Apo2L/TRAIL Avi Ashkenazi * Department of Molecular Oncology, Genentech Inc., 1 DNA Way, South San Francisco, CA 94080-4918, USA *corresponding author tel: 650-225-1853, fax: 650-225-6443, e-mail: [email protected] DOI: 10.1006/rwcy.2000.05012. SUMMARY and lymphotoxin (cid:11) indicate that this region contains aseriesof10(cid:12) strandsthatformthebasicfoldofthe ECD. TNF-related ligands bind to specific receptors Apo2L/TRAILisamemberoftheTNFligandfamily that belong to the TNF receptor (TNFR) gene that is closely related to FasL. Many tissues express superfamily (Smith et al., 1994). Most TNFR family the Apo2L/TRAIL mRNA, and T cells upregulate members are type 1 transmembrane proteins; their Apo2L/TRAIL expression upon T cell receptor stim- common structural feature is the presence of 2–6 ulation. The distinct biologic function of Apo2L/ cysteine-rich domains in the extracellular portion. TRAIL has yet to be defined. There is evidence that The crystal structure of the complex between Apo2L/TRAILmight play arolein T cell activation- lymphotoxin-(cid:11) and the ECD of TNFRI reveals that induced cell death (AICD), in target-killing by cyto- three receptor molecules bind to the homotrimeric toxic lymphocytes and macrophages, and perhaps in ligand, each docking in the groove between two the immune-privilege of certain tumors. Apo2L/ ligand subunits (Banner et al., 1993). TNFR family TRAIL bindsto a complex system of receptors: DR4 proteins fall into two subgroups on the basis of and DR5 have death domains that signal apoptosis, intracellular sequence: receptors that contain death whereas DcR1, DcR2, and OPG lack functional domains (a 70 amino acid homophilic protein deathdomainsandcaninhibitapoptosisinductionby interaction motif), and receptors that lack death the ligand. Apo2L/TRAIL potently activates apop- domains. The receptors that have death domains, tosisin awide variety oftumor cell lines, butitis not dubbed‘deathreceptors’,triggercellularresponsesby cytotoxic towards most normal cell types studied so engaging specific adapter molecules such as TRADD far. Experiments in mouse models of cancer suggest (TNFR-associated death domain) or FADD (Fas- that Apo2L/TRAIL might be useful as an anticancer associated death domain) through homophilic death agent that kills tumor cells without damaging normal domain interactions (Ashkenazi and Dixit, 1998). tissues. TNFR family members that lack death domains signal by engaging specific adaptor molecules called TRAFs (TNFR-associated factors). BACKGROUND The best characterized death receptor and ligand systems (Simonet et al., 1997)are thoseofFas/Apo1/ The members of the tumor necrosis factor (TNF) CD95 with Fas ligand (FasL), and the p55 TNF gene superfamily modulate diverse biological func- receptor (TNFRI) with TNF. There is a growing tions, including cell differentiation, proliferation, and interest in a third, more recently discovered apop- apoptosis (Gruss and Dower, 1995). Most TNF tosis-inducing ligand, called Apo2 ligand (Apo2L) familyligandsareexpressedastype2transmembrane or TNF-related apoptosis-inducing ligand, which is proteins, some of which are processed by specific structurally related to FasL and TNF. Much of the proteases into soluble homotrimeric cytokines. The interest stems from the existence of an unusually sequence homology between TNF and its known complex system of death and decoy receptors that relatives occurs primarily in the C-terminal extra- interact with this ligand (Ashkenazi and Dixit, 1998, cellulardomain(ECD).ThecrystalstructuresofTNF 1999). 