinoic acids (Gillner, M. et al., unpublished). However, it cannot be excluded that natural ligands of the dioxin receptor are xenobiotic. For instance, polycyclic aromatic hydrocarbons from forest fires have always been present in the environment. Alternatively, the natural l&and may occur in the diet. Photolysis of tryptophan and t~tamine yields products that bind to the dioxin receptor with high affiniv’. Certain rutaecarpine alkaloids that can be chemically derived from tryptarnine bind to the dioxin receptor with a relatively high affinity, whereas the parent compound less rutaecarpine appeared activej*. Moreover, certain indole derivatives (such as indolo[3,2-b] carbazole) bind with high affinities to the dioxin recepto$9,40.
;Ji+-+-J-J-+J~; Cl~O-~~C,, 3
2,3,7,8*tetrachlo~ibe~~~ioxin
indolo(3,2-b)carbaxole
clofibric acid
arachidonic acid
f&. 3. Exmpfes of x~~c abbe ~~~~~ @istza&of the dim& weepfor &f~) and the pemxisome pm/iferator-activatedreceptor (right).
Thus, these heterocyclic compounds represent a new class of dioxin receptor ligands distinct from the two classes previously known, halogenated aromatic hydrocarbons and polycyclic aromatic hydrocarbons. Indolo[3,2-b] carbazole (Fig. 3) occurs as a derivative of indoles found in Brussels sprouts and other dietary cruciferous plants (reviewed in Refs 39,40) and may thus qualify as a candidate for a physiolo~c~ receptor ligand. Finally, it was recently observed that other foodborne compounds, such as heterocyclic amines, can activate a dioxin-responsive promoter construct in viva and activate the latent dioxin receptor to a DNAbinding form in an in vitro reconstituted assay (M. Kleman et al., unpublished}. PPAR The peroxisome proliferatoractivated receptor (PPAR) is a recently cloned novel member of the steroid receptor family, which was shown to be activated by a diverse class of xenobiotic compounds (typified by clofibrate; Fig. 3) that all induce peroxisome proliferation41. Thus, this protein is the second example of a nuclear receptor exhibiting specificity toward foreign chemicals and for which no physiological ligand is known. Comparison of the Cterminal, putative liga~d-bind~g domain of the WAR with the corresponding regions from repmembers of the resentative superfamily receptor steroid shows that the PEAR falls into a subfamily of receptors with,
among others, vertebrate receptors for thyroid hormone & vitamin D and retinoic acid ar, and the Drosophila ecdysone receptor (reviewed in Ref. 42). Interestingly, clofibrate and other peroxisome proIiferators induce transcription of a distinct subset (the P45OIV family) of genes of the cytochrome P-450 superfamily of drug-metabolizing enzymes. Cytochrome P4!%IV enzymes catalyse ~-hy~~~tion of fatty acids and, together with peroxisomal fl-oxidation reactions, constitute a fatty acid metabolic pathway independent of the mitochond~~ B-oxidation system. Microsomal and peroxisomal metabolism appear to be of particular importance for longchain fatty acids, which are poor substrates in the mitochondrial oxidative pathway (reviewed in Ref. 43). These physiological aspects gave rise to the hypothesis that fatty acids may contribute to the induction of their own degradation and could represent the physiological counterpart to manmade, exogenous compounds that induce peroxisome proliferation. Studies using a chime& receptor construct encompassing the Ntruns-activating and terminal, central DNA-binding domains of the glucocorticoid receptor linked to the putative ligand-binding, C-terminal domain of the PPAR, revealed that fatty acids such as arachidonic, linoleic or lauric acid could activate the ligand-binding domain of the PPAR (Ref. 44). The mechanism of activation of the PPAR, however, remains
TiPS -June
244 unclear. For instance, it remains to be established whether clofibrate, other peroxisome proliferators or fatty acids such as arachidonic acid directly interact with the putative ligand-binding domain of the receptor or, alternatively, whether they activate the receptor by induction of an as yet unmessenger second identified pathway. Therefore the identification of compounds that ultimately bind to the PPAR will be important. In any case, activation of this receptor by physiologically occurring concentrations of fatty acids suggesk it has a potential role in regulating lipid homeostasis. Future considerations The dioxin receptor and the intriguing represent PPAR examples of proteins mediating signal transduction of xenobiotic compounds, some of which are environmental pollutants. It will be a challenge to determine whether these receptors are activated by physiological signals. A detailed understanding of dioxin receptor function will be greatly facilitated by cloning ik gene. Equally important in future work wiIl be the biochemical chamcterization of the ligand-induced receptor activation process, the posttranslational modification of the receptor by phosphorylation and the possible interplay between the ligand-occupied receptor and distinct non-ligand-binding proteins (such as Amt) that may be important for cellular localization, function or both properties of the receptor. If the dioxin receptor carries a basic helix-loophelix motif (as opposed to the so-called zinc finger motif, which is the common structural denominator among members of the steroid hormone receptor superfamily45), to mediate interaction with target DNA and cofactors, it should be possible to clone the receptor via techniques that have recently resulted in identification of proteins associated with the helix-loop-helix motif of c-myt+46. Another interesting aspect of dioxin receptor function relates to the presence of a recently identified nuclear factor that is distinct from the receptor and constitutiveiy binds to dioxin response elemenk4’&. The presence of the
constitutive factor correlates with a constitutively open chromatin structure at the dioxin response of the cytochrome elements P450IAl gene ‘@, indicative of constitutive occupation of these elements by a factor in viva. It will now be important to establish the function of this factor and to determine whether this factor with the ligandcompetes activated receptor for target DNA sequences or whether it complements receptor function, possibly by the formation of a heteromeric complex. In the case of the PPAR, target transcriptional control elements that may interact with the activated form of the receptor have not yet been identified. It also remains to be established whether peroxisome proliferators or fatty acids activate the receptor by direct interaction with its ligandbinding site or whether intermediate signals are involved. For instance, phosphorylation of the chicken progesterone receptor appears to activate receptordependent target gene transcription even in the absence of progesterone49. c7
0
5 6 7
8 9 10 11
12 13 14 15 16 17
cl
18
Both the dioxin receptor and PPAR have been detected as receptors activated by xenobiotic compounds, which, in turn, stimulate transcription of distinct cytochrome P-450 target genes. By this regulatory pathway the dioxin receptor may modulate metabolism of its own ligands, whereas the PPAR together with the clofibrate-inducible members of the cytochrome P45OIV family and other peroxisomal enzymes may constitute an autoregulatory loop in lipid or fatty acid homeostasis. Acknowledgements Work carried out in the authors’ laboratory is supported by grants from the NIH (ES03954) and the Swedish Cancer Society. M.G. is supported by a fellowship from EMBO and the Swedish Forest and Agricultural Research Council. References 1 Poland, A. and Knutson,1. C. (1982) Annu. Rev. 517-554
Pharmacol.
