death-inducing functions of ligands of the tumor necrosis factor family: a sanhedrin verdict

279

Death-inducing functions of ligands of the tumor necrosis factor family: a Sanhedrin verdict David Wallach, Andrew V Kovalenko, Eugene E Varfolomeev and Mark P Boldin

Members of the tumor necrosis factor ligand family can kill cells in a rather straightforward manner. They induce their receptors to recruit and activate caspases, enzymes that are critically involved in the death process, and this activation is further amplified by intracellular mitochondria-associated mechanisms. The potentially hazardous expression of the ligands occurs widely in the body; it is antigen-restricted only in the lymphocytes. Yet, in addition to control modes affecting ligand expression, there are numerous inhibitory mechanisms that act within target cells, to make doubly sure of avoiding an undue 'death verdict', while allowing the cells to exhibit other, noncytocidal effects of the ligands.

Address Department of Biological Chemistry, The Weizmann Institute, Rehovot, 76100, Israel

Current Opinion in Immunology 1998, 10:279-288

http://biomednet.com/elecref/Og52791501000279

© Current Biology Ltd ISSN 0952-7915

Abbreviations BIR Baculovirus inhibitor of death DD death domain ER endoplasmic reticulurn LT lymphotoxin NGF nerve growth factor TNF tumor necrosis factor

I n t r o d u c t i o n The Sanhedrin (the Jewish supreme court in Israel in the Roman period) is said to have introduced multiple safeguards into its judgement procedure in order to ensure that there would be no mistakes in verdicts involving capital punishment. Even after the death sentence was pronounced, or even while the accused was being led to execution, if he or anyone else was able to recall a new fact that might cast doubt on the verdict, the trial was resumed. Such re-evaluation could be repeated up to five times. The receptors of the tumor necrosis factor/nerve growth factor (TNF/NGF) family seem to act just as carefully when imposing the death penalty. As discussed below and illustrated in Figure 1, studies of their signaling activity have revealed mechanisms that restrict death induction by these receptors at almost every step along their signaling pathways.

Studies of the ligands of the T N F family and their cell-killing activities have been accompanied, from the outset, by attempts to determine the nature of specificity

in these toxic activities. Because these cytotoxic ligands were first detected in the culture media of activated lymphocytes [1,2] the attempts were initially focused on the role of the ligands in the functioning of cytotoxic T lymphocytes and on ways in which the cytotoxic activity could be restricted in an antigen-specific manner (e.g. [3,4]). Since then, along with the vast increase in our acquaintance with the members of this ligand family and of the receptor family through which they act (the T N F / N G F receptor family, see [5,6] for reviews), there have also been great changes in our concepts of the physiological relevance of the cytotoxic function of these molecules. Consistent with the early hypotheses, the cell-killing activity of at least one member of this ligand family, the Fas/Apol ligand is now known to contribute to the antigen-restricted activity of cytotoxic T lymphocytes (reviewed in [7]). It is also known, however, that expression of this ligand and of the other cytotoxic members of the T N F family is not restricted to lymphocytes, nor even just to cells of the immune system. Besides, these cytotoxic ligands have also turned out to be capable of inducing various noncytocidal effects. Most notable in this respect are TNF-c~ and lymphotoxin-c~ (LT-~), which in addition to acting as killer molecules also regulate a wide range of activities involved in inflammation. As a result of these new findings, much of the initial interest in how the formation of these molecules is regulated has been diverted to a search for the nature of specificity in their action. It now seems obvious that cells should possess mechanisms that restrict their responses to the cytotoxic ligands of the T N F family; if they did not, these widely occurring ligands would act as juggernauts throughout the body.

L i g a n d - r e c e p t o r i n t e r a c t i o n s As with various other cell-surface proteins, triggering of signaling by the receptors of the TNF/NGF family occurs as a consequence of juxtaposition of the receptor molecules, resulting from their binding to their ligands. The effectiveness of triggering is proportional to the local concentrations of receptor and ligand at the cell surface. The TNF-related ligands occur as homotrimers, and it is this trimeric structure which apparently accounts for their ability to impose juxtaposition of the receptors to which they bind. Another unique structural feature of the ligands is their occurrence (except for LTot) in a cell-bound form. Some can be proteolytically shed, but the shed forms are less effective than the cell-bound forms [8]. The soluble form of the Fas/Apol ligand actually blocks signal induction by the membrane-bound form [9°°].

280 Lymphocyte activation and effector functions

Figure 1

Decoy receptors

(b)

Effector cell surface O Cytochrome c

(a) Soluble h and ~b • Bcl-2 cleavage products "~c g Vita' ligand

,1,

'"q•Soluble receptor, I Vital ligand receptor BB

Target cell surface ? Caspase ~ i proenzyme I~

w- IB P / ; u \, , i . Q

/ ~ ~" O ~ k ( i ) ~ ~ ~ Mltochond ....

Nucleus

~ l ~ n t a c t DNA '~, Fragmented DNA " ~ = #

Current Opinion in }mmunology

Diagrammatic representation of some of the numerous levels at which death induction by ligands of the TNF family (expressed by effector cells) is inhibited by mechanisms acting within the target cell. Death-inhibitory processes are shown in dark shading; death-inducing processes are shown in light shading. (a) Shedding of ligands (e.g. mediated by TACE) and receptors (by unknown enzymes); (b) decoy receptors (like TRAIL-R3); (c) receptor-associated inhibitors of signaling (like FAP1); (d) adapter-protein-associated inhibitors of signaling (like A20); (e) adapter-protein-associated specific inhibitors of death (like clAP); (f) signaling cascades leading to synthesis of protective proteins (like the NIK-IKK-NF-KB cascade); (g) inhibitors of caspase recruitment to the signaling complex (like FLIP/Casper/CASH/MRIT); (h) effects of Bcl-2 family members on mitochondrial function, particularly cytochrome c release; (i) Inhibitory effects of Bcl-2 family members on post-cytochrome c release mechanisms, particularly inhibition of cytochrome-c-induced caspase processing by Apaf-1 ; (j) downregulation of the function of pro-apoptotic members by receptor-induced phosphorylation of these proteins (e.g. through the phosphatidyl inositol [PI] 3-kinase-Akt cascade); (k) consequently to phosphorylation, BAD binds to 14-3-3, thus becoming incapable of binding to Bcl-2 and neutralizing its protective effect; (I) control of the function of death-mediating molecules (like p28Bap31) in the ER and nuclear membranes by Bcl-2 family members; (m) inhibition of the function of death-mediating proteins like CAD by caspase-sensitive inhibitors (like I-CAD). Vital ligands include insulin-like growth factor. P, phosphorylation. S/T kinase, serine/threonine kinase.

Signaling can also be blocked by formation of soluble forms of the receptors to which the ligands bind. These soluble forms are created either (as in the case of the ligands) by proteolytic cleavage or by the translation

of specific transcripts formed by alternative splicing mechanisms. An enzyme responsible for the shedding of TNF-(x, a cell-associated protease of the Adamylisin family ( 'TACE'), was recently identified [10°',11"']. Indirect

Death-inducing functions of ligands of the tumor necrosis factor family Wallach et aL 281

evidence suggests that a similar enzyme may participate in the shedding of the T N F receptors [12]. Studies of the mechanisms of action of TRAIL/Apo2 ligand revealed another way in which the signaling activity of members of the T N F ligand family can be blocked at the level of ligand-receptor interaction. Apart from two types of receptors through which TRAIL/Apo2 ligand triggers cytotoxicity (TRAIL-R1 and TRAIL-R2), there exists a 'decoy' receptor (TRAIL-R3), that lacks an intracellular or transmembrane domain and is linked to the cell membrane by a phosphoinositide bond [13°-16°]. Some cells also express a fourth type of receptor (TRAIL-R4) w h i c h - - e v e n though it has an intracellular domain and can signal for certain noncytocidal e f fec t s - - is incapable of inducing death [17°,18"]. Expression of either of the latter two receptors results in greatly decreased responsiveness to the cytocidal effect of TRAIL/Apo2 ligand, probably because of competition for ligand binding to the other two receptors (reviewed in [19]). The resistance conferred by TRAIL-R4 may also be mediated in part by the differential activation of the transcription factor NF-KB by this receptor (see below).

