CELL DEATH AT THE MILLENNIUM

HEPATOLOGY A CENTURY OF PROGRESS

1089-3261 /00 $8.00

+ .OO

CELL DEATH AT THE MILLENNIUM Implications for Liver Diseases Neil Kaplowitz, MD

The past 5 years or so have witnessed an explosion of interest and information concerning the processes that participate in the demise of cells. For many years, in the field of hepatology, two morphological patterns of lethal injury have been recognized: zonal coagulative necrosis and spotty shrinkage necrosis (Councilman bodies). Little significance was attributed to this morphological distinction until the work of Kerr in the early 1970s.Kerr recognized that hepatic atrophy following ligation of the portal vein was accompanied by loss of cells through a process of condensation and fragmentation of cells,50followed by their separation from neighbors and phagocytosis. Kerr and his associates subsequently referred to this process as apoptosis,5' a Greek word referring to the dropping of petals from flowers or leaves from trees. Recently, the mechanisms and regulation of the process became accessible through the tools of modern molecular and cellular biology. Now the study of cell death has become one of the most rapidly advancing fields of biology, and translational applications in all fields of medicine are likely by the beginning of the next millenium. Two fundamental types of cell death have been distinguished: necrosis and apoptosis. Necrosis involves loss of ATE swelling of cells, lysis, and secondary inflammation. Apoptosis involves preservation of ATP (at least initially), shrinkage, cytoplasmic and nuclear condensation and fragmentation, and rapid phagocytic removal by macrophages and epithelial cells, with little inflammation. In the liver, necrosis tends to be zonal, whereas apoptosis tends to be spotty. The question arises as to whether these two types of cell death are as distinct

From the University of Southern California Research Center for Liver Diseases; and the University of Southern California-University of California Los Angeles Research Center for Alcoholic Liver and Pancreatic Diseases, University of Southern California School of Medicine, Los Angeles, California

CLINICS IN LIVER DISEASE

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VOLUME 4 NUMBER 1 FEBRUARY 2000

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as their morphology would suggest. Although this issue will be discussed in detail later, the short answer is that the manner of cell death appears to represent a spectrum of responses to the same stimuli, such as cytokines and toxins. Although apoptosis is a fundamental and specific mode of cell death in certain normal biologic processes, such as development and tissue turnover (so-called progranzmed cell death), in disease processes it is less certain that the manner of cell death is as important as its cause. Undoubtedly there exist certain noxious events (mild hypoxia or oxidative stress) that are relatively mild but that activate the quiescent components of the cell-death program. In contrast, there are more massive assaults (e.g., marked oxidative stress or anoxia) that kill the cells quickly by necrosis. There are also, however, intermediate situations which appear to induce apoptosis preferentially, but that lead to necrosis when the apoptosis execution program is blocked, thus demonstrating the inevitability of cell death. Indeed, both apoptosis and necrosis may be seen in different cells in the liver at the same time. This observation is further complicated because massive apoptosis can overwhelm the phagocytic removal of fragmented cells, which then may remain in situ and undergo morphological changes indistinguishable from coagulative necrosis (i.e., secondary necrosis), making it difficult for the pathologist to determine which type of cell death had occurred originally. In summary, apoptosis occurs physiologically in development and tissue turnover, and mild to moderate exposure to reactive oxygen metabolites, hypoxia, or toxins can activate the built-in death program. Severer exposures may be directly lethal, causing necrosis while inhibiting the apoptosis death program. Intermediate situations can occur in which the cell is committed to death whether the apoptosis machinery is active or inhibited.

THE CASCADE OF APOPTOSIS Death Receptors and Proximal Signaling

The machinery of apoptosis includes death receptors, adaptor proteins, and proteolytic enzymes (caspases) (Fig. l).35 As discussed later, mitochondria participate in many forms of cell death, both apoptotic and necrotic.

Chemicals DNA damage

e C sa p A P ,'c

I

Cerarnide, Oxidants, /Cac2, Bax Mitoch.

i+ Bcl,

3A7-

1 Apoptosis

Cyt. c apaf-1 Apoptosorne Procaspase 9

1

Figure 1. Cell death signaling cascades (see text for details).

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Death receptors belong to the tumor necrosis factor (TNF) receptor gene s~perfamily.~~ The cytoplasmic portion of these polypeptides contain an amino acid sequence termed the death domain whch binds adaptor proteins, which then transmit the receptor-binding signal by binding and activating caspases.3Among these receptors, TNF receptor-1 (TNFRl) and Fas (CD95)are the most extensively characterized, and both are abundantly expressed in liver. When Fas ligand (FasL) binds to Fas or TNF binds to TNFR1, the individual receptor molecules trimerize or aggregate, leading to clustering of the death domains. This clustering allows the death domain to bind an adaptor present in the cytosol, either Fas-associated death domain (FADD) or TNF receptorassociated death domain (TRADD) whch in turn binds FADD. FADD then binds procaspase-8, a cytoplasmic zymogen, an event that drives the conformational self-activation of procaspase-8 to caspase-8 by proteolytic cleavage. The complex of Fas, FADD, and procaspase-8 is referred to as the death inducing signaling complex (DISC).Fas ligand is expressed on cytotoxic T cells and sites of immune privilege, such as the eye. Neutrophils also express FasL. Aberrant expression of FasL occurs in epithelial cells in certain diseases, leading to the killing of neighboring cells (fratricide). Tumor necrosis factor is produced by activated macrophages and T cells. When TNFRl is engaged by TNF and undergoes trimerization, TRADD associates with the death domain and serves as a platform for recruitment of a number of molecules, including FADD, TNFR-associated factor-2 (TRAF2), and receptor interacting protein (RIP). FADD can then bind and activate procaspase-8, signaling apoptosis. The binding of RIP and TRAF2 to TRADD promotes activation of transcription factors that lead to the activation of survival genes which oppose apoptosis. Receptor interacting protein signals through a kinase cascade, involving NIK and IKK leading to activation of NFKB (phosphorylation of I-KB), and TRAF2 signals through MAP kinases (JNKK and JNK) lead to cJun/AP-1 activation.12These activated transcription factors (NF-KBand AP-1) then translocate from the cytoplasm to the nucleus, where they promote transcription of genes that oppose apoptosis but promote inflammation ( e g , chemokines, adhesion molecules, N O S ) . Besides FADD, TNFR1-TRADD-RIP can bind another adapter called RAIDD, which activates caspase-2.zoThus, TNF promotes two opposing pathways. In most cell types TNF does not induce cell death unless the cell is sensitized, which can be accomplished by inhibition of protein or RNA synthesis (presumed inhibition of survival gene expression)'2or by GSH dep1eti0n.l~The proinflammatory effects of TNF through the activation of transcription factors are universal. TRAIL is a TNF family member resembling FasL. It binds to DR4 (TRAILR1) and DR5 (TRAIL-R2), both widely expressed members of the TNFRl family.z3 These have death domains but induce apoptosis in FADD knockout mice, indicating signaling mechanisms distinct from TNFRl and Fas. Decoy receptors with absent or truncated cytoplasmic tails, DcRl (TRAIL-R3) and DcR2 (TRAIL-R4), bind TRAIL but do not propagate a death-signaling These receptors are also widely expressed, including in the liver. Thus, presumably because of decoys, TRAIL appears to exert little damage to tissues; however, tumors seem to exhibit high expression of DR4 and DR5 but low expression of decoys, a finding that might be exploited in cancer treatment.lo8 Some anticancer drugs (e.g., topoisomerase inhibitors) have been shown to bypass the engagement of Fas by an agonistic ligand, inducing clustering of Fas and recruitment of FADD to activate a p o p t ~ s i s .lD4 ~ ~Other , anticancer drugs 76 Recently, bile acids have been increase the Fas and FasL in certain cancers.46,