Toxicoi.
22;
19 20 21 22
23 24 25 26 27 28 29 30 31 32
1992 [Vol. 131
Safe, S. (1986) Annu. Rev. Pharmacol. Toxicol. 26, 371-399 Landers. 1. P. and Bunce. N. (1991) Biochem: i 276,273-287 . ’ Poellinger, L., Lund, J., GiBner, M., Hansson, L-A. and Gustafsson, J-A. (1983) 1. Biol. Chem. 258,13535-13542 Poland, A., Glover, E., Ebertino, F. H. and Kende, A. S. (1986) J. Biol. Chem. 261.6352-6365 Poland, A., Glover, E. and Bradfield, C. A. (1991) Mol. Pharmacol. 39,20-26 Poellinger,~L. et al. (1991) in Biological Basis for Risk Assessment of Diotins and Related Compounds (Banbuy Report 35) pp. 311-320, Cold Spring Harbor Laboratory Press Cuthill, S., Wilhelmsson, A. and Poelhnger, L. (1991) Mol. Cell. Biol. 11, 401-411 Denison, M. S., Fischer, J. M. and Whittock, J. P., Jr (1988) PYOC.Nat1 Acad. Sci. USA 85,25%2532 Fujisawa-Sehara, A., Yamane, M. and Fujii-Kuriyama, Y. (1988) PYOC. NutI Acad. Sci. USA 85, 5859-5863 Hapgood, J., Cuthill, S., Denis, M., Poellinger, L. and Gustafsson, J-A. (1989) PYOC. Natl Acad. Sci. USA 86, 60-64 Neuhold, L. A., Shirayoshi, Y., Ozato, K., Jones, J. E. and Nebert, D. W. (1989) Mol. Cell. Biol. 9, 2378-2386 _~ Puiisawa-Sehara, A.. Sotzawa, K.. Yamane, M. and Fujii-KuGyama, Yi (1987) Nucleic Acids Res. 15,4179-4191 Paulson, K. E., Damell, J. E., Rushmore, T. and Pickett, C. B. (1990) Mol. Cell. Biol. 10,1841-1852 Beato. M. (1989) Cell 56. 335-344 Lenaido, M. J. and BalBmore, D. (1989) Cell 58,227-229 Gilmore, T. D. (1991) Trends Genet. 7, 318-322 Decker, T., Lew, D. J., Mirkovitch, I. and Dame& J. E., Jr (1991) EMBO- i. 10, 927-932 Hoffman, E. C. et al. (1991) Science 252, 954-957 Weintraub, H. et al. (1991) Science 251, 761-766 Pongratz, I., Strcimstedt, P-E., Mason, G. G. F. and Poellinger, L. (1991) 1. Biof. Chem. 266,16813-16817 Denis, M., Cuthill, S., WikstrGm, A-C., Poellinger, L. and Gustafsson, J-A. (1988) Biochem. Biophys. Res. Commun. 155,801-807 Perdew, G. H. (1988) J. Biol. Chem. 263, 13802-13805 Wilhelmsson, A. et al. (1990) EMBO J. 9, 69-76 Denis, M., Poetlinger, L., Wikstrom, A-C. and Gustafsson, J-A. (1988) Nature 333,686688 Bresnick, E. H., Dalman, F. C., Sanchez, E. R. and Pratt, W. B. (1989) J. Biol. Chem. 264,4992-4997 Nemoto, T., Ohara-Nemoto, Y., Denis, M. and Gustafsson, J-A. (1990) Biochemistry 29,1880-1886 Picard, D. et al. (1990) Nature 348, 166-168 Nemoto. T. et al. (1990b) 1. Biol. Chem. 265,2269-2277 ’ ’n Prokipcak, R. D. and Okey, A. B. (1988) Arch. Biochem. Biophys. 267,811-828 Elferink, C. J., Gasiewiw, T. A. and Whitlock, J. P., Jr (1990) J. Biol. Chem. 265,20708-20712 Hankinson, 0. (1983) Somatic Cell Genet. 9,497-514
TiPS -June
1992 [Vol. 131
33 Blackwood. E. and Eisenman. R. N. (1991) Science 251,1211-1217 ’ 34 Tsai, S. Y. et al. (1988) Cell 55, 361369 35 Kumar, V. and Chambon, P. i1988) Cell 55,145-156 36 Glass, C. K., Devary, 0. V. and Rosenfeld, M. G. (1990) Cell 63,729-738 37 Rannug, A. et al. (1987) J. Biol. Chem. 262,15422-E&27 38 Gillner, M., Bergman, J., Cambillau, C. and Gustafsson, J-A. (1989) Carcinogenesis 10,651-f&% 39 GiUner, M., Bergman, J., Cambillau, C.,
245
40
41 42 43
44