504 Avi Ashkenazi Discovery Third, Chinese hamster ovary cells transfected with an expression plasmid encoding full-length Apo2L/ TRAIL release a soluble form of the ligand that Investigators (Wiley et al., 1995; Pitti et al., 1996) migrates on SDS-PAGE gels under reducing condi- identifiedexpressedsequencetags(ESTs)thatshowed tions as a 24kDa protein band (Figure 2). homology to the TNF ligand family. On the basis of the ESTs, a full-length cDNA was isolated that encoded a previously unknown member of the TNF Main activities and family. The novel protein, which showed highest homologytoFas/Apo1ligand,wasdesignatedApo2L pathophysiological roles (Pitti et al., 1996) or TRAIL (Wiley et al., 1995). ThebiologicfunctionsofApo2L/TRAILarenotfully understood; however, the ligand’s ability to trigger Alternative names apoptosis in a variety of transformed cell lines sug- gests that it may be a physiologic modulator of Alternative names are TL2 (Tan et al., 1997) and apoptosis. Apo2L/TRAIL also is capable of activat- TNFSF10. ing the proinflammatory transcription factor NF(cid:20)B; however, in comparison to TNF, it is a very weak NF(cid:20)B activator (Sheridan et al., 1997; Ashkenazi Structure et al., 1999). Several lines of evidence suggest that Apo2L/TRAIL may play a role in the AICD of peri- The human Apo2L/TRAIL cDNA predicts a poly- pheralTcells,aprocessinwhichFasLplaysacentral peptide of 281 amino acids. Hydropathy analysis role (Nagata, 1997). suggests that Apo2L/TRAIL has the topology of a First, a subset of peripheral T cells acquires sen- type 2 transmembrane protein, with a 15–17 amino sitivity to Apo2L/TRAIL-induced apoptosis after a acid-longN-terminalcytoplasmicregion,followedby 3-day stimulation by IL-2 (Marsters et al., 1996) or a a transmembrane region of 21–23 residues, and an 5-day stimulation by IL-2 and PHA (Snell et al., extracellular C-terminal region. The protein has one 1997). potential N-linked glycosylationsite (Asn109).Align- Second, Apo2L/TRAIL mRNA expression is in- ment of the extracellular region with that of other duced upon the stimulation of CD4+ and CD8+ family members suggests that the (cid:12) strand region peripheralbloodTcellswithPHAorwiththeprotein starts around amino acid 120 (Figure 1). FACS kinase C activator phorbol 12-myristate 13-acetate staining of cells, transfected with the full-length (PMA) and the calcium ionophore ionomycin Apo2L/TRAIL cDNA tagged by a C-terminal Myc (Jeramiasetal.,1988;Screatonetal.,1997;Martinez- epitope, with anti-Myc antibody indicates that the Lorenzo et al., 1998). molecule is expressed at the cell surface as a type 2 Third, neutralizing anti-Apo2L/TRAIL antibody protein(Pittietal.,1996).FACSstainingofcellsthat reduces the cytotoxicity of the supernatants of PHA- express endogenous Apo2L/TRAIL with polyclonal stimulated peripheral blood mononuclear cells or antiligand antibodies also indicates cell surface ex- JurkatTcellstowardsunstimulatedTcells(Martinez- pression (Mariani and Krammer, 1998a, 1998b), as Lorenzo et al., 1998). In addition, T cells from HIV- does immunohistochemical staining of tissue sections infectedpatientsshowincreasedsensitivitytoApo2L/ with monoclonal anti-Apo2L/TRAIL (Ashkenazi TRAIL,whichsuggestsapotentialrolefortheligand et al., 1999). The full-length Apo2L/TRAIL protein in the killing of virus-infected cells (Jeramias et al., migrates on SDS-PAGE gels as a 32–33kDa protein 1998). (Mariani and Krammer, 1998a, 1998b). There