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reported to do the same thing in hepatocytes." The mechanism for spontaneous clustering of death receptors in the absence of ligand is not yet understood. In addition, death effector filaments (DEF), which are perinuclear filament structures that recruit FADD and caspase-8 triggered by chemotherapeutic agents (e.g., cycloheximide), may have a role in transducing death signals in the absence of DISC.49T h s is an alternative mechanism for ligand-independent death signaling which presumably does not require the receptor at all. Further complicating the picture are studies in caspase-&deficient cells which are resistant to Fasmediated killing, indicating that the artificial oligomerization of FADD kills certain cell lines by necrosis; thus, a caspase-independent FADD-mediated death signal exists that induces necrosis, in addition to caspase-dependent signaling that induces ap0ptosis.4~

Caspases

Caspases, cysteine-containing proteases which cleave at aspartate residues, exist as zymogens that are activated by proteolytic cleavage.'07 These enzymes have very restricted substrates. In contrast, other proteases stored in lysosomes or activated in cytosol by calcium have a wide spectrum of nonspecific substrates. Thus, apoptosis is accompanied by very selective and specific proteolysis. Caspases can be divided into three groupslo7: 1. Interleukin converting enzyme (ICE)-like,which are involved in cytokine production (e.g., caspase-1, -4, -5, -13) 2. death-signaling or initiator caspases, that is, caspases which bind to adaptors and propagate but do not execute death (e.g., caspase-2, -8, -9, -10); these caspases activate the executioner caspases 3. executioner or effector caspases (e.g., caspase-3, -6, -7). Although a cascade from initiator to executioner caspases is critical in apoptosis, the system can be self-amplifying, with effector caspases activating initiator caspases. Obviously, the selective substrates of these various proteolytic reactions are crucial in the final phases of the process. Among the proteolytic targets of the executioner caspases are the inhibitory protein of caspase-activated deoxyribonuclease (CAD), Bcl, poly (ADP-ribose) polymerase (PAW), nuclear lamins, gelsolin, focal adhesion kinase, and proteins involved in DNA repair, mRNA splicing, and DNA replication. The attack on all these targets disables repair while disrupting the structure of the cytoskeleton and nucleus, leading to disassembly of the cell. Inhibition of executioner caspases protects against Fasand TNFRI-mediated liver injury.58 Analogous to the coagulation and complement systems of proteolysis, the caspases have decoys and inhibitory counterparts in cells. FADD-like ICE inhibitory proteins (FLIPS)resemble procaspase-8 minus a catalytic domain and are believed to function as inhibitors that compete with procaspase-8 for binding to FADD. Viruses produce caspase inhibitors that subvert immune killing of virusinfected cells; for example, cowpox virus CrmA is a member of the serpin family that potently inhibits initiator caspases. Apoptosis inhibitor proteins (IAPs) inhibit effector caspases-3 and caspase-7, but not initiator caspase-S?' The IAPs are a family that includes NAIP, cIAP1, cIAP2, X-IAP, and su~vivin.~, 16, lo9 In addition, cIAP2 has also been shown to destabilize IkB, thereby activating NFkB,

CELL DEATH AT THE MILLENNIUM

Death

5

Cell

Mitochondria PPT

cyt.c apaf&TP

CasDase 9

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\ Oxidative Stress

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Figure 2. Key role of mitochondria in cell death. The release of cytochrome c marks the commitment to cell death. Depending on the availability of adenosine triphosphate (ATP) and extent of oxidative stress, the cell will die by apoptosis or necrosis.

with prosurvival consequences.16Precisely how the FLIPS and IAPs control the threshold for apoptosis is not yet well understood. Recently, a variant of caspase9, caspase-9S, has been identified which lacks catalytic activity and blocks apoptosis by competing for binding to apoptosis associated factor-1 ( a ~ a f - l ) , ~ ~ analogous to the competition between cFLIP and procaspase-8 for binding to FADD.

Role of Mitochondria

A picture has emerged in recent years of the mitochondria as pivotal in controlling cell life and death. Indeed, mitochondria play a crucial role in both apoptotic and necrotic cell death. (Fig. 2).56, 94, 95, Io3 In apoptosis, a controlled release of cytochrome c from the intermembrane space by a Bcll inhibitable process is critical in either initiating or amplifying the death program.37,86 Cytochrome c then associates with two cytosol proteins, apaf-1 and procaspase-9; in the presence of ATE activated caspase-9 is generated. This complex, referred to as the apoptosome, is a signaling or initiating complex similar in function to the death receptor-adaptor-procaspase-8complex (DISC) at the plasma membrane. Thus, apaf-1 functions as a scaffold in the cytosol, analogous to FADD and TRADD at the plasma membrane. Caspase-9 then activates procaspase-3 and procaspase-7, leading to the execution phase of apoptosis. Caspase-3 can also act proximally, in an indirect fashion, to activate caspase-8 and self-amplify the death-inducing signals, or it can cleave Bcl, removing its protection against cytochrome c release and membrane permeability transition (MPT). The details of the postmitochondrial caspase activation cascade have been recently clarified (Fig. 3).98 The mechanisms of cytochrome c release are not fully understood but appear to be closely related to the mitochondria1 MPT, that is, the opening of a

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“T.c;P apaf-1 Caspase 9

Caspase 3

Caspase 6

Caspase 7

Caspase 2

Caspase 8, 10

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1

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Figure 3. Postmitochondrial,cytochrome-c-dependent cascade of caspase activation. Caspase 9 is the focal point, with subsequent activated caspase 3 exhibiting the ability to activate both initiator and effector caspases. The initiator caspases are 2, 8, 9, and 10; the executioner caspases are 3,6,and 7.

complex protein mega~hannel,’~ although this is not universally ac~epted.,~ The factors that influence the open or closed state of the MPT pore are

Open Decreased membrane potential Protonophores Calcium Decreased growth-stimulating hormone reduced glutathione (GSW Decreased GSH oxidized glutathione (GSSG) Lipid peroxides Oxidative stress Ceramide Atractyloside Bid, Bax

Closed ADP, ATP Thiol compounds Bongkrekic acid Cyclosporin A Bcl, Bcl-X,

The opening of this pore permeabilizes the inner membrane, leading to swelling of the mitochondria and breach of the outer membrane. The release of cytochrome c and the MPT are modulated by Bcl, family members, which either promote or inhibit the process. Apoptosis as a consequence of these events requires the continued availability of ATP and therefore, presumably, the functional integrity of some proportion of mitochondria. If most of the mitochondria undergo MPT, the widespread collapse of mitochondria membrane potential, loss of ATP, and extensive release of cytochrome c lead to collapse of ion gradients, cell swelling, increased cytosol Ca+, with activation of nonspecific hydrolases, and increased reactive oxygen metabolites (caused by interruption of electron transport resulting from release of cytochrome cI5). Mitochondria participate in cell death induced by death receptors and by internal triggers such as toxins and oxidative stress (Fig. 4).Io1 In the former case, caspase-8 cleavage of a Bcl, family member, Bid, results in translocation and insertion of the latter into mitochondria, promoting MPT (Fig. l).38, 69, 71 Alternatively, deathreceptor signaling may target mitochondria through translocation and insertion of Bax1Io, or the action of ceramide derived by initiator-caspase-dependent activation of acidic sphingomyelina~e~~; ceramide interrupts electron transfer at complex 111, thereby promoting oxidative stress.31Other intracellular sources of oxidative stress distinct from death-receptor signaling may also induce MPT.

