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TRAIL Inhibits Tumor Growth but Is Nontoxic to Human Hepatocytes in Chimeric Mice

Departments of ¹ Laboratory Medicine and Pathology, ² Surgery, and ³ Medical Microbiology and Immunology, University of Alberta, Edmonton, Alberta, Canada

	ABSTRACT

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Tumor necrosis factor (TNF) family ligand TNF- and Fas ligand (FasL) can trigger apoptosis in solid tumors, but their clinical usage has been limited by hepatotoxicity. TNF-related apoptosis-inducing ligand (TRAIL) is a newly identified member of the TNF family, and its clinical application currently is under a similar debate. Here, we report a recombinant soluble form of human TRAIL (114 to 281 amino acids) that induces apoptosis in tumor cells but not human hepatocytes. We first isolated human hepatocytes from patients and showed that the human hepatocytes expressed Fas but no TRAIL death receptor DR4 and little DR5 on the cell surface. Antibody cross-linked FasL, but not TRAIL, triggered apoptosis of the human hepatocytes through cleavage of caspases. We then examined TRAIL hepatotoxicity in severe combined immunodeficient/Alb-uPA chimeric mice harboring human hepatocytes. Intravenous injection of FasL, but not TRAIL, caused apoptotic death of human hepatocytes within the chimeric liver, thus killing the mice. Finally, we showed that repeated intraperitoneal injections of TRAIL inhibited intraperitoneal and subcutaneous tumor growth without inducing apoptosis in human hepatocytes in these chimeric mice. The results indicate that the recombinant soluble human TRAIL has a profound apoptotic effect on tumor cells but is nontoxic to human hepatocytes in vitro and in vivo.

	Introduction

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Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is a recently identified tumor necrosis factor (TNF) family member (1 , 2) and currently is under development as a potential chemotherapeutic agent because it kills many types of tumor cells but spares normal cells in cultures and experimental animals (3 , 4) . However, a polyhistidine-tagged human TRAIL was reported to kill isolated human hepatocytes (5) , raising the same concern as noted with TNF- and Fas ligand (FasL; ref. 6 ). Contradictory to this was a recent study reporting that nontagged human TRAIL (114 to 281 amino acids) was not toxic to isolated human hepatocytes and was well tolerated in nonhuman primates (7) . In this study, we generated chimeric mice with human hepatocytes (8) and observed that recombinant nontagged form of human TRAIL (114 to 281 amino acids) inhibited tumor growth without inducing apoptosis of the human hepatocytes, whereas antibody cross-linked Flag-tagged human FasL triggered apoptotic death in the human hepatocytes and thus killed the chimeric mice.

	Materials and Methods

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Human Hepatocyte Isolation and Culture.
For human hepatocyte isolation, segments of human liver tissue were obtained from specimens following surgical removal. The University of Alberta Faculty of Medicine and Dentistry Research Ethics Board approved this study. Hepatocytes were isolated by collagenase-based perfusion using 0.38 mg/mL Liberase CI (Boehringer, Ingelheim, Germany) in perfusate (DuPont, Wilmington, DE; ref. 8 ). Cells were plated in Dulbecco’s modified Eagle’s medium (Invitrogen, Carlsbad, CA) with 10% fetal bovine serum and 1% penicillin-streptomycin (Invitrogen) or William’s E medium (supplemented with 5% fetal bovine serum, 16 ng/mL glucagon, 20 ng/mL dexamethasone, 200 µmol/L glutamine, 5 m-units/mL insulin, 20 ng/mL epidermal growth factor, and 1% antibiotics). Human melanoma cell line WM793 (Meenhard Herlyn; Wistar Institute, Philadelphia, PA) and human lung adenocarcinoma cell line H460 (American Type Culture Collection, Manassas, VA) were cultured in Roswell Park Memorial Institute 1640 (Invitrogen) supplemented with 10% fetal bovine serum and antibiotics.