Inh ib i tory m o l e c u l e s assoc ia ted wi th the receptors and the i r speci f ic dock ing prote ins The members of the T N F / N G F receptor family that have the greatest ability to induce cell death do so via a shared intracellular domain motif, the death domain (DD). This domain serves to recruit adapter proteins which, either directly or through binding to other adapter proteins, bind to enzymes whose activity initiates the death process. Two kinds of such adapter proteins have been identified. The first kind are adapter proteins which themselves contain DDs and bind them to the receptors and to each other. These proteins serve to recruit precursors (proenzymes) of the family of caspases, cysteine proteases whose function is central to all known processes of programmed cell death (caspase 8, caspase 10 and caspase 2; reviewed in [20,21]). Daxx is an example of the second kind of adapter protein, which is associated with Fas/Apol and lacks a DD. This protein activates a Jun amino-terminal kinase, which leads to death in a manner that is still unknown [22].

Whereas Fas/Apol seems to involve only one DD- containing adapter protein in signaling for death, the main death-inducing T N F receptor (p55R, also known as CD120a) is believed to recruit at least four such adapter molecules, TRADD, MORT1/FADD, RIP and RAIDD/CRADD, that bind sequentially to each other.

In addition to their suspected role in death induction, T R A D D and RIP are also responsible for recruitment of TRAF2, a docking protein devoid of DDs, that plays a role in restricting the cytocidal activity of T N F and also in inducing some of its noncytocidal activities. It does this through interaction with at least three different proteins or groups of proteins that bind to it, each acting in a different manner. The first is A20, a protein

containing a zinc finger, which inhibits several different effects of TNF, including its cytotoxic function, by an unknown mechanism [23,24°,25°]. The second is clAP1 and clAP2, two structurally homologous proteins that belong to a family of death inhibitors sharing a motif found initially in a Baculovirus inhibitor of death (the BIR motif, [26,27°,28"]; these two proteins have a direct inhibitory effect on the function of caspases [29°°], mediated by specific BIR motifs in the IAP proteins [30°]. The third protein that restricts T N F activity is NIK, a protein kinase responsible for activation of the transcription factor NF-KB. This kinase activates two other kinases, IKKc~ and IKKI3, which in turn phosphorylate the NF-KB inhibitory protein IKB and thereby target IKB for degradation (reviewed in [31]). Gene activation by NF-~:B endows cells with resistance to T N F cytotoxicity [32°-35"]. The identity and mechanisms of action of the NF-~:B-regulated proteins that convey this resistance are still unknown.

The ability of TRADD and RIP, adapter proteins that participate in death induction, to also signal for cellular resistance to death seems to account for the fact that exposure of cells to TNF, under conditions allowing protein synthesis, can make them resistant to T N F cytotoxicity (reviewed in [20]). This 'Janus-like' coexistence of cytotoxic and protective potential is not restricted to the T N F receptors, as other receptors of the T N F / N G F family display similar duality of function. CD40, for example, promotes both the survival and the death of B lymphocytes ([36], and references therein) and was likewise found to exert both cytotoxic and protective effects on cells of tumor lines [37,38]. As with T N E it seems to involve the A20 protein, that contains a zinc finger and whose synthesis is induced by CD40, in its anti-apoptotic effects [37]. GITR, a recently identified T N F / N G F family member, is so far known only to be able to endow cells with resistance to death (induced by the T cell receptor; [39]).

Recent findings in connection with the function of MORT1/FADD, an adapter protein that participates in death induction both by Fas/Apol and by TNF, indicate that this protein too is not solely death-associated. Ablation of its function in lymphocytes, either by targeted disruption or by the use of a MORT1/FADD dominant- negative mutant, results not only in complete lack of responsiveness to the cytocidal effect of Fas/Apol and T N F but also in a markedly reduced rate of growth of the lymphocytes [40",41"]. The mechanisms that determine whether MORT1/FADD will signal for death or for growth are unknown, as are the mechanisms determining whether TRADD and RIP will signal for death or for resistance to death induction.

In the case of Fas/Apol, not only proteins that bind to the DD have the ability to restrict death induction. A tyrosine phosphatase called FAP1, which binds to Fas/Apol downstream of its DD, has also been found to


suppress death induction by the receptor. The mechanism of suppression is not yet known [42].

serves a more subtle role than mere inhibition of death induction [52°-57°].

Inhibitors of the initiation of the apoptotic process As mentioned above, the receptors of the T N F family initiate the death process by recruiting members of the caspase cysteine protease family through a series of DD-containing adapter proteins. This recruitment promotes the processing and hence the activation of the caspases, which then process and activate other caspases in the cell. Cell culture studies point to the involvement of three caspases in initiation of the death process. Two of them, caspase 8 and caspase 10, bind to MORT1/FADD through association of a conserved motif, the death effector domain (DED), found at the amino-terminus of MORTI /FADD and duplicated at the amino-termini of the caspases [43-46]. The third, caspase 2, binds to RAIDD/CRADD through hetero-association of another death-related sequence motif, the CARD domain, found in the amino-termini of both proteins [47,48].

The in vivo relevance of these findings has so far been confirmed only partly. As mentioned above, lymphocytcs derived from mice expressing a M O R T I / F A D D dominant negative mutant do not respond to the cytocidal effect of Fas/Apol [40°]. Similarly fibroblasts derived from mice with targeted disruption of the M O R T I / F A D D gene are resistant to the cytocidal effect of Fas/Apol, as well as to that of T N F and of another death-inducing receptor of the T N F / N G F family, DR-3; they can, however, be killed by triggering TRAIL-R4 ([49]; confirming prior in vitro studies suggesting that MORT1/FADD is not involved in death induction by TRAIL/Apo-2-1igand). Targeted disruption of the Caspase 8 gene also fully blocks the induction of death by ligation of either the T N F receptors, Fas/Apol or DR-3 (EE V, unpublished data). In contrast, targeted disruption of RIP in mice, as well as mutational ablation of RIP function in cultured cells, while abolishing the activation of NF-KB by TNF, did not interfere with death induction by T N F (in fact, they potentiated it, as would be expected in view of the death-inhibitory effect of NF-~cB-induced proteins), nor by Fas/Apol, raising doubts as to the significance of caspase-2 activation through the RIP-RAIDD/CRADD axis for death induction in the tested cells [50°°,51°°].

A splice variant of caspase 8 with an incomplete protease motif was found to act as a natural inhibitor of death induction by T N F and Fas/Apol [43]. Recent studies have revealed the existence of a protein homolog of caspase 8, which is devoid of several of the residues required for proteolytic function. This protein (which has a number of names, including FLIP, Casper, CASH and MRIT) also has inhibitory effects on signaling for death by either Fas/Apol or the T N F receptors. In some cells, however, it is capable of triggering cytotoxicity, probably through association with functional caspases, suggesting that it

Inhibitors of the amplification of the apoptotic process At an early stage of the studies of death induction by T N F and of macrophage cytotoxicity (which is largely mediated by T N F ) it has been noted that mitochondria in the apoptosing cells endure severe structural and functional changes [58,59]. It is now known that mjtochondrial damage is common to many apoptotic processes (reviewed in [60]) although there is still room for debate about whether the given manifestations are indeed sine qua

non features of the program of cell death. The most salient death-related mitochondrial changes are an early dissipation of voltage gradient across the inner membrane and onset of the so-called permeability transition, the latter, apparently being a cause of mitochondrial 'swelling' that results in rupture of the outer membrane. The proteins released from the damaged mitochondria initiate in cells mechanisms that result in amplification of the death process, and various suppression mechanisms are entrusted to modulate the activity/accessibility of such proteins. Among those proteins, the best characterized one is cytochrome c [61,62°°]. Its binding to a cytoplasmic protein called Apaf-1 [63] endows the latter with an ability to activate caspase 9 [64]. This activation mode is a conserved death mechanism that displays a conserved mode of negative regulation. A nematode protein related to Apaf-1, CED4, activates a caspase molecule, CED3, and this activation is subject to negative regulation by a protein called CED9 that binds to CED4 [65°°-67 °°] reviewed in [68]). Mammalian cells contain a whole family of regulators of death related to CED9, the prototype of which is Bcl-2. Some, like Bcl-2 and BcI-XL, are death-inhibitory; others, like Bax and Bad, exhibit death-inducing effects (reviewed in [69]).