CELL DEATH AT THE MILLENNIUM

TNF, FasL

1 1

Death Receptors

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Chemicals, ROS ?Bax, etc. DNAdamage

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:/ ?/

-'"'-Executione;Casp&es

bATP

Cvtochrome c

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Necrosis

(3,6, 7)

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Apoptosis Figure 4. Apoptosis induced by death receptors versus chemicals. Both types of cell death stimuli activate initiator (signaling) caspases such as caspases 8 and 9. Cytochrome c release in death receptor-induced apoptosis is initiator caspase dependent (by way of activation of Bid), but in chemical induced apoptosis, it is initiator caspase independent. Broad spectrum caspase inhibitors can inhibit apoptosis in either case, but often convert apoptosis to necrosis in the chemical-induced situation because of caspase-independent loss of mitochondria1 function (not shown). In death receptor-induced apoptosis, effects on mitochondria usually require the action of initiator caspases.

Recently, five potentially apoptogenic proteins have been shown to be released from mitochondria following MPT cytochrome c, apoptosis inducing factor (AIF), DNAse, and procaspases -2 and -9.Io2 The latter initiating caspases are believed to be kept in the intermembrane space in inactive form, separated from the Bc1,-apaf-1 complex which may be on the surface of mitochondria. There may be two fundamentally different death pathways from the Fas receptor which are characteristic of an individual cell type (Fig. 5).93In type I cells, the death-inducing signalling complex (DISC, i.e., Fas-FADD-procaspase8) activates large amounts of caspase-8. In type I1 cells DISC formation and caspase-8 activation are markedly decreased because of the decreased recruitment of FADD and procaspase-8. Proteins that inhibit DISC formation probably determine the type I1 phenotype. In type I cells the death signal is transmitted directly from initiator to executioner caspases. In type I1 cells, the mitochondria participate through sufficient signal production from DISC to mitochondria (presumably through Bid, Bax, or ceramide) to promote cytochrome c release, formation of the apoptosome, and caspase-3 activation. This set of events then can proceed backwards to self-amplify by cleaving procaspase-8 (it is unclear which caspase does ths, because it seems not to be caspase-3) or forward to execute the apoptosis process. Antiapoptotic members of the Bcll family (e.g., Bcl, Bcl-X,), which reside in the mitochondria and inhibit MPT, protect against apoptosis in type I1 cells but not in type I cells. Lymphocytes and thymocytes have a type I phenotype, whereas hepatocytes have a type 11 phenotype, as reflected in the finding that overexpression of the Bcl, transgene in mouse liver protects against agonistic anti-Fas monoclonal antibody-induced fulminant hepatic failure. Thus, in type I1 cells, participation of mitochondria represents an internal amplification system for death-receptor signaling. It remains to be established whether apoptosis induced by other death receptors can be separated into these two phenotypes.

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Fas L

FL

U

Fas

Fas

u

FADD

I

Caspase 8

"-+Mitoch.

CasDase 3

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1

FADD

C a s e8

BC,2

;&

Mitoch.

T

U U

MPT

CyI. c

Caspase 9 casdase 3

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Apoptosis

Figure 5. Cell death signaling in type I and II cells. In type I cells, the cell membrane death receptor recruits and activates sufficient caspase 8 to directly transmit the signal to executioner caspases. In type II cells, insufficient caspase 8 is activated to directly transmit the death signal and the participation of mitochondria is required to amplify the signal. In type II cells (e.g. hepatocytes), but not type I cells, Bcl, prevents apoptosis by blocking the mitochondria1 membrane permeability transition (MPT) and cytochrome c release.

Membrane permeability transition plays a key role in necrotic and in many forms of apoptotic cell death.41,s2 A key question is what determines the ultimate manner of cell death. A crucial factor is the preservation of ATP; thus, if sufficient mitochondria remain intact or glycolytic ATP production is available, . 5 ~ 87 ~ Alternatively, , if ATP is profoundly depleted, apoptosis will be the apoptosis program at the level of the apoptosome cannot propagate, and the cell may die by necrosis. Executioner caspases can also be inactivated by reactive 68 If the MPT has occurred in the face oxygen species39,92 and by nitric of these events, which inhibit propagation of the apoptotic death program, cell death will occur by necrosis.66Lemasters has coined the term necraptosis to describe this condition in which a common death signal or toxic stress leads to MPT and inevitable cell death, with the manner of cell death determined by the status of ATP and the extent of inactivation of caspases (Fig. 2).67This hypothesis does not preclude the possibility that pure forms of necrosis and apoptosis occur but emphasizes that, in many circumstances, the initiating events are common to both processes, and the outcome is death, no matter which pathway predominates. The lethal action of death-receptor ligation by TNF or FasL can be expressed as either necrosis or apoptosis, depending on the conditions. In some cell types the two forms of cell death have the same kinetics, simply switching from one to the other based upon availability of ATE oxygen radicals, nitric oxide, and the activity of executioner caspases66;in other cell types, inhibition of early apoptosis leads to delayed necrosis.38

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INTRACELLULAR FACTORS INVOLVED IN CELL DEATH Several intracellular factors are involved in cell death, and they are described in tlus section. Factors Which Promote or Inhibit Apoptosis

Promote Growth factor withdrawal DNA damage Regeneration Death Receptors (e.g., TNFR1, Fas, etc.) Oxidative stress Drugs and toxins P53 Bid, Bax, Bak, Bad Ceramide Granzyme1porin

Inhibit NFKB Nitric oxide Heat shock protein (HSP) 70 cFLIP Oxidative stress IAPs Silencer of death domains (SODD) Bcla Bcl-XL

P53 p53 is a transcription factor which activates a subset of genes that promote apoptosis, including Fas and oxidoreductases which increase reactive oxygen metabolites. p53 plays an important role in apoptosis after genomic stress such as radiation, chemotherapy, and h y p o ~ i a . ~ ~ , Ceramide Ceramide, produced by sphingomyelinases (SMases), is a second messenger whose role in cell death is controversial.42,96 Neutral SMase is activated at the plasma membrane by a death domain-independent sequence in TNFR1, neutral sphingomyelinase domain (NSD), which acts through an adaptor, factor associated with natural sphingomyelinases (FAN). In addition, normal GSH levels inhibit neutral SMase, and GSH depletion activates this enzyme. Ceramide generated from neutral SMase directs signaling through a kinase cascade, leading to activation of extracellular signal-regulated kinases (ERKs). This pathway does not appear to participate in cell death. In contrast, TNFRl signaling through TRADD and FADD and an unidentified caspase leads to activation of acidic (endosomal-lysosomal)SMase. Ceramide generated in this compartment somehow targets mitochondria to interrupt electron transport at complex 111, thereby promoting oxidative stress and MPT?’ It is believed that this pathway is normally not an important determinant of cell death, particularly in hepatocytes, because of the ability of mitochondria to detoxify reactive oxygen metabolites. When depleted of GSH, however, for example, after alcohol feeding, hepatocytes exposed directly to TNF, acidic sphingomyelinase, or ceramide undergo necrosis.17,~ 5 26* , 31 Recent evidence suggests that ceramide directly binds to cytochrome c, somehow favoring its release32;this evidence supports the view that ceramide-induced oxidative stress in mitochondria is caused by the release of cytochrome c. Depletion of GSH would favor oxidation of cytochrome c, which enhances binding by ceramide. Because ceramide generated from neutral or acidic SMase has distinct effects,

