Meeting Report

Apoptosis and the Liver: A Mechanism of Disease,Growth Regulation, and Carcinogenesis

TUSHAR PATEL,1 CLIFFORD J. STEER,2 AND GREGORY J. GORES3

Our understanding of the mechanisms of apoptosis hasdramatically increased over the past few years. This increas-ing knowledge is being translated into efforts to developtherapeutic strategies for a wide range of human diseases.Although apoptosis has been recognized to occur in the liverfor several decades, the role and relevance of apoptosis tohuman diseases remains undetermined. However, there isaccumulating evidence that liver cell apoptosis is involved inviral, toxin, and cholestatic injury to the liver. In addition,there is an increased awareness of the role of apoptosis inseveral pathophysiological processes relevant to the liver,such as inflammation and fibrogenesis, as well as physiologi-cal responses to injury, such as regeneration. Thus, under-standing the mechanisms of apoptosis in the liver is highlygermane to our understanding of human liver diseases. Forthese reasons, the American Association for the Study of LiverDiseases organized a Basic Research Single Topic Conferenceat Airlie House, Warrenton, VA from June 17 to 20, 1999. Thegoals of this meeting were to discuss current information onthe basic cellular mechanisms of apoptosis; to discuss the roleof apoptosis in viral, toxic, metabolic, cholestatic, andmalignant diseases; and to explore potential therapeuticopportunities for exploiting this knowledge for the treatmentof human liver diseases.

After an introduction by the course codirectors to outlinethe objectives of the meeting, several topics were discussed.These included the mechanisms of apoptosis, apoptosis inliver diseases, carcinogenesis, regeneration, inflammationand fibrogenesis, and therapeutic opportunities.

MECHANISMS OF APOPTOSIS

Tushar Patel (Temple, TX) presented a brief overview ofapoptosis and discussed some of the current approaches toidentifying the presence of apoptosis in the liver. The processof apoptosis is a fundamental mechanism for the deletion of

unwanted, senescent, or damaged cells. Apoptosis plays acritical role in several physiological as well as pathophysiologi-cal processes.1 Studies on Caenorhabditis elegans have out-lined critical genetic components of the apoptotic mecha-nism.2 As these are conserved during evolution, identificationof their human homologues has enhanced our knowledge ofthe core intracellular mechanisms leading to apoptosis. Thesemechanisms are controlled by internal signals as well as bysurvival and death signals from the environment, which serveto maintain tissue integrity.3 Dysregulation of apoptosis isimportant in the pathogenesis of a wide range of liver diseasesranging from autoimmune disease, viral infections, tissueinjury, and malignancy.4 It was emphasized that the reversiblesteps in apoptosis remain to be identified. Furthermore, morestudies are needed to elucidate the potential link betweenapoptosis and liver inflammation.

John Cidlowski (NIEHS, Research Triangle Park, NC)discussed the role of volume regulation in apoptosis. Eventhough core components of the apoptotic machinery, such asproteases or endonucleases, are present in the cell, these areconstitutively inactive. Recent studies from his laboratoryhave shown that the biochemical events of apoptosis, such asDNA fragmentation, ribosomal RNA degradation, decreasedmitochondrial membrane potential, and caspase activation,occur only in cells that have shrunk.5 Cell shrinkage ispreceded by alterations in plasma membrane potential andoccurs by compensatory volume regulation following low-ered intracellular potassium concentrations. The activity ofcaspases and endonucleases in the cell is inhibited at physi-ological potassium concentrations but is markedly enhancedat low concentrations. Furthermore, modulation of the intra-cellular potassium concentration alters the susceptibility ofthe cell to undergo apoptosis, with lower concentrationsenhancing and higher concentrations suppressing apoptosisin response to a variety of apoptotic stimuli.6 These studieshave led to the hypothesis that cell shrinkage is a controllingstep during apoptosis and that cells actively suppress apopto-sis by maintenance of high ionic strength and regulation ofcell volume.7 The effect of death ligands on ion transport is aconcept quite germane to the epithelial cells in the liver(hepatocytes and bile duct epithelial cells) and one that hasbeen neglected but likely is important.