Cell Death and Flow Cytometry Assay.
Isolated human hepatocytes and WM793 and H460 cell lines were seeded in 96-well plates at 3 x 10⁵ cells/well and treated with the recombinant soluble human TRAIL (114 to 281 amino acids, M_r 19,600; provided by PeproTech, Inc., Rocky Hill, NJ) or antibody cross-linked FasL (Alexis, San Diego, CA). Cell death was determined by the crystal violet assay, and the results were presented as the percentage cell death: 1 – (absorbance of cells treated/absorbance at 550 nm of cells untreated) x 100 (9) . Cellular apoptosis was examined under phase contrast light microscopy. Cell surface expression of DR4 and DR5 was analyzed by flow cytometry (9) . In brief, 0.1 µg/mL of phycoerythrin-conjugated antihuman DR4 and DR5 (mouse IgG1; eBioscience, San Diego, CA) or mouse IgG1 (a negative control; eBioscience) were added to the 10⁶ cells in 200 µL of immunofluorescence buffer, and 10,000 cells were analyzed using a Becton Dickinson FACScan (Mountain View, CA), and the data were processed using Cell Quest software (Becton Dickinson).

Generation and Treatment of Severe Combined Immunodeficient/Alb-uPA Chimeric Mice.
The chimeric mice were generated through crossing hemizygous Alb-uPA transgenic mice (Jackson Laboratory, Bar Harbor, ME) with homozygous severe combined immunodeficient (SCID)/bg mice (strain C.b-17/GbmsTac-SCID/bgN7; Taconic Farms, Germantown, NY), and selective backcrosses bred the SCID trait to homozygosity (8) . The litters of homozygous SCID mice carrying the homozygous Alb-uPA transgene were injected with freshly isolated human hepatocytes into inferior splenic pole. Successful engraftment of human hepatocytes was confirmed by serum levels of human -1 antitrypsin >100 µg/L at 8 weeks after transplantation. The animals were housed in the Veterinary Animal Facility, following Canadian Council on Animal Care Guidelines. Two-month-old chimeric mice were treated with TRAIL or FasL before or after inoculation with tumor cells, as described in Results. Necropsy was performed to examine the mice for tumors. During the necropsy, approximately one third of each chimeric liver was snap frozen in liquid N₂ and stored at –80°C for Western blot analysis. The remaining chimeric liver, other organs, and tumors were fixed in 10% formalin and embedded in paraffin. Sections were either stained with H&E or with mouse monoclonal antibody to human hepatocytes (OCH1E5, 1:25; Dako, Carpinteria, CA) and rabbit antibody to cleaved caspase-3 (Cell Signaling Technology, Beverly, MA) and visualized by Super Sensitive Immunodetection System (BioGenex, San Francisco, CA).

Human 1-Antitrypsin and Alanine Aminotransferase Assays.
For antihuman 1-antitrypsin (hAAT) assay (8) , 96-well plates (Corning Inc., Corning, NY) were coated overnight at 4° with goat anti-hAAT antibody (DiaSorin, Saluggia, Italy), and 50 µL of chimeric serum dilution were added and incubated for 2 hours. Standard curves were generated from serial dilutions of human reference serum (Calibrator 4; DiaSorin). The plates were incubated with horseradish peroxidase-linked goat anti-hAAT antibody (Pierce, Rockford IL) for 2 hours, washed, and added with substrate solution (3,3',5,5'-tetramethyl benzidine dihydrochloride; Sigma, St. Louis, MO). Horseradish peroxidase activity was measured spectrophotometrically, and hAAT levels were calculated with the Softmax software (Molecular Devices, Sunnyvale, CA). The alanine aminotransferase (ALT) in the chimeric serum was analyzed using the ALT Flex reagent cartridge (Dade Behring Inc., Newark, DE) and Dade Dimension chemistry analyzer (Dade Behring Inc.).

Gel Electrophoresis and Western Blot Analysis.
The recombinant soluble human TRAIL was treated with 100 mmol/L DTT (Sigma) and subjected to SDS-PAGE and stained with Silver Stain Plus kit (Bio-Rad, Hercules, CA). For Western blot analysis, the chimeric liver tissue was homogenized in radioimmunoprecipitation assay buffer [10 mmol/L Tris (pH 8), 140 mmol/L NaCl, 1% deoxycholate, 0.1% SDS, 1% Triton X-100, 0.025% NaN₃ with the addition of protease inhibitors], and cultured cells were lysed in lysis buffer (50 mmol/L Tris, 150 mmol/L NaCl, 2 mmol/L EDTA, 10% glycerol, 1% Triton X-100, 1% protease inhibitor mixture, and 1 mmol/L phenylmethylsulfonylfluoride). Fifty micrograms of total proteins from each lysate were separated through 15% SDS-PAGE and transferred to nitrocellulose membranes. The membranes were incubated overnight at 4°C with mouse monoclonal antihuman caspase-8 (1:1000; Medical & Biological Laboratories, Nagoya, Japan), DNA fragmentation factor 45 (DFF45; 1:1000; StressGen, Victoria, British Columbia, Canada), rabbit antihuman caspase-3 (1:1000; Cell Signaling Technology), DR4, DR5 (1:500; ProSci, Inc., Poway, CA), and extracellular signal-regulated kinase 1/2 (1:1000; StressGen) and developed by chemiluminescence (Amersham Biosciences, Piscataway, NJ).