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compartmentation must be critical. The internalization of the death receptorligand complex and targeting to acidic endosomes may play an important role in activating acidic SMase in TNF signaling. Granzyme B

Cytotoxic T cells release perforin and granzyme B, which are internalized to endosomes of target cells. Perforin then permeabilizes the vesicles, releasing granzyme into the cytosol where it cleaves the propeptide of caspase-7, which is then accessible to further cleavage-activation by granzyme B.l16 Granzyme B is also capable of cleaving and activating 71 a proapoptotic Bcl, family member, which targets mitochondria and promotes cytochrome c release. Apoptosis-inducing Factor

JSroemer and co-workers have identified a mitochondria1 matrix protease, AIF, which is not a caspase.loz,Io3 Apoptosis-inducing factor is released during MPT and is activated by caspases. Apoptosis-inducing factor can activate caspase-3 and can also propagate the nuclear changes of apoptosis. The molecular characterization of AIF recently has been reported,lo3”and its relative importance versus cytochrome c release remains uncertain. Nitric Oxide

Nitric oxide inhibits most caspases by direct S-nitrosylation of the catalytic cysteine residue.@Nitric oxide also protects against TNF cytotoxicityby a mechanism involving oxidation of GSH and induction of heat shock protein (HSP) 70 and by a cyclic guanosine monophosphate (cGMP)-dependent inhibition upstream of caspase-3 and S-nitrosylation of caspase~.~~, 53 NFKB

NFKB is a redox-sensitive transcription factor, which, as noted, is activated in TNF signaling and by reactive oxygen metabolites generated within the cell. Activation involves phosphorylation of the inhibitory protein (IKBc~) and its degradation by the proteosome, liberating NFKB for translocation to the nucleusz9where it transactivates the transcription of inflammatory genes (cytokines, chemokines, adhesion molecules) and survival genes (TRAF1, TRAF2, cIAP1, and c I A P ~ ) . ~Io9 , IAPs induced by NFKBcan bind to TRAF, and overexpression of both IAP and TRAF are required to inhibit TNF-induced caspase-8 activation in NFKB-deficient cells.I6 Silencer of Death Domains

Silencer of death domains is a widely expressed 60-kd protein that is associated with the death domain of TNFRl and DR3. It is believed to prevent spontaneous signaling by blocking the binding of TRADD and TRAF2. When TNF binds to the receptor, SODD is released.” Overexpression of SODD protects

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against TNF-induced cell death.47It is possible that the reassociation of SODD with the activated receptor is important in shutting down death signaling. It is likely that SODD-related proteins will also be identified for Fas, DR4, and DR5.

Bcl, Family Members of the large Bcl, family can induce or inhibit cell death1,56,86:

Bcl, Family

Death Antagonists Bcl-2 Bcl-XL Bcl-w Mcl-1 Al/Bf 1-1 Boo

Death Agonists Bax Bak Bcl-Xs Bad Bid Bik Hrk Bim Bok

Many family members reside in the outer membrane of mitochondria and in the nuclear membrane and endoplasmic reticulum. In mitochondria, their distribution is focal, at the contact sites between the outer and inner membrane. Three types of functions have been ascribed to these proteins: (1) dimerization, (2) pore or ion channel activity and (3) binding of other proteins. Heterodimerization between death agonistic and antagonistic members may inhibit apoptosis by neutralizing the agonists or promote apoptosis by displacing proapoptotic factors bound to antagonists, such as apaf-1. Alternately, heterodimerization may promote apoptosis by interfering with pore function of Bcl,. Bcl, can be cleaved by caspase-3, which inactivates protection and may generate a proapoptotic peptide. Phosphorylation of Bcl, by stress kinases, as seen with taxol treatment, inactivates Bcl, and promotes cell death. Phosphorylation renders Bad inactive, sequestered, and bound to 14-3-3.29, 117 Dephosphorylation of Bad (e.g., growth-factor withdrawal) frees Bad to dimerize with Bcl-X,. Several features of these proteins are of particular interest. Recent work indicates that the channel-forming portion of Bcl, is necessary for its protective action," but Bax, which also can form channels, promotes death even when its channel-forming portion is deleted." A protective role for nuclear membrane Bcl, has been proposed. Overexpression of Bcl, increases nuclear GSH, which inactivates a serine protease (AP24) responsible for internucleosomal DNA fragmentation.'I2 The mechanism for the Bc1,-induced increase in nuclear GSH is not known. Recently, Bcl, has been shown to block nuclear penetration of granzyme/ perf0rin.4~ The major apoptotic antagonists, Bcl, and Bcl-XL,are membrane localized, particularly in mitochondria. Bcl, is normally expressed in cholangiocytes and is increased in hepatocytes in chronic chole~tasis,~~ suggesting a role in limiting parenchymal liver injury. Bcl-XL is normally expressed in hepatocytes. The proapoptotic family members are usually found in cytosol and, upon activation of apoptosis, translocate to the mitochondria. Thus, Bax and Bak interact with the permeability pore constituent, adenine nucleotide translocator, leading to MPT.79 The mechanism for activation of Bax translocation is not understood."O, 11* Bid, another proapoptotic family member, is present in cytosol in an inactive form

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whch is cleaved by caspase-8, after which truncated Bid translocates to mito71 Thus, Fas and TNFRl chondria and induces MPT and cytochrome c release.69, signaling from DISC can proceed through an initiator caspase activation of Bid. Bcl-X, and Bcl, prevent Bid-induced release of cytochrome c from mitochondria but do not prevent insertion of truncated Bid in the mitochondria1 membrane. In some cell types, necrosis still proceeds, despite the prevention of cytochrome c release that inhibits apoptosis.38 Recently, additional actions of Bcl, have been identified, such as enhanced phosphorylation and degradation of I K B with ~ release of active NFKB.,] The precise mechanisms are not understood, but it is probable that the interaction of Bcl, with certain kinases is involved. Other studies, however, have reported just the opposite, that is, indirect stabilization of IKBCY and prevention of NFKB activationP These different results may depend on cell type. Bcl, also inhibits transcriptional activity of p53 without affecting its nuclear suggesting that Bcl, sequesters a factor needed for the action of p53. A key unresolved issue is whether proapoptotic Bcl, proteins promote the release of cytochrome c by inducing MPT or by an MPT-independent permeabilization of the outer membrane. Although most of the evidence supports the close association of MPT and cytochrome c release, evidence for MPT independence indicates that Bax induces cytochrome c release, which activates caspases, which then secondarily act on mitochondria to induce MPT.27 Reactive Oxygen Species