Femstrijm, B. and Gustafsson, J-A. (1985) Mol. Pharmacol. 28, 353-363 Bjeldanes. L. F., Kim, J-Y., Grose, K. R., Bartholomew, J. C. and Bradfield, C. A. (1991) Droc. Natf Acad. Sci. USA 88, 9543-9547 Isseman, I. and Green, S. (1990) Nature 347,645-650 Koelle, M. R. et al. (1991) Cell 67,59-77 Lock, A. B., Mitchell. A. M. and Elcokbe, C.. R. (1984) Annrc. Reo. Pharmacol. Toxicol. 29, 145-163 Giittlicher,M., Widmark, E., Li, Q. and
Cell death by apoptosis and its protective role against disease Wilfried Bursch, Franziska Oberhammer and Rolf Schulte-Hermann The involvement of cell death in control of tissue growth has long been neglected, but the description of apoptosis as cellular ‘suicide’, the functional opposite of mitosis, is now attracting more attention to this phenomenon. Physiologically unwanted cells are removed by apoptosis, and toxic chemicals and drugs may enhance or inhibit this type of cell death. These findings are providing new insights into the pathophysiology of a variety of diseases, and suggesting new therapeutic strategies. Traditional thinking in toxicology and pharmacology considers cell death as a passive degenerative phenomenon consequent to toxic injury. This view was revolutionized by the concept of apoptosis introduced in 1972 by Kerr, Wyllie
and Currie’. A nontoxic type of cell death has long been known to occur in the embryo or during metamorphosis, governed by the developmental program’,‘, but its general occurrence in organism5 including adults, and its relevance for health and disease, are only gradually being recognized (see Box for terminology and criteria of apoptosis). Apoptosis is a phylogenetically old phenomenon that he5 been observed in a wide variety of animal species. It occurs in specific developmental stages, contributing to the shaping of organs. Apoptosis is the complement of mitosis, and in concert with it determines maintenance, growth involution of or tissues24. Furthermore, apoptosis is considered a process whereby orW. Bursch, F. Oberhammer and R. SchwlteHermann (Head) are all at the Institut fiir Tumorbiologie-KrebsJonchlcngder Universitiif Wien, Borschkegasse 8a, A1090 Wien, Austria.
ganisms eliminate ‘unwanted (damaged, precancerous or excessive) cells. Apoptosis occurs through a series of morphologically distinct alteration5 including condensation, fragmentation and phagocytosis (Fig. 1). Internal and external membranes appear to be well preserved during apoptosis, so that cellular contents are safely sealed within dying cells until phagocytosis, and no inflammatory responses are seen around apoptotic cells. Apoptosis seems to be a genetically programmed event that requires active gene transcription and translation’. Necrosis, on the other hand, according to Kerr, Wyllie and Currie, is restricted to cell death after massive tissue damage leading to rapid collapse of internal homeostasis of the cell’. It is associated with membrane lysis and inflammation4. However, in practice, discrimination between apoptosis and necrosis is not always easy (see below). The term apoptosis is sometimes used synonymously with ‘programmed cell death’. However, the latter term was originally coined by embryologists to describe cell death occurring at de-
Gustafsson, J-A. Proc. Nat1 Acad. Sri.