The extent of involvement of the Bcl-2-related death inhibitors in the regulation of cell killing by the receptors of the T N F family has been a matter of debate. In vivo assessment of the effect of constitutive expression of Bcl-2 as a transgene in mice have shown conclusively, however, that Bcl-2 does have a protective effect against Fas/Apol cytotoxicity, yet in a highly cell-specific manner. It is incapable of protecting lymphocytes from death induction by Fas/Apol (though strongly protecting them from various other cytotoxic agents; [70]), but effectively protects hepatocytes Fas/Apol cytotoxicity [71"].

A detailed comparative study of Fas/Apol cytotoxicity in different cultured cell lines suggests that protection by Bcl-2-related proteins against this toxicity is restricted to those cells in which death depends on amplification of the caspase activation cascade beyond that occurring as a direct consequence of receptor triggering [72]. Indeed, although Bcl-XL seems to be capable of associating with caspase 8 ([65°°]; probably due to the ability of CASH/MRIT to

Death-inducing functions of ligands of the tumor necrosis factor family Wallach et al. 283

bind simultaneously to the two proteins [57°]), it seems incapable of blocking the triggering of its activation. Yet the Bcl-2-related death inhibitors do have the ability to block events further downstream in the death process [73°]. They apparently do so in more than one w a y - - i n the mitochondria, following release of cytochrome c from the mitochondria or elsewhere in the cell.

Blockage of apoptosis in the mitochondria Anti-apoptotic Bcl-2 analogs can block the death-related changes in volume, proton flux and membrane potential in the mitochondria, as well as the release of cytochrome c [74°-77°,78"°]. The exact mechanism of this protective effect is not clear. Several features of the inhibitors have been suggested to have a bearing on these changes-- inhibi tor localization, effects on the phospholipid bilayer, effects on membrane proteins and association with cytosolic proteins.

L oealiza tion Parts of the Bcl-2 molecules are anchored to the outer mitochondrial membrane, while other parts are associated with other cellular membranes. The kinds of effects mediated by the molecules seem to differ according to their localization (for example, see [79°]; and below).

Effects on the mitochondrial phospholipid bilayer The arrangement of or-helical regions in members of the Bcl-2 family resembles the membrane translocation domain in bacterial toxins [80°]. This feature endows several Bcl-2-1ike molecules with an ability to form, at least in vitro, ion-conductivity channels in lipid membranes, in a pH- and voltage-dependent manner [81°-84°]. This intrinsic channel-forming property of Bcl-2 and Bcl-XL differs from that of the pro-apoptotic Bax, especially with respect to its pH-dependence, allowing Bcl-2 and Bcl-XL to block the pore-forming activity of Bax at physiological pH [83°1.

Effects on membrane proteins When expressed in yeasts, Bax associates with the mitochondrial membrane and can cause cytochrome c release and death. Genetic studies indicate that this Bax effect involves the activity of FoF1-ATPase, a proton pump located within the inner mitochondrial membrane [85], and that it can be overridden by BI-I, a transmembrane protein of otherwise unknown function, which is located mainly in the endoplasmic reticulum (ER) and the nuclear envelope and to a lesser extent in the mitochondrial membranes [86].

Association with cytoso/ic proteins The Bcl-2-related proteins form homo- and hetero-dimers. These associations are subject to modulation by phosphorylation of the proteins. Bad is found to be phosphorylated in response to survival factors. The phosphorylated Bad molecules bind to the protein 14-3-3, thus becoming incapable of binding to Bcl-2 or BcI-X L and neutralizing

their protective effect. This phosphorylation is mediated in part by the serine threonine kinase Akt, whose function is regulated by phosphatidylinositide-3'-OH kinase (PI 3-kinase; [87°-89°]). Bad can also be phosphorylated by Raf-1. The latter kinase binds to Bcl-2 and can thus be targeted to the mitochondria together with another Bcl-2- binding-protein, BAG-l, which activates Raf-1. Expression of active Raf-1 in association with the mitochondrial membrane indeed endows cells with increased resistance to apoptosis [90°]. Conversely, phosphorylation of Bcl-2 seems to decrease its protective effect. This change seems independent, however, of the effectiveness at which this protein binds pro-apoptotic proteins like Bax [91°].

Blockage of apoptosis following release of cytochrome c Death can be blocked by anti-apoptotic Bcl-2 analogs even after cytochrome c has been released to the cytoplasm [92°°,93°°]. This protection is at least partly due to an ability of these molecules to bind to Apaf-1 and block the activation of Caspase-9, in a manner similar to the inhibitory effect of CED9 on CED3 activation by CED4 [94°].

Blockage of apoptosis in the ER Bcl-family members are also associated with the ER and the nuclear membrane, where they seem to mediate site-specific protective mechanisms [79°]. A clue to one kind of such mechanism has been gained with the recent discovery of an ER-anchored Bcl-2-binding protein, p28Bap31. This protein binds the caspase-8 precursor, and the product of its cleavage by this caspase or by caspase-1 promotes the apoptotic process by an unknown mechanism. This cleavage and the resulting apoptotic effect are blocked by Bcl-2 and by BcI-X L [95°].

Inhibi tors of e f fector m e c h a n i s m s in apoptos is The proteolytic function of the caspases is not the direct effector mechanism of cell destruction. It is believed that these proteases, by cleaving a restricted set of specific target proteins, set in motion the function of some other effector mechanisms. Although several of the caspase substrates have been identified, there is still little understanding of their exact role in the apoptotic process. Two of the most recently identified substrates, however, provide a hint as to a general principle by which cleavage of these proteins can activate the effector stage of the death process. One of them, called DFF or I-CAD, acts as an inhibitor of a DNase (CAD) that can degrade nuclear DNA [96°°-98°°]. The other is Bcl-2.

Common to the caspase-mediated cleavage of both proteins is the fact that, in both cases, cleavage results in inactivation of the cleaved proteins and consequently in unlatching of destructive mechanisms that are involved in the apoptotic process. The cleavage of DFF/I-CAD allows CAD to cleave the nuclear DNA, yielding the 'ladder-like' pattern of DNA fragments characteristic of


the apoptotic process [97•',98"•]. T h e cleavage of Bcl-2, which yields an apoptotic fragment simultaneous with release of various apoptotic functions from the Bcl-2 inhibitory effect, apparently serves as a general positive feedback mechanism that amplifies the death process [99]. Inhibitory molecules thus also appear to serve important regulatory roles subsequent to the caspase-activation step, providing an additional means of slowing down the death process, via mechanisms that result in the production of increased amounts of these molecules.

Conclusion As in the careful Sanhedrin trial procedure, the multiplicity of inhibitory mechanisms that restrict death induction by the TNF-re la ted ligands at almost every step in the signaling pathways may constitute a safeguard against unjustified death verdicts. Indeed, while the death of some cells, such as lymphocytes or granulocytes, seems to occur quite freely, it appears that nature exerts the utmost care to assure that the death of liver or kidney cells, for example, will occur only at times of extreme needs, and that some neurons will never die.

Still, such a profusion of safeguard mechanisms and their expression at all levels of the signaling cascades seems superfluous for an 'all-or-none' effect like death induction, particularly in view of the fact that at least some of the mechanisms discussed previously affect not the quality but the quantity of the response. It is tempting to speculate that this abundance of mechanisms serves more than mere reversal of an unjustified verdict of death to the cell. Perhaps, when partially induced, the same T N F ligand-induced effector mechanisms that can lead to cell death confer on cells some other, 'rehabilitative' changes that arc beneficial for its further survival.