Peter Galle (Mainz, Germany) discussed the role of thedeath receptors CD95 (Fas/Apo-1) in human liver diseases.Hepatocytes highly express CD95 and are extremely sensitiveto apoptosis in response to CD95 receptor stimulation byagonistic antibodies or CD95 ligand (CD95L).8 The mecha-nisms and implications of CD95 receptor and ligand expres-sion were discussed in conditions such as Wilson’s disease,chemotherapy-induced apoptosis, and hepatocellular cancer.

Abbreviations: CD95L, CD95 ligand; NFkB, nuclear factor kappa B; TNF, tumornecrosis factor; TGFb, transforming growth factor beta; HCV, hepatitis C virus;I-FLICE, Flice inhibitory protein; FADD, Fas-associated death domain; NO, nitricoxide.

From the 1Division of Gastroenterology, Scott and White Clinic, Texas A&MUniversity System Health Science Center College of Medicine, Temple, TX; 2Departmentof Medicine, Division of Gastroenterology, University of Minnesota Medical School,Minneapolis, MN; and 3Division of Gastroenterology and Hepatology, Mayo Clinic,Rochester, MN.

Received July 9, 1999; accepted July 12, 1999.Address reprint requests to: Tushar Patel, M.D., Division of Gastroenterology, Scott

and White Clinic, 2401 South 31st Street, Temple, TX 76508. E-mail: tpatel@swmail.sw.org; fax: 254-724-8276.

Copyright r 1999 by the American Association for the Study of Liver Diseases.0270-9139/99/3003-0030$3.00/0

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In Wilson’s disease, CD95L is up-regulated by a mechanisminvolving reactive oxygen species and activation of nuclearfactor kappa B (NFkB).9 In addition, DNA damage inresponse to copper or reactive oxygen species results in anincrease in p53 and in CD95 expression. Hepatocyte expres-sion of both CD95 and CD95L results in autocrine-and/orparacrine-induced suicide. A different model was presentedfor treatment with chemotherapeutic drugs. Chemotherapy-induced DNA damage also results in CD95 and CD95Lexpression and subsequent autocrine- or paracrine-mediatedsuicide. Up-regulation of CD95 is mediated via a transcription-ally mediated accumulation of p53, whereas up-regulation ofCD95L messenger RNA is independent of p53.10,11 Finally,the role of CD95 expression in hepatocarcinogenesis wasdiscussed. Hepatocellular carcinomas exhibit a loss of CD95,and an increased expression of CD95L.12 Expression ofCD95L could lead to apoptosis of activated T lymphocytesthat bear the CD95 receptor. This has led to the developingconcept of tumor attack of T lymphocytes as a mechanism ofimmune privilege.

David Hockenberry (Seattle, WA) described the role ofBcl-2 family members in apoptosis. Members of this familyare potent intracellular regulators of apoptosis.13 Althoughthey are expressed in most cells and can regulate apoptosis,members of this family may possibly have a fundamentalnonapoptotic function. Their localization to the mitochon-drial membrane is linked to their involvement in mitochon-drial structure and function. Several different functional rolesof Bcl-2 were discussed, such as the regulation of iontransport, osmotic gradients, and voltage gradients by apore-like activity and by modulation of membrane permeabil-ity. In addition, Bcl-2 may be involved in modulating mito-chondrial assembly during proliferation.14,15 In particular, thedata suggesting that Bcl-2 may enhance mitochondrial prolif-eration provide a new paradigm for the study of thiscytoprotective molecule.