USA (in press) 45 Hird, T. et al. (1990) Science 249,157-160 46 Prendergast, G. C., Lawe, D. and Ziff, E. B. (1991) Cell 65, 395407 47 Saatcioglu, F., Perry, D. J., Pasco, D. S. and Fagan, J. B. (1990) Mol. Cell. Biol. 10, 6408-6416 48 Hapgood, J. et al. (1991) Mol. Cell. Biol. 11,4314-4323 49 Denner, L. A., Weigel, N. L., Maxwell,
B. L., Schrader, W. T. and O’MaBey, B. 0. (1990) Science 250,1740-1743
fined stages of the developmental program2,3. Indeed, cell death during development frequently shares morphological features with apoptosis, but sometimes does not?. It is unclear whether cell death during terminal differentiation of tissues should be considered as apoptosis. This review attempts to restrict use of the term apoptosis to unequivocal cases, as originally defined (see also Box). An important characteristic of apoptosis is that it can be prevented by trophic hormones and other growth stimuli (cell ‘rescue’) (Table I). Such examples provide a strong argument for a discrimination between apoptosis and necrosis, because necrosis after severe damage is not expected to be prevented by mitogens. Inhibition of apoptosis may have deleterious consequences for the organism, e.g. tumour promotion or teratogenesis (see below). In addition these findings illustrate that both cell replication and cell death by apoptosis are regulated in concert to produce regression or growth of organs. These relationships are illustrated in Fig. 2. The histologically visible pati apoptosis has a short duration similar to that of cell division, as determined by the following method. Initiation of apoptosis was inhibited by a mitogen, and the disappearance of histological signs of apoptosis was followed. In rat liver the half-life was about 2 hours and the average duration was 3 hours5 (Fig. 1). These estimates allow calculation of the rate of apoptosis from histological counts. In regressing liver and in preneoplastic liver foci, rates were 1% and 34% per hour, estimates Such respectively. should make mathematical modelling of growth kinetics of normal
2992 [Vol. 131
(1988) Pesticide Biochem. Physiol. 32,217-223 34 Pauron, D. et al. (1989) Biochemistry 28, 1673-1677 35 Liu, M-Y. and Plapp, F. W., Jr fl991) Pesticide Biochem. Physiol. 41, l-7
241 36 Klis, S. F. L., Vijverberg, H. P. M. and van den Bercken, J. (1991) Pesticide Biochem. Physiol. 39,210-218 37 Deecher, D. C. and Soderhmd. D. M. (1991) Pesticide Biochem. Physiol. 39, 130-137
The dioxin and peroxisome proliferator-activated receptors: nuclear receptors in search of endogenous ligands Lorecz Poellinger, Martin Gijttlicher and Jan-Ake Gustafsson Dioxins and peroxisome proliferators represent two diverse classes of xenobiotic compounds that induce transcription of specific genes encoding cytochrome P-450 drug-metabolizing enzymes. Signal transduction by these chemicals is mediated by two distinct nuclear receptors, one of which has recently been demonstrated to be a member of the steroid hormone receptor superfamily of ligand-activated transcription factors. However, no endogenous ligand has so far been identified for either of these nuclear receptors. Lorenz Poellinger, Martin Gattlicher and Jan-&e Gustafsson review properties of both these xenobiotic receptor systems and discuss how the molecular details in the receptor activation pathways compare with those of nuclear hormone receptors. ’ Nuclear receptors mediate signal transduction by steroid hormones, thyroid hormones and retinoic acid. In addition, xenobiotic compounds such as dioxin and clofibrate (a peroxisome proliferator) bind to or activate specific nuclear receptors. These two xenobiotic receptor systems share many mechanistic similarities with the nuclear hormone receptors. However, no endogenous ligands have so far been detected for the two xenobiotic receptors. The dioxin receptor Dioxin (used here to refer specifically to 2,3,7,%tetrachlorodibenzop-dioxin) and related halogenated aromatic hydrocarbons are a class of environmental pollutants that give rise to a plethora of biochemical and toxic responses, including the induction of transcription of genes encoding drugmetabolizing enzymes such as L. Poellinger is Associate Professor, M. Giittlicher is Research Associate and 1-A. Gustafsson is Professor and Chairman at the Department of Medical Nutrition, Karolinska Institutet, Huddinge University Hospital F-60, Novum, S-141 86 Huddinge, Sweden.
cytochrome P450IAl (reviewed in Refs 1, 2). Signal transduction by dioxin is mediated by the intracellular dioxin receptor protein. The dioxin receptor was deusing tected, ligand-binding assays, as an intracellular protein that could bind dioxin with high affinity and specificity and is found in virtually all rodent tissues examined (reviewed in Ref. 3). Noncovalent and covalent labelling studies4s5 as well as im-
-1022
*
GGCTCTTCTCACG&ACTCCGG
-1097
Cccncc~nGcGTG~~c'~'~
38 Salgado, V. L. (1990) Pesticide Sci. 26, 389-411 RHX?k methyl I-(IV-&a,&-hifluoro-ptolyl)carbamoyl-3-(4chlorophenyl)-4 methyl-2-pyrazolin-4-yl)carboxylate
munochemical experiments6 have revealed a molecular mass of about 100 kDa for the receptor monomer, which is present in very small amounts and has thus far confounded conventional cloning attempts. In the absence of extracellular stimulus or ligand, the dioxin receptor appears to be present in the cytoplasmic compartment of target cells in a latent, inactive (i.e. in which it doesn’t bind DNA) configuration (reviewed in Refs 3, 7). The process of ligand binding, however, induces an apparent translocation of the receptor to the cell nucleus and initiates the activation of the receptor to a DNAbinding form. Activation of the receptor in vitro’ is stimulated by ligands in a manner that reflects their relative binding affinities for the receptor protein in vitro and their relative potencies to induce cytochrome P45OIAl transcription in vivo. Several studies have established that the ligandactivated dioxin receptor exhibits sequence-specific DNA-binding to the motif TCACGC (Refs 3-12). This motif is conserved in dioxininducible, positive transcriptional control elements (Fig. I), which confer dioxin responsiveness to target promoters in vivo’3,14. The above model implicates the dioxin receptor directly in signal transduction. Thus, the dioxin
-1001
ITg. 1. DNA-regulatory sequences recognized by the ligand-activated dioxin receptor. Sequences are from dioxin response elements identitied in the rat cytochrome P45OlAl gene”(top
and middle)
and rat glutathione-Stransferase Ya gene’= (bottom).
Large
letters
and arrows indicate the position and oriintation, respective/~ of a conserved sequence motif. Small letters indicate deviating bases. Numbers indicate distance in base pairs relativeto tie start site of tmnsmiption.