Reactive oxygen species (ROS) exert contradictory effects in apoptosis, both promoting and inhibiting Oxidative stress can initiate apoptosis by inducing DNA damage (e.g., by activating p53), by increasing expression of FasL in cells which also express Fas, or by promoting MPT. Reactive oxygen species can also amplify apoptosis, once initiated, or promote survival by activating redoxsensitive transcription factors, such as NFKB,or by inactivating caspases. Oxidative stress frequently accompanies apoptosis because of the effects of apoptosis on mitochondria (e.g., TNF- and ceramide-induced inhibition of electron transport)?l,34 As a consequence, electrons build up and are transferred to 02. In many forms of apoptosis there is a massive efflux of GSH from cells,1o7awith loss of antioxidant defense which then may contribute to oxidative stress. Calpains

The calpains are a family of calcium-dependent cysteine proteases; p- and m-calpain are ubiquitous. Unlike caspases, they do not have strict sequence requirements for substrate cleavage and are active, to a limited extent, in normal cellular metabolism. In some cell types, increased calpain activity has been associated with the degradative phase of apoptosis, leading to disruption of the cytoskeleton and cell ~ignaling.5~ Calpain activation can be mediated by caspases in some cells; this mediation involves proteolytic cleavage by caspases of the endogenous calpain inhibitor, calpastatin.I1l Akt Kinase

Akt kinase is activated by survival (growth) factors, such as insulin-like growth factor 1, and by neurotropins which signal through intermediate kinases

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to activate Akt kinase. Akt phosphorylates Bad and caspase-9, suppressing their proapoptotic function.’5a Akt regulates transcription factors (e.g., FKHRLl) whch control cell death genes, such as FasL. Akt-dependent phosphorylation of FKHRLl causes its retention in the cytoplasm bound to 14-3-3 protein~.’~ Withdrawal of survival factors triggers dephosphorylation and relocation of FKHRLl to the nucleus. APOPTOSIS IN LIVER DISEASE

In recent years, through increased understanding of the molecular mechanisms of apoptosis, the original observations of Kerr50,51 have begun to be translated to both experimental and human liver diseases. As listed, a major role for apoptosis is now recognized in a wide variety of liver diseases, including immune, viral, malignant, drug-induced, alcohol-induced, ischemic, and copperstorage diseases. Some highlights of the importance of this are touched upon in the following sections. Apoptosis in Liver Disease 1. Autoimmune liver disease: chronic hepatitis, primary biliary cirrhosis, allograft rejection 2. Acute and chronic viral hepatitis 3. Regeneration (promotes both proapoptosis and antiapoptosis) 4. Liver cancer (resistance to apoptosis) 5. Cancer chemotherapy 6. Drug-induced liver disease 7. Endotoxin / TNF 8. Alcoholic liver disease 9. Hypoxia and preservation injury 10. Wilson’s disease Role of the Fas system

The possibility of immune-mediated apoptosis was suggested by seminal observations showing that agonistic monoclonal antibody administered to mice induced fulminant hepatic failure caused by massive hepatocyte a p o p t ~ s i s . ~ ~ ” Although hepatocytes in human liver normally express low levels of Fas, increased Fas expression in liver diseases makes hepatocytes more susceptible to FasL-induced a p o p t o s i ~Lymphocytes, .~~ and possibly neutrophils, that express surface FasL need to reach the hepatocyte to act. Studies in cultured rat cells have shown that FasL is expressed in Kupffer’s cells and sinusoidal endothelial cells.n Exposure to lipopolysaccharide (LPS) rapidly increases FasL and subsequently increases Fas expression in hepatocytes.” Dexamethasone increases FasL in hepatocytes.n These FasL-regulatory mechanisms may serve to protect the liver from immunologic attack, although the full significance remains to be established. Fas expression has been reported to be increased in chronic hepatitis B and C.30,40 Interaction of cytotoxic T lymphocytes with HBV-infected hepatocytes increases the expression of lymphocytic FasL, which can kill the hepatocytes even when granzyme-porin is id1ibited.5~In contrast, FasL expression by hepatocytes has been reported in patients with alcoholic cirrhosis3oand fulminant Wilson’s disease.looIn the latter, high expression of both Fas and FasL were

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described in hepatocyte membranes. Copper overload induces oxidative stress; consequent DNA damage activates p53, which increases the expression of Fas. Oxidative stress also increases the expression of FasL.lo0Expression of both Fas and FasL in hepatocytes can lead to a process known as fratricide in which neighboring hepatocytes could kill each other. Chronic liver allograft rejection is associated with increased centrilobular hepatocyte expression of Fas, FasL, and CD40. In vitro, CD40L induces FasL in hepatocytes or HepG2 cells, which then causes fratricide (2). In mice, both agonistic anti-Fas monoclonal antibody-induced and TNF / galactosamine-induced fulminant hepatic failure can be prevented with systemic administration of large doses of a nonselective caspase inhibitor.90Transgenic mice overexpressing Bcl, are highly resistant to agonistic monoclonal anti-Fasinduced liver inj~ry.5~ This finding has been exploited by showing that transplanted hepatocytes overexpressing Bcl, proliferate and repopulate the liver in the face of anti-Fas-induced a p o p t ~ s i s ~ the ~ ; implications for gene therapy or treatment of acute liver failure are obvious. Role of Tumor Necrosis Factor Although TNF promotes receptor-mediated signaling events in hepatocytes, the antiapoptosis pathway normally prevents cell death. Under some circumstances, however, hepatotoxins impair activation of survival mechanisms, and TNF then promotes cell death. Thus, toxins such as ethanol, carbon tetrachloride, acetaminophen, and cw-amanitin may induce hepatic failure by sensitization to the lethal effects of TNF-cx.~~ Two mechanisms of sensitization have been identified: inhibition of protein or RNA synthesis, and GSH depletion sufficient to impair mitochondria1defense against TNF-induced oxidative stress. Liver regeneration depends on TNF. Conversely, interference with TNFinduced NF-KBactivation impairs regeneration and promotes a p o p t o s i ~Thus, .~~ regeneration accompanied by DNA synthesis is a stimulus to apoptosis which is controlled by counter-regulatory pro-survival events. Both TNF and interleukin-6 (IL-6) are needed for regeneration, favoring DNA synthesis while promoting hepatocyte viability (Fig. 6). Blockade of these cytokines both inhibits regeneration after partial hepatectomy and increases death of liver cells. Among the protective mechanisms of these cytokines is the induction of iNOS (Fig. 6). Partial hepatectomy in iNOS-knockout mice results in hepatocyte apoptosis, necrosis, and organ failure.83 Tumor necrosis factor may play a role in fulminant and chronic viral hepatitis. The hepatitis C core protein interacts with the death domain of TNFR1, although it is uncertain whether this interaction promotes119or inhibits cell deaths4and whether sufficient core protein is produced in hepatocytes to play any role. The sequence of events that leads to liver injury and the role of apoptosis and inflammation have been extensively studied using two experimental models: galactosamine plus LPS (endotoxin) and concanavalin A-mediated hepatic injury. Endotoxin alone, at doses which do not cause profound shock, stimulates the release of cytokines (e.g., TNF) which have a nonlethal proinflammatory action, such as increasing the expression of adhesion molecules on sinusoidal endothelial cells and hepatocytes, thereby causing sequestration of neutrophils in the sinusoids. If galactosamine is co-administered, the resulting transcriptional arrest renders hepatocytes susceptible to apoptosis.61Following apoptosis, some-