John Lemasters (Chapel Hill, NC) described the involve-ment of mitochondria in apoptotic and necrotic cell deathand reviewed the role of the mitochondrial pore and mem-brane permeability transition. In a series of several carefullyperformed single-cell studies, activation of the membranepermeability transition was observed during hypoxia, oxida-tive stress, or calcium ionophore treatment and preceded celldeath in each instance.16-18 Thus, the membrane permeabilitytransition may represent a typical rather than an exceptionalevent during necrotic and apoptotic cell death.19 The involve-ment of a structural and functional cellular perturbationcommon to both apoptosis (morphologically defined, ac-tively controlled cell death) and necrosis (accidental deathafter disruption of plasma membrane integrity in response tocellular stresses) emphasizes that the distinction betweenthese processes is arbitrary and that they represent ends of aspectrum by which a cell could die.20

Yuri Lazebnik (Cold Spring Harbor, NY) presented anoverview of the caspases associated with apoptosis by review-ing their hierarchy, function, and activation as well as theirrole in mediating cell death processes.21 Biochemical studieswith cell free systems led to the identification of a moleculeidentical to apoptotic protease-activating factor-1 (APAF-1)in apoptosis-inducing extracts from transformed fibroblasts.This molecule is important in the activation of caspase 9,which is activated only when it forms a complex with amultimer of APAF-1 and cytochrome c in the presence of

adenosine triphosphate, which is likely as a result of allostericinteractions.22 The roles of perturbations in APAF-1 or incaspase 9 activation in the pathogenesis of cancer areunknown.

APOPTOSIS IN LIVER DISEASES

Michael Lai (Los Angeles, CA) described the role of viralproteins in modulating apoptosis. Viruses have evolved avariety of mechanisms for the modulation of apoptosis.23

These include binding to p53, acting as Bcl-2 homologues,blocking tumor necrosis factor (TNF) or Fas signal transduc-tion pathways, and inhibiting caspases such as CrmA andp35. An increase in hepatocyte apoptosis is seen in chronichepatitis B and C. Induction of apoptosis may be due toactivation of p53 or c-myc, up-regulation of Fas and TNFreceptor, or alteration in TNF/Fas pathways. Apoptosis inresponse to a viral infection most likely involves the interac-tion of both host and viral factors. Host factors includecytotoxic T cells and proapoptotic cytokines such as TNFa,transforming growth factor beta (TGFb), and FasL, whereasviral factors include proteins such as the hepatitis B X proteinand the hepatitis C core protein (NS5A). Clearly, the hostcytotoxic T lymphocyte response is involved in apoptosis.The involvement of Fas and FasL is suggested by thecorrelation of their expression with the degree of liverpathology and inflammation. Hepatitis B X protein hasvariable effects on apoptosis and may sensitize cells to TNF,induce apoptosis, abrogate effects of TNF, or have no effects.Thus, the effect of hepatitis B X protein is likely to bedependent on specific conditions. Although it is a structuralprotein, the hepatitis C virus (HCV) core protein may have aregulatory function in modulating apoptosis by either inhibit-ing it, for example in response to c-myc, cisplatin, or TNF, ormay enhance it, for example in response to Fas, TNF, andlymphotoxin. Modulation of apoptosis may involve bindingof the HCV core protein to the intracellular signal transduc-ing portion of death receptors such as Lymphotoxin b, TNF,or Fas and displacement of signaling molecules such asTNF-associated factor (TRAF-2), and TNF-associated deathdomain (TRADD).24 Although HCV core protein binding hasbeen shown to inhibit NFkB activation and induce apoptosis,this has not been observed in human-derived epithelial cellssuch as HepG2, Hep3B, or 293 cells. The effects of the HCVcore protein in modulating the sensitivity to cytokines arevariable. Some groups have shown enhanced sensitivity,whereas others have shown decreased sensitivity or nochange. The core protein may also modulate promoteractivity, and bind to other molecules such as helicases.Furthermore, recent biochemical studies have shown that thecore protein interacts in vitro with and inhibits the interferon-induced, RNA-dependent protein kinase, PKR, which isinvolved in apoptosis. The notion that apoptosis may repre-sent a mechanism for viral shedding rather than a mechanismfor viral elimination was suggested in the discussion by Dr.Lai. This provocative idea deserves further attention andraises the concept that pharmacological inhibition of apopto-sis could ameliorate this difficult to treat disease.