is evidence also for the involvement of There is evidence that cells can release Apo2L/ Apo2L/TRAILintarget-killingbyCD4+cytotoxicT TRAIL in soluble form, apparently through proteo- lymphocytes (CTLs) and by natural killer (NK) cells, lytic processing of the ECD. First, supernatants from bothofwhichexpressApo2L/TRAILmRNA(Thomas phytohemagglutinin (PHA)-stimulated human Jurkat and Hersey, 1998; Zamani et al, 1998). Further T cells contain cytotoxic activity that can be blocked evidence suggests that Apo2L/TRAIL is involved in partially by a neutralizing monoclonal anti-Apo2L/ tumor cell killing by macrophages (Griffith et al., TRAIL antibody (Martinez-Lorenzo et al., 1998). 1999).Humanastrocyticbraintumors,butnotnormal Second, the surface expression of Apo2L/TRAIL on glia, express Apo2L/TRAIL mRNA (Rieger et al., Jurkat cells increases upon treatment with inhibitors 1998),asdoseveralcelllinesderivedfromothertypes ofcysteineproteases(Mariani andKrammer,1998a). of cancer (Ashkenazi et al., 1999); this expression Apo2L/TRAIL 505 Figure 1 Alignment of the putative extracellular (cid:12) strand region of Apo2L/TRAIL with that of other TNF family members. The (cid:12) strand regions are marked above with lines and letters. suggestsapotentialinvolvementofApo2L/TRAILin transcription of the Apo2L/TRAIL gene (Screaton the immune-privilege of tumors. et al., 1997; Martinez-Lorenzo et al., 1998). The apparent involvement of Apo2L/TRAIL in T cell AICD, in target-killing by cytotoxic lympho- Accession numbers cytes,andintheimmune-privilegeoftumorssuggests that this ligand resembles FasL not only in sequence, Human mRNA: HSU37518, HSU57059 but also in certain functions. Recent work suggests Mouse mRNA: MMU37522 that Apo2L/TRAIL, but not FasL or TNF, plays a key role in the killing of dendritic cells by auto- Chromosome location logousCD4+CTLs(Wanget al.,1999),suggestinga potentially unique role for Apo2L/TRAIL in mod- ulating the immune system. Human chromosome 3q26 (Wiley et al., 1995). Relevant linkages GENE AND GENE REGULATION Apo2L/TRAIL receptors PublishedinformationonthestructureoftheApo2L/ Osteoprotegerin TRAIL gene is not yet available. The PHA- FasL stimulation of peripheral blood T cells induces TNF 506 Avi Ashkenazi Figure2 Westernblotanalysisofsol- PROTEIN uble Apo2L/TRAIL shed by Chinese hamster ovary cells after transfection Accession numbers with cDNA encoding the full-length protein. Cell supernatants were col- lected 24 hours after transfection, Human protein: GEN13977 resolvedbySDS-PAGE,andanalyzed Human protein: P_W19777 byimmunoblotwithmonoclonalanti- Mouse protein: GEN13978 Apo2L/TRAIL antibody 5C2. Sequence See Figure 3. 60 Description of protein Apo2L/TRAIL is a 281 amino acid, type 2 trans- 42 membraneprotein,withacalculatedmolecularweight of 32.5kDa and an isoelectric point of 7.63. The full- lengthproteinmigratesasa32–33kDabandonSDS- PAGE gels (Mariani and Krammer, 1998a, 1998b); 30 a shed form of the protein migrates as a band of approximately 24kDa (see Figure 2). Several recom- binant soluble versions of the protein have been 22 generated. A polyhistidine-tagged soluble form of Apo2L/TRAIL (amino acids 114–281) forms homo- trimers and is biologically active (Pitti et al., 1996; 17 Ashkenazi et al., 1999). In contrast, a Flag-epitope- tagged soluble form of the protein (amino acids 95– 281) is poorly active, and requires oligomerization by anti-Flag antibody for potent biologic activity (Wiley et al., 1995; Walczak et al., 1999). A fusion