CELL DEATH AT THE MILLENNIUM

15

/KC

TNF-TNFRI

I

1

Proliferation

Others

4

N F - k Hepatocyte B l x ! r -IL-6 3

J-

Survival Figure 6. Role of cytokines in hepatic regeneration. Liver regeneration requires tumor necrosis factor (TNF), which acts on Kupffer cells to promote IL-6 release and on hepatocytes to promote NF-kB-stimulated prosurvival genes and proliferation. Interleukin-6 also promotes survival genes. Knockout of TNFRl, IL-6, iNOS, and inhibition of NF-kB all impair regeneration and promote liver failure with increased hepatocyte death. Exogenous IL-6 reverses these effects in both TNF-R1 and IL-6 knockouts. KC = Kupffer cells.

thing signals the transmigration of neutrophils w h i c h then augment the liver injury by inducing massive necrosis (Fig. 7). Although current thinking holds that apoptosis does n o t promote inflammation (and it does not, in the case of Fas-mediated fulminant hepatic failure), the endotoxin/ TNF model is unique in that the neutrophils are already sequestered in sinusoids without liver injury. The added TNF-induced apoptosis after galactosamine sensitization seems to be able to signal nearby adherent sinusoidal neutrophils to transmigrate to hepatocytes w h i c h also have increased adhesion molecule expression induced by endotoxin/ TNF. The activated neutropluls, n o w in direct contact with hepatocytes, produce free radicals and proteolytic enzymes w h i c h induce necrosis of hepatocytes. The model of concanavalin A-induced liver injury is characterized by im-

Endrin - jtAdhesion KC -+ TNF

Endothelial Cell

1

Molecules

Hepatocyte'.--t Activated PMN Adherence (galactosamine) ?Adhesion

\Apoptosis

'2Transmigration

Necrosis Figure 7. Interplay of cells in the endotoxin/galactosamine model of liver injuty. Tumor necrosis factor (TNF) released from Kupffer cells (KC) enhances adhesion molecule expression in sinusoidal endothelial cells (with polymorphonuclear (PMN) sequestration) and in hepatocytes. TNF also induces apoptosis in galactosamine sensitized hepatocytes. The moderate extent of apoptosis somehow triggers PMN transmigration and attack on hepatocytes. Features of this model may be also relevant to alcoholic hepatitis.

16

KAPLOWITZ

mune-mediated apoptosis through activation of CD4 cells which produce cytokines. Tumor necrosis factor and IFN-7 promote apoptosis, which is followed Io6 by IL-&mediated pr~liferation.'~~, Ethanol ingestion is an important cause of liver disease throughout the world. In experimental models of ethanol feeding, increased numbers of perivenular apoptotic hepatocytes have been described using morphological analysis." 8* 33, 78, 99, 114 Although only a small percentage of hepatocytes are apoptotic in liver sections from ethanol-fed animals, it is important to recognize that apoptotic cells are rapidly cleared, so a snapshot can markedly underestimate the rate of apoptosis. The cause and significance of this increase in apoptosis is uncertain. Nevertheless, a scenario can be proposed that involves increased gut permeability to endotoxin, endotoxin-stimulated release of cytokines such as TNF, and subsequent hepatocyte death. Considerable evidence exists that the liver of alcohol-fed animals is sensitized to the toxic action of endotoxin or TNF.17,l9 Thus, hepatocytes from alcohol-fed rats are killed by TNF, whereas in control 25, 26 A possible mechanism animals, these cells exhibit the expected re~istance.'~, for the sensitization is depletion of mitochondrial GSH,18 rendering hepatocytes sensitive to mitochondrial oxidative stress induced by TNF or its mediators, acidic SMase and ceramide.I7,~ 5 , The manner of cell death in vitro under these conditions appears to be necrosis. This observation, however, may reflect the extent of oxidative stress and inactivation of caspases. Thus, GSH depletion in mitochondria sensitizes to death; the type of cell death depends on the extent of TNF signaling and, presumably, on the status of ATP and caspase activity (inactivation of oxygen radicals). It is conceivable that the increase in basal apoptosis in livers of alcohol-fed animals reflects TNF actions under conditions which favor apoptosis, whereas in alcoholic hepatitis the balance favors necrosis and inflammation (greater TNF signaling and oxidative stress). It is also conceivable that the initial ethanol-induced apoptosis triggers neutrophil transmigration, as noted previously, extending the injury and causing necrotic cell death in hepatocytes which are more vulnerable (e.g., because of ethanol-induced mitochondrial GSH depletion). Greater neutrophil-induced injury to the liver has been observed in glutathione peroxidase-knockout mice treated with galactosamine and TNEU Glutathione peroxidase is needed to catalyze the reduction of H20, by GSH in mitochondria. Another potential source of oxidative stress in ethanol-fed animals is the induction of cytochrome P450 2E1 (CYP2E1). In HepG2 cells expressing CYP2E1, ethanol induced apoptosis, which was inhibited by antioxidants and B c ~ ~ . ~ This occurrence suggests that extramitochondrial oxidative stress can act either directly or indirectly on mitochondria, which then release cytochrome c. Mitochondrial GSH depletion would be expected to potentiate this phenomenon as well.

Death-receptor-independent Processes

Cell death in the liver is of clinical relevance in hypoxia and in the context of organ preservation for orthotopic liver transplantation. In the liver, mainly hypoxia produces necrosis of hepatocytes but may also selectively target sinusoidal cells for apoptosis. Nevertheless, transfection and overexpression of Bc12 protects against hypoxic ne~r0sis.I~~ This approach may be of benefit in reperfusion after cold ischemia, although cyclosporin A seems to accomplish the same outcome,62namely inhibition of MPT. An important role for calpain activation