Sheri Krams (Stanford, CA) discussed the role of apoptosisin rejection and tolerance after transplantation.25 There is acorrelation between apoptosis and rejection in animal modelsas well as in clinical transplantation.26 In experimental livertransplantation, increased expression of apoptosis-regulatoryproteins, such as perforin, granzyme B, Fas, and FasL, is

812 PATEL, STEER, AND GORES HEPATOLOGY September 1999

noted in allografts as compared with isografts. Furthermore,the expression of granzyme B may be used as a marker ofrejection. Thus, apoptosis may have a role in rejection-mediated graft damage. Furthermore, natural killer cells wereidentified as being a major source of FasL in the rejectinggraft. Apoptosis may be detected in vivo using technetium-labeled annexin V during experimental graft rejection. Thecaspase inhibitor zVAD-fmk decreases apoptosis but does notprevent histological features of rejection. Finally, the use ofDNA microarray techniques and dendrogram analysis toidentify genes that are over- and under-expressed duringrejection may be a useful approach to identifying new orcritical genes involved in the process.

Craig McClain (Lexington, KY) discussed the evidence fora role of apoptosis in experimental alcohol-related liverdisease. In addition he discussed studies of dysregulated TNFsignaling in response to alcohol as well as studies onTNF-induced apoptosis in HepG2 cells. A large body ofevidence suggests that cytokines such as TNF may mediatemany metabolic activities in alcohol-related liver injury aswell as non–alcohol-related liver injury.27 Apoptosis wasidentified in sinusoidal endothelial cells and in hepatocytes inan animal model of alcohol-related liver injury.28 Further-more, an increase in FasL was observed in animals fed theLieber-diCarli diet compared with pair-fed animals. In amodel of lethal lipopolysaccharide-induced liver injury, ad-ministration of the pan-caspase inhibitor IDUN 1965 for 24hours completely prevented cell death and attenuated liverinjury, although death rapidly ensued when the inhibitor wasnot given. A useful model of cytokine-induced liver injuryand hepatocyte apoptosis is the administration of TNF aftersensitization with actinomycin D. TNF injury can be blockedby selective inhibition of caspase 3. Pretreatment with smalldoses of TNF prior to giving actinomycin D results intolerance to TNF/actinomycin D–induced cell death in vitro.Proteosomal inhibition sensitizes to TNF killing and blocksthis tolerance effect. Inhibition of NFkB induces proteolyticcleavage of caspase 3. Although proteosomal inhibitioninhibits NFkB proteolysis, other TNF-related transcriptionevents, such as activation of AP-1, are not blocked. The datashowing Fas-mediated apoptosis of sinusoidal endothelialcells by ethanol provides a new direction for research inalcohol-related liver disease by raising the question: Isalcohol-related liver disease promoted by a microvascularinjury?

Gregory Gores (Rochester, MN) outlined the role ofhepatocyte injury in cholestatic liver injury. Retention ofbiliary constituents such as bile salts during cholestasis mayresult in hepatocyte damage from apoptosis.29 Bile salt–induced hepatocyte apoptosis is decreased in lpr mice, butnot in gld mice suggesting that the process is dependent onFas but independent of FasL. Glycochenodeoxycholate in-duces Fas oligomerization in the absence of a ligand.30 Thereis an increase in cell surface Fas expression after treatmentwith glycochenodeoxycholate that is inhibited by brefeldin A(disruptor Golgi trafficking) or with the use of nocadozole(a microtubule poison). Thus, bile salts likely affect theshuttling of Fas from an intracellular compartment to the cellsurface. These observations have been correlated in vivo inbile duct ligated rats. Thus, the accumulation of toxic bilesalts in the liver during cholestasis results in Fas-dependenthepatocyte apoptosis. These data suggest a new mechanismfor Fas-related liver injury during toxin exposure.