References and recommended reading Only papers that are of particular interest with regard to the mechanisms by which death is inhibited and which have been published within the annual period of review have been highlighted as:

• of special interest • - of outstanding interest

1. Ruddle NH, Waksman BH: Cytotoxicity mediated by soluble antigen and lymphocytes in delayed hypersensitivity. II1. Analysis of mechanisms. J Exp Med 1968, 128:1267-1279.

2. Granger GA, Kolb WP: Lymphocyte in vitro cytotoxicity: mechanisms of immune and non-immune small lymphocyte mediated target L cell destruction. J/mmuno/1968, 101:111 - 120.

3. Amino N, Linn ES, Pysher TJ, Mier R, Moore GE, DeGroot L J: Human lymphotoxin obtained from established lymphoid lines: purifcation characteristics and inhibtion by anti- immunoglobulin. J Immuno/1974, 113:1334-1345.

4. Yamarnoto RS, Hiserodt JC, Granger GA: The human LT system V. A comparison of the relative lytic effectiveness of various MW human LT classes on 51 Cr-labeled allogeneic target cells in vitro: enhanced lysis by LT complexes associated with Ig- like receptor(s), eel//mmuno/197g, 45:261-2?5.

5. Gruss H J, Dower SK: Tumor necrosis factor ligand superfamily: involvement in the pathology of malignant lymphomas. Blood 1995, 85:3378-3404.

6. Wallach D: A decade of accumulated knowledge and emerging answers. The 6th international congress on TNF Rhodes, Greece. May 1996. Eur Cytokine Netw 1996, 7:713-724.

7 Nagata S: Apoptosis by death factor. Cell 1997, 88:355-365.

8. Grell M, Douni E, Wajant H, L~hden M, Ciauss M, Baxeiner B, Georgopoulos S, Lesslauer W, Kollias G, Pfizenmaier K, Scheurich P: The transmembrane form of tumor necrosis factor is the prime activating ligand of the 80 kDa tumor necrosis factor receptor. Cell 1995, 83:793-802.

9. Tanaka M, Itai T, Adachi M, Nagata S: Downregulation of Fas e, ligand by shedding. Nat Med 1998, 4:31-36. The soluble form of the Fas/Apol-ligand, which is created by proteolytic cleavage of the cell-bound form, is shown in this study to act as competitive inhibitor of the function of the cell-bound form.

10. Black RA, Rauch CT, Kozlosky C J, Peschon J J, Slack JL, Wolfson • • MF, Castner B J, Stocking KL, Reddy P, Srinivasan Set al.: A

metalloproteinase disintegrin that releases tumour-necrosis factor-alpha from cells. Nature 1997, 385:729-733.

References by Black eta/., 1997 and by Moss et a/., 1997 [11 " ] describe for the first time the isolation and cloning of the protease that is associated with the cell surface and is responsible for shedding of cell-surface protein(s).

11. Moss ML, Jin SL, Milla ME, Burkhart W, Carter HL, Chen WJ, • e Clay WC, Didsbury JR, Hassler D, Hoffman CR et aL: Cloning

of a disintegrin metalloproteinase that processes precursor tumour-necrosis factor-alpha. Nature 1997, 385:?33-736.

References by Moss eta/., 1997 and by Black eta/., 1997 [10 °°] describe for the first time the isolation and cloning of the protease that is associated with the cell surface and is responsible for shedding of cell-surface protein(s).

12. Mullberg J, Durie FH, Otten-Evans C, Alderson MR, Rose-John S, Cosman D, Black RA, Mohler KM: A metalloprotease inhibitor blocks shedding of the IL-6 receptor and the p60TNF receptor. J Immunol 1995, 155:5198-5205.

13. Degli Esposti MA, Smolak PJ, Walczak H, Waugh J, Huang • CP, DuBose RF, Goodwin RG, Smith CA: Cloning and

characterization of TRAIL-R3, a novel member of the emerging TRAIL receptor family. J Exp Med 1997, 186:1165-1170.

References by Degli Esposti et aL, 1997, Pan et aL, 1997 [14°], Sheridan et aL, 1997 [15 °] and MacFarlane et aL, 1997 [16 °°] describe the cloning of a decoy receptor for TRAIL/Apo2 ligand.

14. Pan G, Ni J, Wei YF, Yu G, Gentz R, Dixit VM: An antagonist • decoy receptor and a death domain-containing receptor for

TRAIL. Science 1997, 277:815-818. References by Pan eta/., 1997, Degli Esposti et aL, 1997 [13°], Sheridan eta/., 1997 [15"] and MacFarlane et a/., 1997 [16 °-] describe the cloning of a decoy receptor for TRAIL/Apo2 ligand.

15. Sheridan JP, Marsters SA, Pitti RM, Gurney A, Skubatch M, • Baldwin D, Ramakrishnan L, Gray CL, Baker K, Wood Wl et al.:

Control of TRAIL-induced apoptosis by a family of signaling and decoy receptors. Science 1997, 277:818-821.

References by Sheridan et aL, 1997, Degli Esposti et aL, 1997 [13°], Pan et aL, 1997 [14 °] and MacFarlane et aL, 1997 [16 °°] describe the cloning of a decoy receptor for TRAIL/Apo2 ligand.

16. MacFarlane M, Ahmad M, Srinivasula SM, Fernandes Alnemri T, • Cohen GM, Alnemri ES: Identification and molecular cloning of

two novel receptors for the cytotoxic ligand TRAIL. J Bio/Chem 199"7, 272:25417-25420.

References by MacFarlane eta/., 1997, Degli Esposti et aL, 1997 [13"l, Pan et al., 1997 [14"] and Sheridan et aL, 1997 [15"] describe the cloning of a decoy receptor for TRAII_/Apo2 ligand.

17. Degli-Esposti MA, Dougall WC, Smolak PJ, Waugh JY, Smith CA, • Goodwin RG: The novel receptor TRAIL-R4 induced NF-•B and

protects against TRAIL-mediated apoptosis, yet retains and incomplete death domain. Immunity 1997, 7:813-820.

References by Degli-Esposti et aL, 1997, and Marsters et a/., 1997 [18"] describe the cloning of a receptor for TRAIL/Apo2 ligand that does not signal for cell death (although it has the ability to signal for other effects) and blocks death induction, apparently by competing with those TRAIL/Apo2 ligand receptors that upon ligand binding signal to cause death.

18. Marsters SA, Sheridan JP, Pitti RM, Hunag A, Skubatch M, • Baldwin D, Yan J, Gurney A, Goddard AD, Godowski P, Ashenazi

A: A novel receptor for Apo2L/TRAIL contains a truncated death domain. Curt Bio/1997, 7:1003-1006.

References by Marsters eta/., 1997, and Degli-Esposti eta/., 1997 [17"] describe the cloning of a receptor for TRAIL/Apo2 ligand that does not signal for cell death (although it has the ability to signal for other effects) and blocks death induction, apparently by competing with those TRAIL/Apo2 ligand receptors that upon ligand binding signal to cause death.


19. Golstein P: Cell death: TRAIL and its receptors. Curt Biol 1998, 7:R75-R753.

20. WaUach D: Cell death induction by TNF: a matter of self control. Trends Biochem Sci 1997, 22:107-146.

21. Nicholson DW, Thornberry NA: Caspases: killer proteases. Trends Biochem Sci 1997, 22:299-306.

22. Yang X, Khosravi Far R, Chang HY, Baltimore D: Daxx, a novel Fas-binding protein that activates ,INK and apoptosis. Ce// 1997, 89:1067-1076.

23. Opipari AW, Hu HM, Yabkowitz R, Dixit VM: The A20 Zinc finger protein protects cells from tumor necrosis factor cytotoxicity. J Biol Chem 1992, 267:12424-12427.