TiPS -June 1992 Wok 231
242
active receptor 200 kDa DNA-binding activity
@and-binding activity no DNA-bindingactivity
i!i~*~ 8 3 r
8 zf =
b z 8 !?! .E :: 5
release of hsp90; homo- or heterodimerization
phosphorylation?
r
0
P
dioxin
TCACGC target DNA activation of the dioxin receptor showing proposed critical steps during the activation process. For ancephral si@city hsp90 is represented as a dimer. The active 200 kDa dioxin receptor form represents either a homodimer of the . _ lcgandbrndbrg 100kDa mceptor or, aftematively, a hetemdimeric complex of the receptor with a distinct, non-ligand-bindiing protein @ethaps Amt? See textj (hatched box). Finally. the receptor appears to require phosphorylatton for DNA-binding activity.
FQ.2 heel for&u&dependent
receptor, like steroid hormone receptors (reviewed in Ref. IS), the NF-KB transcription factor (reviewed in Refs 16, 17) or yinterferon-activated transcription factors18, acts as a messenger to transmit the gene induction signal from the cytoplasm to the nucleus and its target transcription unit. Moreover, the dioxin receptor together with most, if not all, members of the steroid receptor superfamily functionally represent gene regulatory proteins that require ligand-dependent conversion from a covert precursor to an active form. Ligand-dependent activation of the dioxin receptor At present the mechanism of activation of the dioxin receptor to a functional species is poorly understood. A non-dioxin-binding factor, AmPg, which bears similarity to the broad class of helix-loop-helix regulatory factors (reviewed in Ref. 20) appears to be required for nuclear translocation of the ligand-activated form of receptor. In addition, preliminary evidence suggests that the DNAbinding activity of the dioxin receptor is regulated by phosphorylation”. Moreover, it has previously been shown that the inactive form of dioxin receptor forms a stable heteromeric complex with the 90 kDa heat shock protein hsp90, which does not
take part in ligand binding (Refs 22, 23). In this context hsp90 appears to function as an inhibitory protein, preventing the receptor from binding to DNArecognition elementsz4. Thus, dioxin-induced activation of the receptor requires release of hsp90. A model that summarizes our current understanding of the ligand-dependent activation of the dioxin receptor is illustrated in Fig. 2. Interestingly, the specific DNA-binding activity of a number of steroid receptors, most notably that of the glucocorticoid receptor, has also beel. shown to be repressed by association with hsp90 (Ref. 25). In addition, hsp90 appears to modulate the steroidbinding activity in vitro26,27and, possibly as a consequence thereof, hormone responsiveness in vivoz8 of the glucocorticoid receptor. In agreement with these observations, when the dioxin receptor is not complexed with hsp90 it appears to form a less stable complex with dioxin”. Therefore hsp90 may serve as a cellular chaperon molecule, which, in addition to repressing receptor activity, directly determines the signal-responsiveness of a distinct subclass of nuclear receptors (including the dioxin and glucocorticoid receptors) by stabilizing and maintaining a ligand-binding conformation of these proteins. The architecture of the activated
form of the dioxin receptor is still an unresolved issue. The in vivo activated form of receptor detected in nuclear extracts of dioxin-treated cells is a 200 kDa protein’g*24,30. Moreover, DNA crosslinking studies have indicated an association between the 100 kDa dioxin receptor and a protein of very similar molecular mass to form a complex of about 200 kDa (Ref. 31). Taken together, these data suggest that the active form of receptor is either a homodimer of the 100 kDa dioxinbinding subunit or that it is a heteromeric complex of the ligand-binding subunit and an unrelated protein of similar molecular mass. The latter possibility is favoured by genetic data from dioxin-resistant, mutant hepatoma cells, showing that three distinct gene products affect functioning of the dioxin receptors’. In this context it is interesting to note that the putative helix-loophelix motif of the Amt factor facilitating nuclear translocation of the dioxin receptor has been implicated in mediating formation of homomeric- or heteromerit complexes and in regulation of the DNA-binding specificity of a broad class of transcription factors (reviewed in Refs 20,33). It is not yet known, however, if the Amt factor modulates dioxin receptor function by directly associating with the receptor to
TiPS -June
1992 Wol. 131
form a heteromeric complex. Moreover, it cannot yet be conclusively ruled out that the activated 200 kDa complex simply represents a homodimeric form of the receptor. In the case of several steroid hormone receptors, homodimerization has been shown to be critical for DNA-binding activity34.35. In contrast, multiple and distinct cell type-specific proteins appear to modulate the functional activity of the crretinoic acid receptor (another member of the steroid receptor superfamily) by the formation of heteromeric protein complexes36. Physiological ligand for the dioxin receptor? Since the known ligands of the dioxin receptor are mainly environmental contaminants of industrial origin (reviewed in Ref. 2), they are most probably not the natural ligands for this receptor. In view of the great number of mechanistic similarities between the dioxin receptor and steroid hormone receptor superfamily systems, it is conceivable that the physiological ligand for the dioxin receptor is an as yet unidentified hormone. By analogy to retinoic acid, dioxin is able to modulate differentiation processes (reviewed in Refs 1,2). Thus, it is also possible that the endogenous dioxin receptor ligand may represent an unknown morphogen. The dioxin receptor binds neither steroid hormones1 nor retinoic acids (Gillner, M. et al., unpublished). However, it cannot be excluded that natural ligands of the dioxin receptor are xenobiotic. For instance, polycyclic aromatic hydrocarbons from forest fires have always been present in the environment. Alternatively, the natural l&and may occur in the diet. Photolysis of tryptophan and t~tamine yields products that bind to the dioxin receptor with high affiniv’. Certain rutaecarpine alkaloids that can be chemically derived from tryptarnine bind to the dioxin receptor with a relatively high affinity, whereas the parent compound less rutaecarpine appeared activej*. Moreover, certain indole derivatives (such as indolo[3,2-b] carbazole) bind with high affinities to the dioxin recepto$9,40.