protein, containingthesameApo2L/TRAILaminoacids(95– 281) fused N-terminally to a modified leucine-zipper Cells and tissues that express that promotes trimerization, forms trimers and is the gene biologically active (Walczak et al., 1999). Northern blot analyses with probes that are based Important homologies upon the Apo2L/TRAIL cDNA reveal a single mRNA transcript of (cid:24)2kb (Wiley et al., 1995; Pitti The C-terminal portion of the Apo2L/TRAIL ECD et al., 1996). The Apo2L/TRAIL message is present shows 28% identity to the FasL ECD, 23% identity constitutively in many tissues, including fetal lung, to the ECDs of TNF and lymphotoxin (cid:11), and 22% liver, and kidney, and adult spleen, prostate, thymus, identity to the ECD of lymphotoxin (cid:12) (see Figure 1). ovary, small intestine, colon, PBLs, heart, placenta, The loop that connects the first and second putative lung, skeletal muscle, and kidney. In situ hybridiza- (cid:12) strands is substantially longer in the ECD of tionanalysisindicatesApo2L/TRAILmRNAexpres- Apo2L/TRAIL ECD than in other family members. sion in human astrocytic brain tumors and in glial The murine Apo2L/TRAIL protein is 291 amino cancer cell lines (Rieger et al., 1998). In addition, the acids long, and it shows 65% identity to the human stimulation of CD4+ and CD8+ peripheral blood protein (Wiley et al., 1995). T cells with PHA or with PMA and ionomycin induces expression of the Apo2L/TRAIL mRNA Posttranslational modifications (Screatonetal.,1997;Jeramiasetal.,1998;Martinez- Lorenzo et al., 1998). Stimulation of macrophages with IFN(cid:13) induces cell surface expression of the Human and murine Apo2L/TRAIL each have one Apo2L/TRAIL protein (Griffith et al., 1999). potential N-linked glycosylation site (Asn109 and Apo2L/TRAIL 507 Figure 3 Amino acid sequence of Apo2L/TRAIL. Sequence MAMMEVQGGPSLGQTCVLIVIFTVLLQSLCVAVTYVYFTNELKQMQDKYSKSGIA CFLKEDDSYWDPNDEESMNSPCWQVKWQLRQLVRKMILRTSEETISTVQEKQQ NISPLVRERGPQRVAAHITGTRGRSNTLSSPNSKNEKALGRKINSWESSRSGHSFL SNLHLRNGELVIHEKGFYYIYSQTYFRFQEEIKENTKNDKQMVQYIYKYTSYPDPIL LMKSARNSCWSKDAEYGLYSIYQCGIFELKENDRIFVSVTNEHLIDMDHEASFFG AFLVG Asn52 respectively). Apo2L/TRAIL is made as a RECEPTOR UTILIZATION type 2 transmembrane protein; a soluble form that contains the ECD can be released proteolytically Apo2L/TRAIL interacts with four cell surface by cells that express the full-length protein (see receptors that form a distinct subgroup within the Description of protein). TNFR gene superfamily (for a review, see Ashkenazi and Dixit, 1998, 1999). Death receptor 4 (DR4) (Pan et al., 1997) and DR5 (Pan and Dixit, 1997; Sheridan et al., 1997) (also called TRICK2, TRAIL-R2 or CELLULAR SOURCES AND KILLER)(Screatonetal.,1997;Walczaketal.,1997; TISSUE EXPRESSION Wuetal.,1997)havecytoplasmicdeathdomainsand signal apoptosis. Decoy receptor 1 (DcR1) (Sheridan Cellular sources that produce et al., 1997) (also called TRID, TRAIL-R3, or LIT) (Degli-Esposti et al., 1997a; Mongkolsapaya et al., 1998; Pan et al., 1998a), is a phospholipid-anchored Apo2L/TRAILappearstobeexpressedconstitutively cell surface protein that lacks a cytoplasmic tail. on the surface of mouse and human tumor cells of DcR2 (Marsters et al., 1997) (also called TRAIL-R4 TandBorigin,includingmouseEL4TcellsandA20 orTRUNDD)(Degli-Espostietal.,1997b;Panetal., B cells, and human Jurkat T cells and Bjab B cells 1998b) has a substantially truncated death domain (Mariani and Krammer, 1998a). Freshly isolated that does not signal apoptosis induction. Upon over- murine