24. Song HY, Rothe M, Goeddel DV: The tumor necrosis factor- • inducible zinc finger protein A20 interacts with TRAF1/TRAF2

and inhibits NF-kappa B activation. Proc Nat/Acad Sci USA 1996, 93:6721-6725.

This provides evidence that A20, which acts as an inhibitor of TNF-receptor signaling, is part of the signaling complex.

25. Jaattela M, Mouritzen H, Elling F, Bastholm L: A20 zinc finger • protein inhibits TNF and IL-1 signaling. J Immuno11996,

156:1166-1173. The authors provide evidence that A20 is a wide-ranging inhibitor of TNF function (as well as of the function of IL-1, whose receptor employs parts of the signaling molecules activated by the TNF receptors).

26. Rothe M, Pan M-G, Henzel WJ, Ayres TM, Goeddel DV: The TNFR2-TRAF signaling complex contains two novel proteins related to baculoviral inhibitor of apoptosis proteins. Cell 1995, 83:1243-1252.

27. Uren AG, Pakusch M, Hawkins CJ, Puls KL, Vaux DL: Cloning and expression of apoptosis inhibitory protein homologs that function to inhibit apoptosis and/or bind tumor necrosis factor receptor-associated factors. Proc Nat/Acad Sci USA 1996, 93:4974-4978.

28. Shu H-B, Takeuchi M, Goeddel DV: The tumor necrosis • factor receptor 2 signal transducers TRAF2 and c-lAP1 are

components of the tumor necrosis factor receptor 1 signaling complex. Proc Nat/Acad Sci USA 1996, 93:13973-13978.

This provides evidence that c-lAP1, an inhibitor of apoptosis that contains a Baculovirus inhibitor of death motif, is recruited to the main death-inducing TNF receptor, the p55 receptor (CD120a).

29. Devereaux QL, Takahashi R, Salvesen GS, Reed JC: X-linked • e lAP is a direct inhibitor of cell-death proteases. Nature 1997,

388:300-304. This reports that apoptosis inhibitors of the lAP family directly inhibit specific caspases.

30. Takahashi R, Deveraux (3, Tamm I, Welsh K, Assa-Munt N, • Salvesen GS, Reed JC: A single BIR domain of XIAP sufficient

for inhibiting caspases. J Biol Chem 1998, 273:7787-7790. The authors demonstrate that caspase inhibition by lAP family members is mediated by specific Baculovirus inhibitor of death (BIR) motifs within them.

31. Stankovski I, Baltimore D: NF-KB activation: the IKB kinase revealed? Cell 1997, 91:299-302.

32. Beg AA, Baltimore D: An essential role for NF-KB in preventing • TNF-a-induced cell death. Science 1996, 274:782-784. References by B~g and Baltimore, 1996, Van Antwerp et al., 1996 [33"], Wang et aL, 1996 [34 °] and Liu et aL, 1996 [35 °] indicate that some NF-KB- induced proteins have a protective effect against TNF cytotoxicity and that various cells that are resistant to TNF depend on the activation of NF-KB by TNF for this resistance.

33. Van Antwerp D J, Martin S J, Kafri T, Green DR, Verrna IM: • Suppression of TNF-(x-induced apoptosis by NF-KB. Science

1996, 274:787-789. References by Van Antwerp eta/., 1996, Beg and Baltimore, 1996 [32°], Wang eta/., 1996 [34 °] and Liu et aL, 1996 [35 °] indicate that some NF-KB- induced proteins have a protective effect against TNF cytotoxicity and that various cells that are resistant to TNF depend on the activation of NF-KB by TNF for this resistance.

34. Wang C-Y, Mayo MW, Baldwin AS Jr: TNF- and cancer therapy- . induced apoptosis: potentiation by inhibition of NF-KB. Science

1996, 274:784-787. References by Wang et aL, 1996, Beg and Baltimore, 1996 [32°], Van Antwerp eta/., 1996 [33 °) and Liu eta/., 1996 [35 °] indicate that some NF-KB-induced proteins have a protective effect against TNF cytotoxicity and that various cells that are resistant to TNF depend on the activation of NF-KB by TNF for this resistance.

35. Liu Z-G, Hsu H, Goeddel DV, Karin M: Dissection of TNF • receptor 1 effector functions: JNK activation is not linked to

apoptosis while NF-KB activation prevents cell death. Ceil 1996, 87:565-576.

References by Liu et al., 1996 [35"], Beg and Baltimore, 1996 [32"], Van Antwerp eta/., 1996 [33"] and Wang et al., 1996 [34 °] indicate that some NF-KB-induced proteins have a protective effect against TNF cytotoxicity and that various cells that are resistant to TNF depend on the activation of NF-KB by TNF for this resistance.

36. Rathmell JC, Townsend SE, Xu JC, Flavell RA, Goodnow CC: Expansion or elimination of B cells in vivo: dual roles for CD40- and Fas (CD95)-Iigands modulated by the B cell antigen receptor. Cell 1996, 87:319-329.

37. Sarrna V, Lin Z, Clark L, Rust BM, Tewari M, Noelle R J, Dixit VM: Activation of the B-cell surface receptor CD40 induces A20, a novel zinc finger protein that inhibits apoptosis. J Bio/Chem 1995, 270:12343-12346.

38. Hess S, Engelmann H: A novel function of CD40: induction of cell death in transformed cells. J Exp Med 1996, 183:159-167.

39. Nocentini G, Giunchi L, Ronchetti S, Krausz LT, Bartoli A, Moraca R, Migliorati G, Riccardi C: A new member of the tumor necrosis factor/nerve growth factor receptor family inhibits T cell receptor-induced apoptosis. Proc Natl Acad Sci USA 1997, 94:6216-6221.

40. Newton K, Harris AH, Bath ML, Smith KGC, Strasser A: A • dominant interfering mutant of FADD/MORT1 enhances

deletion of autoreactive thymocytes and inhibits proliferation of mature T lymphocytes. EMBO J 1998, 18:706-718,

References by Newton et al., 1998 and Zsrnig et al., 1998 [41°] present evidence suggesting that MORT1/FADD, in addition to its role in death induction by receptors of the TNF/NGF family, also has a role in signaling for growth.

41. Zsrnig M, Hueber A-O, Evan G: p53-dependent impairment of • T-cell proliferation in FADD dominant-negative transgenic mice.

Curr Bio11998, 8:467-470. References by Zsrnig et al., 1998 and Newton et al., 1998 [40 °] present evidence suggesting that MORT1/FADD, in addition to its role in death induction by receptors of the TNF/NGF family, also has a role in signaling for growth.

42. Sato T, Irie S, Kitada S, Reed JC: FAP-I: a protein tyrosine phosphatase that associates with Fas. Science 1995, 268:411 - 415.

43. Boldin MP, Goncharov TM, Goltsev YV, Wallach D: Involvement of MACH, a novel MORT1/FADD-interacting protease, in Fas/APO1- and TNF receptor-induced cell death. Ceil 1996, 85:803-815.

44. Muzio M, Chinnaiyan AM, Kischkel FC, O'Rourke K, Shevchenko A, Ni J, Scaffidi C, Bretz JD, Zhang M, Gentz M, Mann Met al.: FLICE, a novel FADD-homologous ICE/CED-3-1ike protease, is recruited to the CD95 (Fas/APO-1) death-inducing signaling complex. Ceil 1996, 85:817-827.

45. Fernandes-Alnemri T, Armstrong RC, Krebs J, Srinivasula SM, Wang L, Bullrich F, Fritz LC, Trapani JA, Tomaseli K`I, Litwack G, Alnernri ES: In vitro activation of CPP32 and Mch3 by Mch4, a novel human apoptotic cysteine protease containig two FADD- like domains. Proc Nat/Acad Sci USA 1996, 93:7464-7469.