;Ji+-+-J-J-+J~; Cl~O-~~C,, 3
2,3,7,8*tetrachlo~ibe~~~ioxin
indolo(3,2-b)carbaxole
clofibric acid
arachidonic acid
f&. 3. Exmpfes of x~~c abbe ~~~~~ @istza&of the dim& weepfor &f~) and the pemxisome pm/iferator-activatedreceptor (right).
Thus, these heterocyclic compounds represent a new class of dioxin receptor ligands distinct from the two classes previously known, halogenated aromatic hydrocarbons and polycyclic aromatic hydrocarbons. Indolo[3,2-b] carbazole (Fig. 3) occurs as a derivative of indoles found in Brussels sprouts and other dietary cruciferous plants (reviewed in Refs 39,40) and may thus qualify as a candidate for a physiolo~c~ receptor ligand. Finally, it was recently observed that other foodborne compounds, such as heterocyclic amines, can activate a dioxin-responsive promoter construct in viva and activate the latent dioxin receptor to a DNAbinding form in an in vitro reconstituted assay (M. Kleman et al., unpublished}. PPAR The peroxisome proliferatoractivated receptor (PPAR) is a recently cloned novel member of the steroid receptor family, which was shown to be activated by a diverse class of xenobiotic compounds (typified by clofibrate; Fig. 3) that all induce peroxisome proliferation41. Thus, this protein is the second example of a nuclear receptor exhibiting specificity toward foreign chemicals and for which no physiological ligand is known. Comparison of the Cterminal, putative liga~d-bind~g domain of the WAR with the corresponding regions from repmembers of the resentative superfamily receptor steroid shows that the PEAR falls into a subfamily of receptors with,
among others, vertebrate receptors for thyroid hormone & vitamin D and retinoic acid ar, and the Drosophila ecdysone receptor (reviewed in Ref. 42). Interestingly, clofibrate and other peroxisome proIiferators induce transcription of a distinct subset (the P45OIV family) of genes of the cytochrome P-450 superfamily of drug-metabolizing enzymes. Cytochrome P4!%IV enzymes catalyse ~-hy~~~tion of fatty acids and, together with peroxisomal fl-oxidation reactions, constitute a fatty acid metabolic pathway independent of the mitochond~~ B-oxidation system. Microsomal and peroxisomal metabolism appear to be of particular importance for longchain fatty acids, which are poor substrates in the mitochondrial oxidative pathway (reviewed in Ref. 43). These physiological aspects gave rise to the hypothesis that fatty acids may contribute to the induction of their own degradation and could represent the physiological counterpart to manmade, exogenous compounds that induce peroxisome proliferation. Studies using a chime& receptor construct encompassing the Ntruns-activating and terminal, central DNA-binding domains of the glucocorticoid receptor linked to the putative ligand-binding, C-terminal domain of the PPAR, revealed that fatty acids such as arachidonic, linoleic or lauric acid could activate the ligand-binding domain of the PPAR (Ref. 44). The mechanism of activation of the PPAR, however, remains
TiPS -June
244 unclear. For instance, it remains to be established whether clofibrate, other peroxisome proliferators or fatty acids such as arachidonic acid directly interact with the putative ligand-binding domain of the receptor or, alternatively, whether they activate the receptor by induction of an as yet unmessenger second identified pathway. Therefore the identification of compounds that ultimately bind to the PPAR will be important. In any case, activation of this receptor by physiologically occurring concentrations of fatty acids suggesk it has a potential role in regulating lipid homeostasis. Future considerations The dioxin receptor and the intriguing represent PPAR examples of proteins mediating signal transduction of xenobiotic compounds, some of which are environmental pollutants. It will be a challenge to determine whether these receptors are activated by physiological signals. A detailed understanding of dioxin receptor function will be greatly facilitated by cloning ik gene. Equally important in future work wiIl be the biochemical chamcterization of the ligand-induced receptor activation process, the posttranslational modification of the receptor by phosphorylation and the possible interplay between the ligand-occupied receptor and distinct non-ligand-binding proteins (such as Amt) that may be important for cellular localization, function or both properties of the receptor. If the dioxin receptor carries a basic helix-loophelix motif (as opposed to the so-called zinc finger motif, which is the common structural denominator among members of the steroid hormone receptor superfamily45), to mediate interaction with target DNA and cofactors, it should be possible to clone the receptor via techniques that have recently resulted in identification of proteins associated with the helix-loop-helix motif of c-myt+46. Another interesting aspect of dioxin receptor function relates to the presence of a recently identified nuclear factor that is distinct from the receptor and constitutiveiy binds to dioxin response elemenk4’&. The presence of the
constitutive factor correlates with a constitutively open chromatin structure at the dioxin response of the cytochrome elements P450IAl gene ‘@, indicative of constitutive occupation of these elements by a factor in viva. It will now be important to establish the function of this factor and to determine whether this factor with the ligandcompetes activated receptor for target DNA sequences or whether it complements receptor function, possibly by the formation of a heteromeric complex. In the case of the PPAR, target transcriptional control elements that may interact with the activated form of the receptor have not yet been identified. It also remains to be established whether peroxisome proliferators or fatty acids activate the receptor by direct interaction with its ligandbinding site or whether intermediate signals are involved. For instance, phosphorylation of the chicken progesterone receptor appears to activate receptordependent target gene transcription even in the absence of progesterone49. c7
0
5 6 7
8 9 10 11
12 13 14 15 16 17
cl
18
Both the dioxin receptor and PPAR have been detected as receptors activated by xenobiotic compounds, which, in turn, stimulate transcription of distinct cytochrome P-450 target genes. By this regulatory pathway the dioxin receptor may modulate metabolism of its own ligands, whereas the PPAR together with the clofibrate-inducible members of the cytochrome P45OIV family and other peroxisomal enzymes may constitute an autoregulatory loop in lipid or fatty acid homeostasis. Acknowledgements Work carried out in the authors’ laboratory is supported by grants from the NIH (ES03954) and the Swedish Cancer Society. M.G. is supported by a fellowship from EMBO and the Swedish Forest and Agricultural Research Council. References 1 Poland, A. and Knutson,1. C. (1982) Annu. Rev. 517-554
Pharmacol.