CELL DEATH AT THE MILLENNIUM

17

in reperfusion-induced necrosis and apoptosis has been demonstrated recently.54 It remains unclear where calpain fits into the cell-death cascade and what activates it in this circumstance. Drug-induced liver disease falls into two major categories: immune-mediated disease and disease caused by the direct toxicity of metabolites. In immunemediated disease, apoptosis induced by cytotoxic T lymphocytes and soluble mediators predominates. In the latter category, necrosis has been traditionally viewed as the mode of injury. It has been impossible to rule out apoptosis, however. Furthermore, despite the manner of cell death, the mechanism may be caused by the action of toxic metabolites or oxidative stress on MPT or by a sublethal sensitization to the toxic effects of soluble mediators, such as TNF. Acetaminophen toxicity is an interesting example in which both necrosis and and trifluoperazine apoptosis inhibition of MPT with cyclosporin prevents necrosis.6The role of TNF in acetaminophen hepatotoxicity is controversial. Kupffer's-cell blockage prevents toxicity,60whereas toxicity is not altered in TNF-knockout mice.l0 Cholestatic liver disease is associated with a range of secretion impairment which, in theory, should correlate with the extent of bile-acid retention in hepatocytes. In isolated liver mitochondria, hydrophobic bile acids induce the MPT, which can be blocked by ursodeoxycholate or cyclosporin A.", 35 In cultured hepatocytes, low concentrations of hydrophobic bile acids lead to mitochondrial oxidative stress,80MPT, and mainly apoptotic cell death; antioxidants block the apoptosissO.There has also been speculation that a bile acid-induced increase in cytosol Ca+*activates a mitochondrial calpain whch induces the MPT.35High concentrations of bile acids induce rapid necrosis, presumably by a similar but more profound oxidative stress and MPT mechanism; necrosis is also prevented by ursodeoxycholic acid.9 Hepatocytes and liver mitochondria isolated from rats with ligated bile duct (that are in chronic cholestasis) are resistant to the toxic actions of hydrophobic bile acid. This resistance has been attributed to an increase in mitochondrial cardiolipin which inhibits the MPT70and to increased expression of B C ~Com~?~ plicating the picture further are data indicating that bile acids may cause FasLindependent trimerization of Fas with activation of DISC followed by activation of initiator and executioner caspases." Most impressive is the fact that hepatocytes from Fas-deficient mice are resistant to bile acid-induced apoptosis, at least in the early stages of cholestasis. Cathepsin B is also activated and contributes to bile acid-induced apoptosis downstream of c a s p a ~ e - 8Hydrophobic .~~ bile acid feeding is also associated with a large increase in Bax translocation to mitochondria,s9 which may be independent of Fas and contribute in the later stages of cholestasis. In a variety of cell types, Ursodeoxycholic acid prevents apoptosis triggered by a variety of events, including death-receptor ligation.88Coupled with the ability of ursodeoxycholate to inhibit both bile acid-induced and chemically induced MPT, ursodeoxycholic acid seems to be a general cytoprotective agent. The precise mechanism for its inhibition of MPT is not known.

4

THE FUTURE

It is anticipated that the rapidly increasing basic knowledge about cell death will be applied to prevent and treat acute and chronic liver diseases. Although long-term tampering with cell death may promote cancer, short-term or cell-

18

KAPLOWITZ

specific targeting of antiapoptotic or antinecrotic strategies holds considerable promise. Conversely, promoting apoptosis within the liver may be useful in suppressing unwanted cells, such as immunocytes (in immune-mediated liver disease) or stellate cells (in fibrotic liver disease), and in limiting or treating the development of hepatocellular carcinoma or the secondary spread of cancer to the liver. In preventing cell death, strategies that act at or upstream of mitochondria are likely to be more effective than attack on distal effector caspases that may cause a switch from apoptosis to necrosis.

Potential Clinical Approaches in Hepatoprotection Use of cytokines that promote regeneration and survival Interleukin-6 Interleukin-10 Human growth factor Inhibitors of MPT Cyclosporin A Ursodeoxycholic acid Novel natural and synthetic compounds (future) Increased expression of Bcl, and Bcl-X, Death receptor antagonists L Depletion of TNF, or blocking FasL with monoclonal antibodies or soluble receptors Decreased surface expression of TNFRl and Fas Endogenous and exogenous antagonists of DISC and signaling caspases Antioxidants References 1. Adams JM, Cory S The Bcl-2 protein family: Arbiters of cell survival. Science

281:1322, 1998 2. Afford SC, Randhawa S, Eliopoulos A, et al: CD40 activation induces apoptosis in cultured human hepatocytes via induction of cell surface Fas ligand expression and amplifies Fas-mediated hepatocyte death during allograft rejection. J Exp Med 189:441, 1999 3. Ashkenazi A, Dixit V Death receptors: Signaling and modulation. Science 281:1305, 1998 4. Badrichani AZ, Srtoka DM, Bilbao G, et al: Bcl-2 and Bcl-XLserve an anti-inflammatory function in endothelial cells through inhibition of NF-KB.J Clin Invest 103:543, 1999 5. Baroni GS, Marucci L, Benedetti A, et al: Chronic ethanol feeding increases apoptosis and cell proliferation in rat liver. J Hepatol 20:508, 1994 6. Beales D, McLean AEM: Protection in the late stages of paracetamol-induced liver cell injury with fructose, cyclosporin A and trifluoperazine. Toxicology 107201, 1996 7. Beg A, Baltimore D: An essential role for NF-KBin preventing TNF-a-induced cell death. Science 274:782, 1996 8. Benedetti A, Brunelli E, Risicato R, et al: Subcellular changes and apoptosis induced by ethanol in rat liver. J Hepatol 6137, 1988 9. Benz C, Anaermiiller, Tox U, et al: Effect of tauroursodeoxvcholic acid on bileacid-induce; apoptosis and 6tolysis in rat hepatocytes. J Heiatol 28:99, 1988 10. Boess F, Bopst M, Althaus R, et al: Acetaminophen hepatotoxicity in tumor necrosis factor/ lymphotoxin-a gene knockout mice. Hepatology 273021, 1998 11. Botla R, Spivey J, Aguilar H, et al: Ursodeoxycholate (UDCA) inhibits the mitochondrial membrane permeability transition induced by glycochenodeoxycholate: A mechanism of UDCA cytoprotection. J Pharmacol Exp Ther 272930, 1995