46. Vincenz C, Dixit VM: Fes-associated death domain protein interleukin-1 ~-converting enzyme 2 (FLICE2), an ICE/Ced-3 homologue, is proximally involved in CD95- and p55-mediated death signaling. J Biol Chem 1997, 272:6578-6583.

47. Duan H, Dixit VM: RAIDD is a new 'death' adapter molecule. Nature 1997, 385:86-89.

48. Abroad M, Srinivasula SM, Wang L, Taianian RV, Litwack G, Fernandes-Alnemri T, Alnemri ES: CRADD, a novel human apoptotic adaptor molecule for caspase-2, and FasL/tumor necrosis factor-interacting protein RIP. Cancer Res 1997, 57:615-619.

49. Yeh W, de la Pompa JL, McCurrach ME, Shu H, Ella A J, Shahinian A, Ng M, Wakeham A, Khoo W, Mitchell K et aL: FADD: essential for embryo development and signaling from some, but not all, inducers of apoptosis. Science 1998, 279:1954-1958.

50. Kelliher MA, Grimm S, Ishida Y, Kuo F, Stanger BZ, Leder P: The • • death domain kinase RIP mediates the TNF-induced NF-KB

signal. Immunity 1 g98, 8:297-303. References by Kelliher et al., 1998, and Ting eta/., 1996 [51 °°] provide evidence suggesting that RIP, a death-domain-containing adapter protein that can cause death when overexpressed in cells, does not serve an important


role in death induction by Fas/Apo-1 or by TNF and actually contributes to cell resistance to TNF cytotoxicity, being crucial for the TNF-induced activation of NF-KB.

51. Ting AT, Pimentel Muinos FX, Seed B: RIP mediates tumor • • necrosis factor receptor 1 activation of NF-KappaB but not

Fas/APO-l-initiated apoptosis. EMBO J 1996, 15:6189-6196. References by Ting et al., 1996, and Kelliher eta/., 1998 [50"'] provide evidence suggesting that RIP, a death-domain-containing adapter protein that can cause death when overexpressed in cells, does not serve an important role in death induction by Fas/Apo-1 or by TNF and actually contributes to cell resistance to TNF cytotoxicity, being crucial for the TNF-induced activation of NF-KB.

52. Irmler M, Thome M, Hahne M, Schneider P, Hofmann K, Steiner V, • Bodmer JL, Schroter M, Burns K, Mattmann C eta/.: Inhibition of

death receptor signals by cellular FLIP. Nature 1997, 388:190- 195.

References by Irmler eta/., 1997, Shu et aL, 1997 [53"], Goltsev et aL, 1997 [54"], Srinivasula et a/., 1997 [55"], Hu et al., 1997 [56"] and Han et al., 1987 [57"] describe the cloning of a protein that displays a marked sequence homology to caspase-8 and -10, but lacks several residues that are crucial for caspase activity. This protein can prevent cell death induction by TNF and Fas/Apol, apparently by competing with caspase-8 and -10 for their binding to the adapter protein MORTI/FADD. In some cells, however, it is capable of triggering cytotoxicity, probably through association with functional caspases, suggesting that it serves a more subtle role than mere inhibition of death induction.

53. Shu HB, Halpin DR, Gceddel DV: Casper is a FADD- and • caspase-related inducer of apoptosis. Immunity 1997, 6:751-

763. References by Shu et aL, 1997, Irmler et al., 1997 [52"], Goltsev et aL, 1997 [54"], Srinivasula et al., 1997 [55"], Hu et al., 1997 [56"] and Han et a/., 1997 [57"] describe the cloning of a protein that displays a marked sequence homology to caspase-8 and -10, but lacks several residues that are crucial for caspase activity. This protein can prevent cell death induction by TNF and Fas/Apol, apparently by competing with caspase-8 and -10 for their binding to the adapter protein MORTI/FADD. In some cells, however, it is capable of triggering cytotoxicity, probably through association with functional caspases, suggesting that it serves a more subtle role than mere inhibition of death induction.

54. Goltsev YV, Kovalenko AV, Arnold E, Varfolomeev EE, Brodianskii • VM, Wailach D: CASH, a novel caspase homologue with death

effector domains. J Biol Chem 1997, 272:19641-19644. References by Goltsev et a/., 1997, Irmler eta/., 1997 [52"], Shu et al., 1997 [53"], Srinivasula et a/., 1997 [55"], Hu et al., 1997 [56"] and Han et aL, 1997 [57"] describe the cloning of a protein that displays a marked sequence homology to caspase-8 and -10, but lacks several residues that are crucial for caspase activity. This protein can prevent cell death induction by TNF and Fas/Apol, apparently by competing with caspase-8 and -10 for their binding to the adapter protein MORTI/FADD. In some cells, however, it is capable of triggering cytotoxicity, probably through association with functional caspases, suggesting that it serves a more subtle role than mere inhibition of death induction.

55. Srinivasula SM, Ahmad M, Ottilie S, Butlrich F, Banks S, Wang • Y, Femandes Alnemri T, Croce CM, Litwack G et aL: FLAME-l,

a novel FADD-like anti-apoptotic molecule that regulates Fas/TNFRl-induced apoptosis. J Biol Chem 199?, 272:18542- 18545.

References by Srinivasula eta/., 1997, Irmler et al., 1997 [52"], Shu et a/., 1997 [53"], Goltsev et al., 1997 [54"], Hu et al., 199? [56"] and Han et al., 1997 [57"] describe the cloning of a protein that displays a marked sequence homology to caspase-8 and -10, but lacks several residues that are crucial for caspase activity. This protein can prevent cell death induction by TNF and Fas/Apol, apparently by competing with caspase-8 and -10 for their binding to the adapter protein MORTI/FADD. In some cells, however, it is capable of triggering cytotoxicity, probably through association with functional caspases, suggesting that it serves a more subtle role than mere inhibition of death induction.

56. Hu S, Vincenz C, Ni J, Gentz R, Dixit VM: I-FLICE, a novel • inhibitor of tumor necrosis factor receptor-I- and CD-95-

induced apoptosis. J Biol Chem 199?, 272:17255-17257. References by Hu et al., 1997, Irmler eta/., 1997 [52"], Shu eta/., 1997 [53"], Goltsev et a/., 199"7 [54"], Srinivasula eta/., 1997 [55"] and Han et al., 1997 [57"] describe the cloning of a protein that displays a marked sequence homology to caspase-8 and -10, but lacks several residues that are crucial for caspase activity. This protein can prevent cell death induction by TNF and Fas/Apol, apparently by competing with caspase-8 and -10 for their binding to the adapter protein MORTt/FADD. In some cells, however, it is capable of triggering cytotoxicity, probably through association with functional caspases, suggesting that it serves a more subtle role than mere inhibition of death induction.

57. Han DK, Chaudhary PM, Wright ME, Friedman C, Trask B, Riedel • R, Baskin D, Schwartz S, Hood L: MRIT, a novel death-effector

domain-containing protein, interacts with caspases and BclXL and initiates cell death. Proc Nat/Acad Sci USA 199"7, 94:11333-11338.

References by Han et aL, 1997, Irmler et aL, 1997 [52°], Shu et aL, 1997 [53"], Goltsev eta/., 1997 [54"], Srinivasula et a/., 1997 [55"] and Hu et aL, 1997 [56"] describe the cloning of a protein that displays a marked sequence homology to caspase-8 and -10, but lacks several residues that are crucial for caspase activity. This protein can prevent cell death induction by TNF and Fas/Apol, apparently by competing with caspase-8 and -10 for their binding to the adapter protein MORT1/FADD. In some cells, however, it is capable of triggering cytotoxicity, probably through association with functional caspases, suggesting that it serves a more subtle role than mere inhibition of death induction.

58. Granger DL, Taintor RR, Cook JL, Hibbs JB Jr: Injury of neoplastic cells by murine macrophages leads to inhibition of mitochondrial respiration. J C/in Invest 1980, 65:357-370.

59. Matthews N: Anti-tumour ¢ytotoxin produced by human monocytes: studies on its mode of action. Br J Cancer 1983, 48:405-410.