Toxicoi.
22;
19 20 21 22
23 24 25 26 27 28 29 30 31 32
1992 [Vol. 131
Safe, S. (1986) Annu. Rev. Pharmacol. Toxicol. 26, 371-399 Landers. 1. P. and Bunce. N. (1991) Biochem: i 276,273-287 . ’ Poellinger, L., Lund, J., GiBner, M., Hansson, L-A. and Gustafsson, J-A. (1983) 1. Biol. Chem. 258,13535-13542 Poland, A., Glover, E., Ebertino, F. H. and Kende, A. S. (1986) J. Biol. Chem. 261.6352-6365 Poland, A., Glover, E. and Bradfield, C. A. (1991) Mol. Pharmacol. 39,20-26 Poellinger,~L. et al. (1991) in Biological Basis for Risk Assessment of Diotins and Related Compounds (Banbuy Report 35) pp. 311-320, Cold Spring Harbor Laboratory Press Cuthill, S., Wilhelmsson, A. and Poelhnger, L. (1991) Mol. Cell. Biol. 11, 401-411 Denison, M. S., Fischer, J. M. and Whittock, J. P., Jr (1988) PYOC.Nat1 Acad. Sci. USA 85,25%2532 Fujisawa-Sehara, A., Yamane, M. and Fujii-Kuriyama, Y. (1988) PYOC. NutI Acad. Sci. USA 85, 5859-5863 Hapgood, J., Cuthill, S., Denis, M., Poellinger, L. and Gustafsson, J-A. (1989) PYOC. Natl Acad. Sci. USA 86, 60-64 Neuhold, L. A., Shirayoshi, Y., Ozato, K., Jones, J. E. and Nebert, D. W. (1989) Mol. Cell. Biol. 9, 2378-2386 _~ Puiisawa-Sehara, A.. Sotzawa, K.. Yamane, M. and Fujii-KuGyama, Yi (1987) Nucleic Acids Res. 15,4179-4191 Paulson, K. E., Damell, J. E., Rushmore, T. and Pickett, C. B. (1990) Mol. Cell. Biol. 10,1841-1852 Beato. M. (1989) Cell 56. 335-344 Lenaido, M. J. and BalBmore, D. (1989) Cell 58,227-229 Gilmore, T. D. (1991) Trends Genet. 7, 318-322 Decker, T., Lew, D. J., Mirkovitch, I. and Dame& J. E., Jr (1991) EMBO- i. 10, 927-932 Hoffman, E. C. et al. (1991) Science 252, 954-957 Weintraub, H. et al. (1991) Science 251, 761-766 Pongratz, I., Strcimstedt, P-E., Mason, G. G. F. and Poellinger, L. (1991) 1. Biof. Chem. 266,16813-16817 Denis, M., Cuthill, S., WikstrGm, A-C., Poellinger, L. and Gustafsson, J-A. (1988) Biochem. Biophys. Res. Commun. 155,801-807 Perdew, G. H. (1988) J. Biol. Chem. 263, 13802-13805 Wilhelmsson, A. et al. (1990) EMBO J. 9, 69-76 Denis, M., Poetlinger, L., Wikstrom, A-C. and Gustafsson, J-A. (1988) Nature 333,686688 Bresnick, E. H., Dalman, F. C., Sanchez, E. R. and Pratt, W. B. (1989) J. Biol. Chem. 264,4992-4997 Nemoto, T., Ohara-Nemoto, Y., Denis, M. and Gustafsson, J-A. (1990) Biochemistry 29,1880-1886 Picard, D. et al. (1990) Nature 348, 166-168 Nemoto. T. et al. (1990b) 1. Biol. Chem. 265,2269-2277 ’ ’n Prokipcak, R. D. and Okey, A. B. (1988) Arch. Biochem. Biophys. 267,811-828 Elferink, C. J., Gasiewiw, T. A. and Whitlock, J. P., Jr (1990) J. Biol. Chem. 265,20708-20712 Hankinson, 0. (1983) Somatic Cell Genet. 9,497-514
TiPS -June
1992 [Vol. 131
33 Blackwood. E. and Eisenman. R. N. (1991) Science 251,1211-1217 ’ 34 Tsai, S. Y. et al. (1988) Cell 55, 361369 35 Kumar, V. and Chambon, P. i1988) Cell 55,145-156 36 Glass, C. K., Devary, 0. V. and Rosenfeld, M. G. (1990) Cell 63,729-738 37 Rannug, A. et al. (1987) J. Biol. Chem. 262,15422-E&27 38 Gillner, M., Bergman, J., Cambillau, C. and Gustafsson, J-A. (1989) Carcinogenesis 10,651-f&% 39 GiUner, M., Bergman, J., Cambillau, C.,
245
40
41 42 43
44
Femstrijm, B. and Gustafsson, J-A. (1985) Mol. Pharmacol. 28, 353-363 Bjeldanes. L. F., Kim, J-Y., Grose, K. R., Bartholomew, J. C. and Bradfield, C. A. (1991) Droc. Natf Acad. Sci. USA 88, 9543-9547 Isseman, I. and Green, S. (1990) Nature 347,645-650 Koelle, M. R. et al. (1991) Cell 67,59-77 Lock, A. B., Mitchell. A. M. and Elcokbe, C.. R. (1984) Annrc. Reo. Pharmacol. Toxicol. 29, 145-163 Giittlicher,M., Widmark, E., Li, Q. and
Cell death by apoptosis and its protective role against disease Wilfried Bursch, Franziska Oberhammer and Rolf Schulte-Hermann The involvement of cell death in control of tissue growth has long been neglected, but the description of apoptosis as cellular ‘suicide’, the functional opposite of mitosis, is now attracting more attention to this phenomenon. Physiologically unwanted cells are removed by apoptosis, and toxic chemicals and drugs may enhance or inhibit this type of cell death. These findings are providing new insights into the pathophysiology of a variety of diseases, and suggesting new therapeutic strategies. Traditional thinking in toxicology and pharmacology considers cell death as a passive degenerative phenomenon consequent to toxic injury. This view was revolutionized by the concept of apoptosis introduced in 1972 by Kerr, Wyllie