splenocytes, concanavalin A/IL-2-activated expression, DcR1 or DcR2 inhibits apoptosis induc- T cells, and lipopolysaccharide-activated B cells were tionbyApo2L/TRAIL.Apo2L/TRAILbindsalsoto analyzedbysurfacestainingwithanti-Apo2L/TRAIL a secreted, soluble member of the TNF receptor antibody (Mariani and Krammer, 1998b). Activated, family, osteoprotegerin (OPG) (Simonet et al., 1997; but not resting, CD3+ cells expressed Apo2L/ Emery et al., 1998). The binding affinity of Apo2L/ TRAIL. Freshly isolated B220+ cells displayed sur- TRAIL for OPG is 3–5-fold lower than the affinity face Apo2L/TRAIL and CD95L that were retained for DR5 or DcR1 (Emery et al., 1998). OPG is not following activation. In CD3+ TCR(cid:11)(cid:12) cells with the closely related to the other four Apo2L/TRAIL CD4+orCD8+phenotype,restimulationwithPMA receptors, and it binds also to another TNF family and ionomycin or an agonistic anti-CD3 monoclonal member, OPGL (also called RANKL or TRANCE). antibody induced a significant upregulation of sur- face Apo2L/TRAIL and CD95L. Apo2L/TRAIL upregulation was inhibited by cyclohexamide and by cyclosporin A. IN VITRO ACTIVITIES In vitro findings Eliciting and inhibitory stimuli, including exogenous and The main in vitro biologic activity of Apo2L/TRAIL so far reported is the induction of caspase-dependent endogenous modulators apoptosis in tumor cell lines from various tissue origins (Wiley et al., 1995; Pitti et al., 1996; Mariani See Gene and gene regulation. et al., 1997; Snell et al., 1997; Jeramias et al., 1998; 508 Avi Ashkenazi Thomas and Hersey, 1998). There is also evidence and Dixit, 1998; Emery et al., 1998). Intracellularly, that Apo2L/TRAIL can induce apoptosis in a subset the c-FLIP molecule, which blocks apoptosis signal- of activated T cells (Marsters et al., 1996; Snell et al., ing by FasL and TNF, is also capable of inhibiting 1997; Martinez-Lorenzo et al., 1998), and in a subset apoptosisinductionbyApo2L/TRAIL(Thomeetal., ofTcellsfromHIV-infectedchildren(Jeramiasetal., 1997). Certain chemotherapeutic drugs, including 1998). In addition, Apo2L/TRAIL is capable of inhibitors of transcription or translation and DNA- activating NF(cid:20)B; the magnitude of this activation is damaging agents, sensitize cancer cell lines to the much less than that of activation by TNF, and it cytotoxic activity of Apo2L/TRAIL (Keane et al., requires a substantially higher concentration of 1999; Mori et al., 1999). In addition, IL-1 protects Apo2L/TRAIL relative to TNF (Sheridan et al., transformed keratinocyte cell lines against apoptosis- 1997; Ashkenazi et al., 1999). Apo2L/TRAIL is also induction by Apo2L/TRAIL (Kothny-Wilkes et al., capable of inducing the activation of c-Jun 1998). N-terminal kinase (JNK): in HeLa cells, but not in Kym-1 cells, the activation can be blocked by the caspase inhibitor zVAD-fmk, which suggests the Bioassays used involvement of caspase-dependent and caspase- independent mechanisms in this effect (Muhlenbeck Apo2L/TRAIL bioactivity can be conveniently et al., 1998). assayed in standard cytotoxicity or cell viability Morethanhalfofthe60tumorcelllinesinapanel assays. Alternatively, Apo2L/TRAIL bioactivity can developed by the US National Cancer Institute, be assayed by measuring apoptosis induction, on the which contains cell lines derived from leukemia, basis of parameters such as DNA fragmentation, or melanoma, non-small cell lung cancer, colon cancer, phosphatidylserine-flip, as measured by FACS stain- central nervous system