60. Petit PX, Susin SA, Zamzami N, Mignotte B, Kroemer G: Mitochondria and programmed cell death: back to the future. FEBS Lett 1996, 396:7-13.

61. Krippner A, Matsuno Yagi A, Gottlieb RA, Babior BM: Loss of function of cytochrome c in Jurkat cells undergoing fas- mediated apoptosis. J Biol Chem 1996, 271:21629-21636.

62. Liu X, Kim CN, Yang J, Jemmerson R, Wang X: Induction of o• apoptotic program in cell-free extracts: requirement for dATP

and cytochrome c. Cell 1996, 86:147-157. This reports that cytochrome c released by the mitochondria during apoptosis can trigger damage in isolated nuclei, similar to that observed in the apoptotic cells. This is a major step forward in definition of the in vitro system that has allowed identification of Apaf-1 and study of the positive and negative regulation of its function.

63. Zou H, Henzel WJ, Liu X, Lutschg A, Wang X: Apaf-1, a human protein homologous to C. elegens CED-4, participates in cytochrome c-dependent activation of caspase-3. Cell 1997, 90:405-413.

64. Li P, Nijhawan D, Budihardjo I, Srinivasula SM, Ahmad M, Alnemri ES, Wang X: Cytochrome c and dATP-dependent formation of Apaf-1/Caspase-9 complex initiates and apoptotic protease cascade. Cell 199?, 91:479-489.

65. Chinnaiyan AM, O'Rourke K, Lane BR, Dixit VM: Interaction of • ,, CED-4 with CED-3 and CED-9: a molecular framework for cell

death. Science 1997, 275:1122-1126. This shows that the anti-apoptotic effect of CED-9 is mediated by its binding to CED-4 and inhibition of CED-3 processing by it. CED-4 also binds in transfected cells to the mammalian caspase 1 and caspase 8.

66. Wu D, Wallen HD, Nuez G: Interaction and regulation of • - subcellular localization of CED-4 by CED-9. Science 1997,

275:1126-1129, The authors demonstrate that the anti-apoptotic effect of CED-9 is mediated by its binding to CED-4.

6"7. Seshagiri S, Miller LK: Caenorhabditis elegans CED-4 stimulates o• CED-3 processing and CED-3-induced apoptosis. Curt Bio/

199"7, 7:455-60. This reports that the anti-apoptotic effect of CED-9 is mediated by its binding to CED-4 which results in inhibition of CED-3 processing.

68. Hengartner MO: Apoptosis. CED-4 is a stranger no more. Nature 1997, 388:714-715.

69. Yang E, Korsmeyer S J: Molecular thanatopsis: a discourse on the BCL2 family and cell death. Blood 1996, 88:386-401.

70. Strasser A, Harris AW, Huang DC, Krammer PH, Cory S: Bcl-2 and Fas/APO-1 regulate distinct pathways to lymphocyte apoptosis. EMBO J 1995, 14:6136-6147.

71. Rodriguez I, Matsuura K, Khatib K, Reed JC, Nagata S, Vassalli • P: A bcl-2 transgene expressed in hepatocytes protects mice

from fulminant liver destruction but not from rapid death induced by anti-Fas antibody injection. J Exp Med 1996, 183:1031-1036.

This reports on in vivo evidence for involvement of Bd-2 in controlling death of hepatocyes induced by Fas/Apol.

72. Scaffidi C, Fulda S, Srinivasan A, Friesen C, Li F, Tomaselli KJ, Debatin K-M, Krammer PH, Peter ME: Two CD95 (APO-1/Fas) signaling pathways. EMBO J 1998, 17:1675-1687.


73. Boise LH, Thompson CB: Bcl-x(L) can inhibit apoptosis in cells • that have undergone Fas-induced protease activation. Proc

Nat/Acad Sci USA 1997, 94:3759-6374. The authors demonstrate evidence that mechanisms acting downstream of the initial caspase activation by Fas/Apol are the targets for the protective effect of Bcl-x(L) against apoptosis induced by Fas/Apol.

74. Yang J, Liu X, Bhalla K, Kim CN, Ibrado AM, Cai J, Peng TI, • Jones DP, Wang X: Prevention of apoptosis by Bcl-2: release

of cytochrome c from mitochondria blocked. Science 199?, 275:1129-1132.

This demonstrates that Bcl-2 can prevent the release of cytochrome c by mitoohondria

75. Kluck RM, Bossy Wetzel E, Green DR, Newmeyer DD: The • release of cytochrome c from mitochondria: a primary site for

Bcl-2 regulation of apoptosis. Science 1997, 275:1132-1136. This shows that cytochrome c release from the mitochondria seems to be independent of dissipation of a voltage gradient in the mitochondrial membrane and can be blocked by Bcl-2.

76. Kharbanda S, Pandey P, Schofield L, Israels S, Ronoinske R, • Yoshida K, Bharti A, Yuan ZM, Saxena S, Weichselbaum Ret

aL: Role for Bcl-xL as an inhibitor of cytosolic cytochrome C accumulation in DNA damage-induced apoptosis. Proc Nat/ Acad Sci USA 1997, 94:6939-6942.

The authors report that cytochrorne c release from the mitochondria can be blocked by Bol-2.

77. Shimizu S, Eguchi Y, Kamiike W, Funahashi Y, Mignon A, • Lacronique V, Matsuda H, Tsujimoto Y: Bcl-2 prevents apoptotic

mitochondrial dysfunction by regulating proton flux. Proc Nat/ Acad Sci USA 1998, 95:1455-1459.

This shows that BcI-2 prevents dissipation of voltage gradient in the mitochondrial membrane, apparently by affecting proton efflux from the mitochondria.

78. Vander Heiden MG, Chandel NS, Williamson EK, Schumacker PT, • • Thompson CB: Bcl-x L regulates the membrane potential and

volume homeostasis of mitochondria. Cell 1997, 91:627-637. The authors demonstrate that cytoohrome c release to the cytoplasm in apoptotic processes seems to result from swelling and rupture of the outer mitoohondrial membrane. Bol-x L inhibits this process by regulating the elec- trical and osmotic homeostasis of mitochondria.

79. Zhu W, Cowie A, Wasfy GW, Penn I.Z., Leber B, Andrews DW: • Bcl-2 mutants with restricted subcellular location reveal

spatially distinct pathways for apoptosis in different cell types. EMBO J 1996, 15:4130-4141.

In this report chimeric Bcl-2 molecules whose membrane-anchoring region was replaced with an anchoring region specific for mitochondria or endoplasmic reticulum endow different cells differentially with resistance to apoptosis, indicating site-specific effects of Bcl-2 within cells.

80. Muchmore SW, Sattler M, Liang H, Meadows RP, Harlan JE, Yoon • HS, Nettesheim D, Chang BS, Thompson CB, Wong SLet al.:

X-ray and NMR structure of human Bcl-xL, an inhibitor of programmed cell death. Nature 1996, 381:335-341.

Here a detailed structural analysis of Bcl-xL reveals arrangement of ~-helical regions in it which resembles the membrane translocation domain in bacterial toxins.

81. Minn AJ, Velez P, Schendel SL, Liang H, Muchmore SW, Fesik • SW, Fifl M, Thompson CB: Bcl-x(L) forms an ion channel in

synthetic lipid membranes. Nature 1997, 385:353-357. References by Minn et aL, 1997, Schendel eta/., 199? [82"] and Schlesinger et al., 1997 [83"] show that the Bcl-2 family members have an intrinsic ability to form pores in artificial membranes in vitro with somewhat different features for the anti- and pro-apoptotic members.

82. Schendel SL, Xie Z, Montal MO, Matsuyama S, Montal M, Reed • JC: Channel formation by antiapoptotic protein Bcl-2. Proc Nat/

Acad Sci USA 1997, 94:5113-5118. References by Schendel et al., 1997, Minn et al., 1997 [81 °] and Schlesinger et al., 1997 [83"] show that the Bcl-2 family members have an intrinsic ability to form pores in artificial membranes in vitro with somewhat different features for the anti- and pro-apoptotic members.