and Currie’. A nontoxic type of cell death has long been known to occur in the embryo or during metamorphosis, governed by the developmental program’,‘, but its general occurrence in organism5 including adults, and its relevance for health and disease, are only gradually being recognized (see Box for terminology and criteria of apoptosis). Apoptosis is a phylogenetically old phenomenon that he5 been observed in a wide variety of animal species. It occurs in specific developmental stages, contributing to the shaping of organs. Apoptosis is the complement of mitosis, and in concert with it determines maintenance, growth involution of or tissues24. Furthermore, apoptosis is considered a process whereby orW. Bursch, F. Oberhammer and R. SchwlteHermann (Head) are all at the Institut fiir Tumorbiologie-KrebsJonchlcngder Universitiif Wien, Borschkegasse 8a, A1090 Wien, Austria.
ganisms eliminate ‘unwanted (damaged, precancerous or excessive) cells. Apoptosis occurs through a series of morphologically distinct alteration5 including condensation, fragmentation and phagocytosis (Fig. 1). Internal and external membranes appear to be well preserved during apoptosis, so that cellular contents are safely sealed within dying cells until phagocytosis, and no inflammatory responses are seen around apoptotic cells. Apoptosis seems to be a genetically programmed event that requires active gene transcription and translation’. Necrosis, on the other hand, according to Kerr, Wyllie and Currie, is restricted to cell death after massive tissue damage leading to rapid collapse of internal homeostasis of the cell’. It is associated with membrane lysis and inflammation4. However, in practice, discrimination between apoptosis and necrosis is not always easy (see below). The term apoptosis is sometimes used synonymously with ‘programmed cell death’. However, the latter term was originally coined by embryologists to describe cell death occurring at de-
Gustafsson, J-A. Proc. Nat1 Acad. Sri.
USA (in press) 45 Hird, T. et al. (1990) Science 249,157-160 46 Prendergast, G. C., Lawe, D. and Ziff, E. B. (1991) Cell 65, 395407 47 Saatcioglu, F., Perry, D. J., Pasco, D. S. and Fagan, J. B. (1990) Mol. Cell. Biol. 10, 6408-6416 48 Hapgood, J. et al. (1991) Mol. Cell. Biol. 11,4314-4323 49 Denner, L. A., Weigel, N. L., Maxwell,
B. L., Schrader, W. T. and O’MaBey, B. 0. (1990) Science 250,1740-1743
fined stages of the developmental program2,3. Indeed, cell death during development frequently shares morphological features with apoptosis, but sometimes does not?. It is unclear whether cell death during terminal differentiation of tissues should be considered as apoptosis. This review attempts to restrict use of the term apoptosis to unequivocal cases, as originally defined (see also Box). An important characteristic of apoptosis is that it can be prevented by trophic hormones and other growth stimuli (cell ‘rescue’) (Table I). Such examples provide a strong argument for a discrimination between apoptosis and necrosis, because necrosis after severe damage is not expected to be prevented by mitogens. Inhibition of apoptosis may have deleterious consequences for the organism, e.g. tumour promotion or teratogenesis (see below). In addition these findings illustrate that both cell replication and cell death by apoptosis are regulated in concert to produce regression or growth of organs. These relationships are illustrated in Fig. 2. The histologically visible pati apoptosis has a short duration similar to that of cell division, as determined by the following method. Initiation of apoptosis was inhibited by a mitogen, and the disappearance of histological signs of apoptosis was followed. In rat liver the half-life was about 2 hours and the average duration was 3 hours5 (Fig. 1). These estimates allow calculation of the rate of apoptosis from histological counts. In regressing liver and in preneoplastic liver foci, rates were 1% and 34% per hour, estimates Such respectively. should make mathematical modelling of growth kinetics of normal