CARCINOGENESIS AND REGENERATION IN THE LIVER

Snorri Thorgeirsson (Bethesda, MD) discussed elegantwork on transgenic models of hepatocarcinogenesis such asthe c-myc/TGFa double transgenic mice.31 These have beenuseful models to determine the cellular biology and geneticlesions during hepatocarcinogenesis, and have allowed adetailed examination of the roles of tumor suppressors,apoptosis, DNA repair processes and other events in theclonal expansion and accumulation of genetic lesions.32,33

Recent studies have shown the applicability of this model tohuman carcinogenesis. During initiation, there is rapid prolif-eration and a high amount of apoptosis along with specificchromosomal abnormalities. In the promotion phase, there isa reduction in TGFbRII expression and an increase in TGFb,TGFa, cyclin D, p21, p53, and other predisposing genes. Inaddition, there is decreased cell death and increased cellproliferation. Studies in oncogene-transformed rat epithelialcells, which are sensitive to TGFbII growth inhibition, showhyperphosphorylation of IkBa resulting in a high level ofNFkB activation. Inhibition of NFkB activity reduces theirsensitive to TGFb1-induced apoptosis. Furthermore, theactivity of kinases that phosphorylate IkB are important forsurvival as well as for transformation because inhibition ofkinase activity reduces the colony-forming capacity in thesecells.

Rolf Schulte-Hermann (Vienna, Austria) observed thatmany tumors show an increase in the rates of ‘‘cell birth’’ aswell as ‘‘cell death’’ over those in the tissue of origin. Anexcess of cell birth over cell death is a crucial characteristic ofpreneoplastic and neoplastic cell populations. Defects inpreneoplastic populations permit enhanced cell death andenhanced cell replication resulting in higher cell turnover.34

Glutathione-S-transferase–positive cells may be used as amarker of early putative stem cells. Single initiated cells showlow proliferation and apoptosis, but enhanced birth anddeath rates appear from the 2-cell stage. Selective growth ofpreneoplastic and neoplastic cells may result from an over-response to mitogenic or survival signals. A shift between thebirth and death balance favors the growth of preneoplasticand tumoral lesions. This balance can be shifted by negativegrowth signals, such as withdrawal of growth factors or foodrestriction, and can induce apoptosis and regression ofpreneoplastic lesions as well as tumors in the liver.35

Florencia Que (Rochester, MN) discussed the role ofdysregulation of apoptosis as a mechanism of cholangiocar-cinogenesis. An increase in Bcl-2 expression is seen incholangiocarcinoma, which may result in their resistance toapoptosis.36 Reduction of Bcl-2 expression by antisenseoligonucleotides renders cholangiocarcinoma cells more likelyto undergo apoptosis. The role of T-lymphocyte–mediatedcell death in cholangiocarcinoma was discussed. Using acoculture model, FasL expression by cholangiocarcinoma celllines induced apoptosis of Jurkat cells. Cholangiocarcinomacells expressed low levels of Fas and were resistant toFas-mediated apoptosis. Flice (caspase-8) inhibitory protein(I-FLICE) can bind to the Fas-associated death domain(FADD) and prevent apoptosis. Antisense to I-FLICE reducedI-FLICE protein expression and increased apoptosis. How-ever, no alterations in components of the Fas-mediatedsignaling pathway, such as FLICE, FADD, or I-FLICE, werenoted in several cholangiocarcinoma cell lines. I-FLICE

HEPATOLOGY Vol. 30, No. 3, 1999 PATEL, STEER, AND GORES 813

antisense treatment could potentially provide an additionaltherapeutic adjunct in the treatment of this otherwise oftenfatal cancer.

Clifford Steer (Minneapolis, MN) discussed the role ofhepatocyte apoptosis in hepatic regeneration. The adult liverhas the ability to regenerate in response to a wide range ofstimuli, including toxins. Partial hepatectomy has been auseful model to study the cellular mechanisms of hepaticregeneration. After partial hepatectomy, a variety of molecu-lar events act in concert to trigger regeneration, and tissuereplication.37 Many genes implicated in the proliferativeprocess can mediate apoptosis. Many of these genes undergopost-transcriptional regulation by changes in messenger RNAstability. In addition, there is a significant uncoupling oftranscript and protein expression. Apoptosis may play animportant role in the process of hepatic remodeling duringregeneration and in fine tuning the regenerative response.38,39