cancer, ovarian cancer, renal ing with annexin V (Pitti et al., 1996). Apoptosis can cancer, prostate cancer, and breast cancer, showed also be assayed on the basis of caspase activation, sensitivitytoApo2L/TRAIL(Ashkenazietal.,1999). measured by the cleavage of synthetic caspase sub- In contrast, several normal cell types were resistant strates, or by the cleavage of cellular caspase sub- in culture to Apo2L/TRAIL (Ashkenazi et al., 1999; strates such as poly-ADP ribose polymerase (PARP) Walczak et al., 1999), with the exception of fetal or caspase 3. astrocytes (Walczak et al., 1999). Neutralizing anti-Apo2L/TRAIL antibodies atten- uate the cytotoxic activity of supernatants from activated T cells towards unstimulated T cells IN VIVO BIOLOGICAL (Martinez-Lorenzo et al., 1998), which implicates ACTIVITIES OF LIGANDS IN Apo2L/TRAIL in the AICD of T lymphocytes. This ANIMAL MODELS notion gains further support from the observation that blocking anti-Apo2L/TRAIL antibody inhibits the AICD of T cells from HIV-infected patients Species differences (Katsikis et al., 1997). Soluble Apo2L/TRAIL recep- tors in the form of an Fc-fusion protein (immuno- Studies on a limited number of cell lines indicate that adhesin) inhibit the killing of target tumor cells by human Apo2L/TRAIL is cytotoxic towards murine macrophages, implicating the ligand in mediating the celllinessimilarlytomurineApo2L/TRAIL,andthat cytotoxic activity of macrophages (Griffith et al., murine Apo2L/TRAIL is cytotoxic towards human 1999). cell lines similarly to human Apo2L/TRAIL (Wiley etal.,1995;Marianietal.,1997;Walczaketal.,1999). Regulatory molecules: Inhibitors Endogenous inhibitors and and enhancers enhancers Extracellular,aswellasintracellular,mechanismsmay modulate apoptosis induction by Apo2L/TRAIL. The mRNA for Apo2L/TRAIL is expressed consti- Extracellularly, three decoy receptors that belong to tutively in many tissues, as are the mRNAs for DR4 the TNFR gene superfamily can bind to Apo2L/ and DR5; hence, there may be endogenous mecha- TRAIL and inhibit the ligand’s apoptosis-inducing nisms that control cellular sensitivity to the cytotoxic activity:theseareDcR1,DcR2,andOPG(Ashkenazi action of Apo2L/TRAIL. One such mechanism may Apo2L/TRAIL 509 involve the expression of the decoy receptors DcR1 injections of the protein in nonhuman primates and DcR2 in normal tissues. Because DcR1 and did not cause detectable toxicity to the tissues and DcR2areexpressedinfrequentlyandgenerallyatlow organs examined. Apo2L/TRAIL exerted cytostatic levels in cancer cell lines, it is unlikely that they or cytotoxic effects in vitro on 32 of 39 cell lines regulate the sensitivity to the ligand in transformed from colon cancer, lung cancer, breast cancer, kidney cells. Whether the decoys are important inhibitors of cancer, brain cancer, and skin cancer. The treatment Apo2L/TRAIL cytotoxicity in normal tissues that of athymic mice with Apo2L/TRAIL shortly after express DcR1 or DcR2 more abundantly remains to tumor xenograft injection markedly reduced tumor be investigated. incidence. The Apo2L/TRAIL treatment of mice Consistent with the latter hypothesis is the bearing solid tumors induced tumor cell apoptosis observation that T cell activation downregulates and tumor regression, suppressed tumor progression, DcR1 expression, perhaps as a mechanism for and improved survival. Apo2L/TRAIL cooperated regulating the sensitivity to Apo2L/TRAIL during synergistically with the chemotherapeutic drugs AICD (Mongkolsapaya et al., 1998). In