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12. Bradham CA, Plumpe J, Manns MP, et al: Mechanisms of hepatic toxicity. I. TNFinduced liver injury. Am J Physiol 275:G387, 1998 13. Bradham CA, Qian T, Streetz K, et al: The mitochondrial permeability transition is required for tumor necrosis factor alpha-mediated apoptosis and cytochrome c release. Mol Cell Biol 18:6353, 1998 14. Brunet A, Bonni A, Zigmond MJ, et al: Akt promoted cell survival by phosphorylating and inhibiting a forkhead transcription factor. Cell 96357, 1999 15. Cai J, Jones DF: Superoxide in apoptosis. J Biol Chem 273:11401, 1998 15a. Cardone M, Roy N, Stennicke H, et al: Regulation of cell death protease caspase 9 by phosphorylation. Science 282:13318, 1998 16. Chu ZL, Mckinsey T, Liu L, et al: Suppression of tumor necrosis factor-induced cell death by inhibitor of apoptosis c-IAP2 is under NF-KBcontrol. Proc Natl Acad Sci U S A 94:10057, 1997 17. Colell A, Garcia-Ruiz C, Miranda M, et al: Selective glutathione depletion of mitochondria by ethanol sensitizes hepatocytes to tumor necrosis factor. Gastroenterology 115:1541, 1998, 18. Colell A, Garcia-Ruiz C, Morales A, et al: Transport of reduced glutathione in hepatic mitochondria and mitoplasts from ethanol-treated rats: Effect of membrane physical properties and S-adenosyl-L-methionine.Hepatology 26:699, 1997 19. Deaciuc IV, Fortunato F, DSouza NB, et al: Modulation of caspase-3 activity and Fas ligand mRNA expression in rat liver cells in vivo by alcohol and lipopolysaccharide. Alcohol Clin Exper Res 23:349, 1999 20. Duan H, Dixit VM: RAIDD is a new 'death adaptor molecule. Nature 385:86, 1997 21. De Moissac D, Mustapha S, Greenberg AH, et al: Bcl-2 activates the transcription factor NFKBthrough the degradation of the cytoplasmic inhibitor I K B ~J .Biol Chem 273:23946,1998 22. Eguchi Y, Shimuzu S, Tsujimoto Y Intracellular ATP levels determine cell death fate by apoptosis or necrosis. Cancer Res 57:1835, 1997 23. Faubion WA, Gores G: Death receptors in liver biology and pathology. Hepatology 29:1, 1999 24. Faubion WA, Guicciardi ME, Miyoshi H, et al: Toxic bile salts induce rodent hepatocyte apoptosis via direct activation of F?s. J Clin Invest 103:137, 1999 25. Femdndez-Checa JC, Kaplowitz N, Garcia-Ruiz C, et al: GSH transport in mitochondria: Defense against TNF-induced oxidative stress and alcohol-induced defect. Am J Physiol 273:G7, 1997 26. Femdndez-Checa JC, Kaplowitz N, Garcia-Ruiz C, et al: Mitochondria1 glutathione: Importance and transport. Semin Liver Dis 18:389, 1998 27. Finucane DM, Bossy-Wetzel E, Waterhouse NJ, et al: Bax-induced caspase activation and apoptosis via cytochrome c release from mitochondria is inhibitable by Bcl-xL. J Biol Chem 274:2225, 1999 28. Froesch BA, Aim6-Sempe C, Leber B, et al: Inhibition of p53 transcriptional activity by Bcl-2 requires its membrane-anchoring domain. J Cell Biol 274:6469, 1999 29. Gajewski T, Thompson C: Apoptosis meets signal transduction: Elimination of a BAD influence. Cell 87589, 1996 30. Galle PR, Hofmann WJ, Walczak H, et al: Involvement of the CD95 (APO-l/Fas) receptor and ligand in liver damage. J Exp Med 182:1223, 1995 31. Garcia-Ruiz C, Colell A, Mari M, et al: Direct effect of ceramide on the mitochondrial electron transport chain leads to generation of reactive oxygen species. J Biol Chem 27211369, 1997 32. Ghafourifar P, Klein SD, Schucht 0, et al: Ceramide induces cytochrome c release from isolated mitochondria. J Cell Biol 274:6080, 1999 33. Goldin RD, Hunt NC, Clark J, et al: Apoptotic bodies in a murine model of alcoholic liver disease: Reversibility of ethanol-induced changes. J Pathol 171:73, 1993 34. Goossens V, Grooten J, De Vos K, et a1 Direct evidence for tumor necrosis factorinduced mitochondrial reactive oxygen intermediates and their involvement in cytotoxicity. Proc Natl Acad Sci USA 9223115, 1995 35. Gores GJ, Miyoshi H, Botla R, et al: Induction of the mitochondrial permeability

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62. Leducq N, Delmas-Beauvieux M-C, Bourdel-Marchasson I, et al: Mitochondria1 permeability transition during hypothermic to normothermic reperfusion in rat liver demonstrated by the protective effect of cyclosporin A. Biochem J 336:501, 1998 63. Leist M, Gantner F, Naumann H, et al: Tumor necrosis factor-induced apoptosis during the poisoning of mice with hepatotoxins. Gastroenterology 112:923, 1997 64. Leist M, Nicotera P: Breakthroughs and views. The shape of cell death. Biochem Biophys Res Commun 236:1, 1997 65. Leist M, Single A, Castoldi AF, et al: Intracellular adenosine triphosphate (ATP) concentration: A switch in the decision between apoptosis and necrosis. J Exp Med 185:1481, 1997 66. Lemaire C, Andrbau K, Souvannavong V, et al: Inhibition of caspase induces a switch from apoptosis to necrosis. FEBS Lett 425:266, 1998 67. Lemasters JJ: Mechanisms of hepatic toxicity v. necrapoptosis and the mitochondrial permeability transition: Shared-pathways io necrosis and apoptosis. Am J Physiol 276:G1, 1999 68. Li J, Billiar TR, Talanian RV, et al: Nitric oxide reversibly inhibits seven members of the caspase family via S-Nitrosylation. Biochem Biophys Res Commun 240:419, 1997 69. Li H, Zhu H, Xu CJ, et al: Cleavage of BID caspase 8 mediates the mitochondrial damage pathways of apoptosis. Cell 94:491, 1998 70. Lieser MJ, Park J, Natori S, et al: Cholestasis confers resistance to the rat liver mitochondrial permeability transition. Gastroenterology 115:693, 1998 71. Luo X, Budihardjo I, Zou H, et al: Bid, a Bc12 interacting protein, mediates cytochrome c release from mitochondria in response to activation of cell surface death receptors. Cell 94481, 1998 72. Marzo I, Brenner C, Zamzami N, et al: Bax and adenine nucleotide translocator cooperate in the mitochondrial control of apoptosis. Science 281:2027, 1998 73. Matsuyama S, Schendel SL, Xie Z, et al: Cytoprotection by Bcl-2 requires the poreforming a 5 and a6 helices. J Biol Chem 273:30995, 1998 74. Micheau 0, Solary E, Hammann A, et al: Fas ligand-independent, FADD-mediated activation of the Fas death pathway by anticancer drugs. J Cell Biol 274:7987, 1999 75. Mignon A, Guidotti JE, Mitchell C, et al: Selective repopulation of normal mouse liver by Fas/CD95-resistant hepatocytes. Nature 4:1185, 1998 76. Miiller M, Strand S, Hug H, et al: Drug-induced apoptosis in hepatoma cells is mediated by the CD95 (APO-1/Fas) receptor/ligand system and involves activation of wild-type p53. J Clin Invest 99:403, 1997 77. Muschen M, Warskulat U, Douillard P, et al: Regulation of CD95 (APO-l/Fas) receptor and ligand expression by lipopolysaccharide and dexamethasone in parenchymal and nonparenchymal rat liver cells. Hepatology 27:200, 1998 78. Nanji A: Apoptosis and alcoholic liver disease. Semin Liver Dis 18:187, 1998 79. Narita M, Shimizu S, Ito T, et al: Bax interacts with the permeability transition pore to induce permeability transition and cytochrome c release in isolated mitochondria. Proc Natl Acad Sci USA 95:14681, 1998 79a. Ogasawara J, Watanabe-Fukunaga R, Adachi M, et al: Lethal effects of the anti-Fas antibody in mice. Nature 364:806, 1993 80. Pate1 T, Gores G: Inhibition of bile-salt-induced hepatocyte apoptosis by the antioxidant lazaroid U83836E'. Tox Appl Pharm 142:116, 1997 81. Polyak K, Xia Y, Zweier JL, -2al: A model for p53-induced apoptosis. Nature 389:300, 1997 82. Qian T, Herman B, Lemasters JJ: The mitochondrial permeability transition mediates both necrotic and apoptotic death of hepatocytes exposed to Br-A23187. Toxic01 Appl Pharmacol 154:117, 1999 83. Rai RM, Lee FYJ, Rosen A, et al: Impaired liver regeneration in inducible nitric oxide synthase deficient mice. Proc Natl Acad Sci USA 95:13829, 1998 84. Ray RB, Meyer K, Steele R, et al: Inhibition of tumor necrosis factor (TNF-a)mediated apoptosis by hepatitis C virus core protein. J Biol Chem 273:2256, 1998 85. Ray SD, Mumaw VR, Raje RR, et al: Protection of acetaminophen-induced hepatocellular apoptosis and necrosis by cholesteryl hemisuccinate pretreatment. J Pharmacol Exp Ther 279:1470, 1996

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