INFLAMMATION AND FIBROGENESIS

Timothy Billiar (Pittsburgh, PA) discussed the role of nitricoxide (NO) in the liver. Although NO has been shown to beboth proapoptotic as well as antiapoptotic, NO acts as aninhibitor of apoptosis in hepatocytes. Stimulation of NO by acytokine mix or the addition of NO donors inhibits apoptosisin hepatocytes. The mechanism of cytoprotection may in-volve NO-mediated regulation of caspase activation.40 NOdonors decrease caspase-3 activity. Furthermore, NO syn-thase up-regulation by a cytokine mix inhibits caspase-3activity. Stimulation of caspase-3 activity by cytokines isinhibited by 2 distinct mechanisms: a direct action of NO oncaspase-3 as well as by the 38,58-cyclic guanosine monophos-phate (cGMP) second messenger system after NO activationof soluble guanylyl cyclase. Both mechanisms inhibit apopto-sis early. Protection via the cGMP-dependent mechanism mayinvolve the AKT kinase.

Giuliano Ramadori (Gottingen, Germany) discussed therole of the hepatic stellate cells during fibrogenesis in theliver. Liver fibrosis results from the excessive secretion ofmatrix proteins by hepatic stellate cells after injury andactivation of these cells. Apoptosis of activated hepaticstellate cells may limit matrix protein deposition. Data fromhis group have identified that apoptosis of activated hepaticstellate cells occurs by a Fas-mediated pathway.41 Althoughthe cytokines TNFa and TGFb have no effect on theproliferation of hepatic stellate cells, they appear to keepstellate cells activated by decreasing apoptosis, thus permit-ting them to continue matrix protein production.42

Mark Czaja (New York, NY) discussed the role of thecellular response to nonalcoholic liver toxins and cell injuryand cell death.43 Kupffer cell–derived cytokines such asTNFa and TGFb are important in liver injury. TNFa canresult in both apoptosis and necrosis, and is important intoxin-induced liver injury.44 However, TNFa is not toxic tonormal cells and can also act as a mitogen after partialhepatectomy. Nevertheless, TNFa is hepatotoxic in vivo, andsoluble TNF will neutralize TNFa and decrease carbontetrachloride–induced injury and mortality after a lethalinjection. Thus, TNFa may produce a protective factor, andthe relative lack of this will cause death. Both actinomycin Dand inhibition of NFkB activation sensitize to TNFa toxicityby a mechanism dependent on FADD but not on TRAF2.However, the timing of apoptosis differs between thesemodels with actinomycin D sensitizing hepatocytes to de-

layed caspase-independent apoptosis, whereas sensitizationby NFkB causes rapid caspase-dependent apoptosis.45

THERAPEUTIC OPPORTUNITIES

Karen Valentino (La Jolla, CA) discussed the potentialtherapeutic implications of characterizing apoptosis and itsrole in human liver disease. Apoptosis is a fundamentalbiological process, and multiple targets have been identified.Thus, modulation of apoptosis is a potentially fruitful area forthe development of new therapeutic agents. The process ofdrug development involves carefully choosing a moleculartarget, developing small molecule or biological therapies,choosing a disease and clinical indication, demonstratingsafety and efficacy, and getting a new agent to the marketexpeditiously. Inhibition of apoptosis in the liver may beuseful in diseases in which apoptosis is thought to contributeto organ damage such as alcohol-induced hepatitis, drug-induced liver disease, or during donor organ storage beforeliver transplantation. A number of small molecule caspaseinhibitors with demonstrated liver efficacy and bioavailabilityhave been identified and are currently being tested in animalmodels.

SUMMARY

Apoptosis is a fundamental biological process that plays arole in a wide range of pathophysiological processes anddiseases of the liver. The precise role of apoptosis in diseasepathogenesis remains to be defined. Nevertheless, enhancedunderstanding of the molecular mechanisms mediating apop-tosis and of the various factors contributing to the modula-tion of this process in disease states may ultimately result inthe development of novel therapeutic approaches.

Acknowledgment: The authors acknowledge the sponsor-ship of the AASLD and the invaluable assistance of Julie Dealfrom the AASLD in organizing this meeting.

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