addition, the 5-fluorouracil and CPT-11 at causing substantial treatment of human umbilical cord endothelial cells tumor regression or complete tumor ablation. These with phosphatidylinositol phospholipase C sensitizes studies suggest that Apo2L/TRAIL may turn out to the cells to apoptosis induction by Apo2L/TRAIL, be useful as an anticancer agent that induces apop- consistent with the removal of GPI-anchored DcR1 tosis in tumors while sparing normal tissues. from the cell surface (Sheridan et al., 1997). Another mechanism to regulate the sensitivity of cells to Pharmacokinetics Apo2L/TRAIL may involve the expression of anti- apoptoticfactorssuchasc-FLIP(Thomeetal.,1997) or BclXL (Mori et al., 1999). It is also possible that The leucine-zipper Apo2L/TRAIL fusion protein thereisregulationoftheApo2L/TRAILproteinitself exhibited a distribution half-life of 1.3 hours and an at the level of translation, cellular transport, and/or elimination half-life of 4.8 hours after intravenous proteolytic processing. Further, in certain tumor cell injection in mice (Walczak et al., 1999). The native lines, activation of the p53 tumor suppressor up- sequence form of recombinant soluble Apo2L/ regulates the expression of DR5 mRNA, perhaps TRAIL exhibited a pharmacologic half-life of 0.5 increasing sensitivity to Apo2L/TRAIL (Wu et al., hours in cynomolgus monkeys (Ashkenazi et al., 1997). 1999). Toxicity IN THERAPY Preclinical – How does it affect The systemic injection of up to 0.5mg of the human leucine-zipper Apo2L/TRAIL fusion protein and disease models in animals? 1mg of the corresponding murine fusion protein in mice had no detectable adverse effects on viability, Apo2L/TRAIL has been studied in mouse-based tissue integrity, or blood cell count (Walczak et al., xenograft models of human cancer (Ashkenazi et al., 1999). Intravenous injection of the native sequence 1999; Walczak et al., 1999). In nude mice, systemic form ofrecombinant soluble Apo2L/TRAILat up to treatment with a leucine-zipper-fused Apo2L/TRAIL 10mg/kg/day for 7 days in cynomolgus monkeys proteinbegunshortlyafterthesubcutaneousinjection showednoevidenceoftoxicityusingacomprehensive of tumor cells substantially reduced tumor incidence set of clinical tests. Apo2L/TRAIL did not affect (Walczak et al., 1999). Treatment with the fusion body weight, body temperature, blood pressure or proteinaftersolidtumorshadbeguntoformreduced heart rate. There were no Apo2L/TRAIL-associated tumor volume and in some cases eliminated the changes in leukocyte count, liver enzyme activity, tumors if started within a few days, delaying tumor coagulation profiles, serum chemistry or urine anal- progression if started later. Histologic examination ysis. Histologic evaluation ofliver, lung, heart, brain, indicated that the systemic injection of the fusion kidney,ovary,testis,spleen,bonemarrow,andlymph protein in tumor-bearing mice induced apoptosis in nodes did not reveal Apo2L/TRAIL-related adverse tumor epithelial cells (Walczak et al., 1999). changes. In particular, there was no morphologic In another study, a native sequence version of evidence of hepatotoxicity or lymphotoxicity, and recombinant solubleApo2L/TRAIL wasinvestigated bone marrow sections displayed normal cellularity (Ashkenazi et al., 1999). Repeated intravenous andprogressivematurationofalllineages(Ashkenazi 510 Avi Ashkenazi et al., 1999). These data suggest that, unlike its Mariani, S., and Krammer, P. (1998a). 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