83. Schlesinger PH, Gross A, Yin XM, Yamamoto K, Saito M, • Waksman G, Korsemeyer S J: Comparison of the ion channel

characteristics of proapoptotic BAX and antiapoptotic BCL-2. Proc Nat/Acad Sci USA 1997, 94:11357-11362.

References by Schlesinger eta/., 1997, Minn eta/., 1997 [81 "] and Schendel eta/., 1997 [82"] show that the Bcl-2 family members have an intrinsic ability to form pores in artificial membranes in vitro with somewhat different features for the anti- and pro-apoptotic members.

84. Antonsson B, Conti F, Ciavatta A, Montessuit S, Lewis S, Martinou • I, Bernasconi L, Bernard A, Mermod J-J, Mazzei Ge t a/.: Inhibition

of Bax channel-forming activity by Bcl-2. Science 1997, 277:370-372.

The authors demonstrate the intrinsic channel-forming property of Bcl-2 family members differs from that of the pro-apoptotic ones. Bcl-2 can prevent channel formation by Bax in vitro.

85. Matsuyama S, Xu Q, Velours J, Reed JC: The mitochondiral F0F 1-ATPase proton pump is required for function of the proapoptotic protein Bax in yeast and mammalian cells. Mol Cell Bio11998, 1:327-336.

86. Xu Q, Reed JC: Bax inhibitor-I, a mammalian apoptosis suppressor identified by functional screening in yeast. Mol Cell Biol 1998, 1:337-346.

87. Zha J, Harada H, Yang E, Jockel J, Korsmeyer S J: Serine • phosphorylation of death agonist BAD in response to survival

factor results in binding to 14-3-3 not BCL-X(L). Ceil 1996, 87:619-628.

The authors show that treating cells with IL-3, which serves as a survival factor, promotes phosphorylation of the pro-apoptotic Bcl-2 homolog Bad. Binding of the protein 14-3-3 to the phosphorylated site blocks the binding of Bad to the anti-apoptotic Bcl-2 homolog BcI-XL. Mutation of the phosphorylation site enhances the pro-apoptotic function of Bax.

88. Del Peso L, Gonzalez Garcia M, Page C, Herrera R, Nunez G: • Interieukin-3-induced phosphorylation of BAD through the

protein kinase Akt. Science 199?, 278:687-689, References by del Peso et al., 1997, and Datta et eL, 1997 [89 °] indicate that survival-factor-induced phosphorylation and inactivation of Bad is mediated by the serine/threonine protein kinase Akt which is activated by the phosphatidylinositide 3-kinase.

89. Datta SR, Dudek H, Tao X, Masters S, Fu H, Gotoh Y, Greenberg • ME: Akt phosphorylation of BAD couples survival signals to

the cell-intrinsic death machinery. Cell 1997, 91:231-241. References by Datta et al., 1997, and del Peso et al., 1997 [88"] indicate that survival factor-induced phosphorylation and inactivation of Bad is mediated by the serine/threonine protein kinase Akt which is activated by the phosphatidylinositide 3-kinase.

90. Wang HG, Rapp UR, Reed JC: Bcl-2 targets the protein kinase he Raf-1 to mitochondria. Cell 1996, 87:629-638.

authors show that the serine/threonine protein kinase Raf-1, which can phosphorylate Bad, binds to Bcl-2 and, when recruited to the mitochondria, enhances survival.

91. Chang BS, Minn A J, Muchmore SW, Fesik SW, Thompson CB: • Identification of a novel regulatory domain in BcI-X(L) and

Bcl-2. EMBO J 1997, 16:968-977. This reports that a region within the Bcl-2 and BcI-X L molecules is required for phosphorylation of these proteins. Its deletion enhances the anti-apoptotic function of Bol-2 and BcI-X L even though it has no effect on their ability to bind to the pro-apoptotic Bcl-2 homolog, Bax. The findings suggest that the function of the anti-apoptotic Bcl-2 analogs is subject to down-regulation by phosphorylation, similarly to the function of the pro-apoptotic ones.

92. Zhivotovsky B, Orrenius S, Brustugun OT, Deskeland SO: Injected

; e f e r e CcYte s~l~ ~ i :o?oivns~y Ce~ Sa ~. ,P ~ P~S, I : n NaRU~s 19egtSal.31914~ 9s h4ohw0 that

not only the release of cytochrome c but also the apoptotic effect which it has when released to the cytoplasm can be blocked by anti-apoptotic Bcl-2 analogs.

93. Rosse T, Olivier R, Monney L, Rager M, Conus S, Fellay I, Jansen • • B, Borner C: Bcl-2 prolongs cell survival after Bax-induced

release of cytochrome c. Nature 1998, 391:496-499. References byRoss~ et a1.,1998, and Zhivotovsky et aL, 1998 [92"'] show that not only the release of cytochrome c but also the apoptotic effect which it has when released to the cytoplasm can be blocked by anti-apoptotic Bcl-2 analogs.

94. Pan G, O'Rourke K, Dixit VM: Caspase-9, Bcl-XL, and Apaf-1 his form a ternary complex. J Biol Chem 1998, 273:5841-5845.

reports that, in analogy with the CED-9-CED-4 interaction in nema- todes, the mammalian CED-9 analog BcI-X L is found to bind to the CED-4 analog Apaf-1 and thereby block the activation of caspase-9 by the latter.

95. Ng FW, Nguyen M, Kwan 1", Branton PE, Nicholson DW, Cromlish • JA, Shore GC: p28 Bap31, a Bcl-2/BcI-XL- and procaspase-8-

associated protein in the endoplasmic reticulum. J Cell Biol 1997, 139:327-338.

The authors report the cloning of an ER-associated protein whose function may allow molecular changes that occur in the ER to trigger apoptosis and in the ER-associated Bcl-2 analogs to control this triggering.

96. Liu X, Zou H, Slaughter C, Wang X: DFF, a heterodimeric • • protein that functions downstream of caspase-3 to trigger DNA

fragmentation during apoptosis. Cell 1997, 89:175-184. References by Liu et a/. 199?, Enari et al., 1998 [97 °°] and Sakahira et al., 1998 [98"'] describe the isolation and cloning of an inhibitor of a DNase


(the cloning of which is also described by Enari et aL and Sakahira et aL) which is apparently involved in apoptotic DNA degradation. Cleavage of this inhibitor by caspases set in motion the function of the DNase.

97. Enari M, Sakahira H, Yokoyama H, Okawa K, iwamatsu A, Nagata • - S: A caspase-activated DNase that degrades DNA during

apoptosis and its inhibitor ICAD. Nature 1998, 391:43-50. References by Enari et al., 1998, Liu et a/. 1997 [96"'] and Sakahira eta/., 1998 [98"'] describe the isolation and cloning of an inhibitor of a DNase (the cloning of which is also described by Enari et al. and Sakahira eta/.) which is apparently involved in apoptotic DNA degradation. Cleavage of this inhibitor by caspases set in motion the function of the DNase.

98. Sakahira H, Enari M, Nagata S: Cleavage of CAD inhibitor in • e CAD activation and DNA degradation during apoptosis. Nature

1998, 391:96-99. References by Sakahira eta/., 1998, Liu eta/. 1997 [96"'] and Enari eta/., 1998 [97"] describe the isolation and cloning of an inhibitor of a DNase (the cloning of which is also described by Enari et aL and Sakahira et a/.) which is apparently involved in apoptotic DNA degradation. Cleavage of this inhibitor by caspases set in motion the function of the DNase.

99. Cheng EH, Kitsch DG, Clern R J, Ravi R, Kastan MB, Bedi A, Ueno K, Hardwick JM: Conversion of Bcl-2 to a Bax-like death effector by caspases. Science 199?, 278:1966.

death-inducing functions of ligands of the tumor necrosis factor family: a sanhedrin verdict

Documents