	Results and Discussion

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TRAIL Is Nontoxic to Isolated Human Hepatocytes.
TRAIL primarily is expressed as a type II membrane protein with an extracellular COOH-terminal of 114 to 281 amino acids (1 , 2) of the receptor binding site (10) . The extracellular domain can be cleaved to yield a soluble and biologically active form of 114 to 281 amino acids (M_r 19,000 to 20,000; ref. 11 ). TRAIL carries a zinc ion bound by a free cysteine residue (Cys²³⁰), which is an essential moiety of TRAIL capacity to induce apoptosis (12) . The recently reported recombinant human TRAIL (114 to 281 amino acids) contained sufficient bound zinc ions and formed TRAIL homotrimers and killed tumor cells but was not toxic to isolated human and nonhuman primate hepatocytes (7) . In contrast, recombinant polyhistidine-tagged human TRAIL (114 to 281 amino acids) implicated in human hepatotoxicity (5) showed poor zinc ion coordination and the formation of free disulfide-linked dimers (7) . We previously have shown antitumor activity of a recombinant nontagged soluble human TRAIL of 114 to 281 amino acids (13) . This human TRAIL was homogenous on nonreducing and reducing SDS-PAGE (Fig. 1A) .

View larger version (74K): [in this window] [in a new window] [Download PPT slide]   Fig. 1. FasL, but not TRAIL, induces apoptosis in human hepatocytes in culture. A, silver-stained SDS-PAGE of recombinant human TRAIL under reduction with or without DTT. B, cell death assay. Human hepatocytes and WM793 cells were exposed to TRAIL (300 ng/mL) or antibody cross-linked Flag-FasL (50 ng/mL FasL plus 2 µg/mL antibody) for the times indicated. Cell death was determined by crystal violet assay. C, Western blot detection of caspase-8, caspase-3, and DFF45 cleavage products in WM793 cells and human hepatocytes treated either with TRAIL or FasL as described in B for the times indicated. Western blot antibodies are indicated to the left, and the proteins detected are indicated to the right. Extracellular signal-regulated kinase (ERK) 1/2 was used as a loading control. D, Phase contrast microscopy observation of cellular apoptosis marked by cell surface blebbing of the human hepatocytes exposed to FasL for 3 hours but not to medium or TRAIL (300 ng/mL) for 16 hours. E, Flow cytometry analysis of cell surface expression of DR4 and DR5 proteins in human hepatocytes, WM793, and H460 cells. The areas indicated by gray lines represent results from isotype control antibodies, and the numbers indicate percent of positive cells. F, Western blot analysis of DR4 and DR5 expression in human hepatocytes, WM793, and H460 cells. Three isoforms of DR5 proteins were detected on Western blot analysis.

Flag-tagged FasL cross-linked with anti-Flag antibody was reported to kill human hepatocytes in culture (18) . We observed caspase-8, caspase-3, and DFF45 cleavage (Fig. 1C) , significant cell death (Fig. 1B) , and cellular apoptosis (Fig. 1D) in the human hepatocytes exposed to the antibody cross-linked Flag-FasL (50 ng/mL FasL mixed with 2 µg/mL antibody). The results indicate that caspase-8-initiated apoptotic machinery exists in human hepatocytes and can be activated by FasL but not by TRAIL.

DR4 and DR5 transcripts were reported in human liver tissue and isolated human hepatocytes (5) . However, studies of DR4 and DR5 proteins have produced controversial results; one showed DR4 and DR5 on human hepatocyte surface (19) , whereas others reported no DR5 (20) . We compared DR4 and DR5 expression between isolated human hepatocytes and tumor cells. DR4 protein was not detected either by flow cytometry (Fig. 1E) or on Western blot analysis (Fig. 1F) , whereas flow cytometry showed a little DR5 expression on human hepatocyte surface (Fig. 1E) . DR5 proteins exist in three isoforms: one p60 intracellular form and two membrane p43 and p49 forms (15) , and Western blot analysis detected a strong p60 band but weak p43 and p49 bands in human hepatocytes; the results confirmed that the hepatocytes express low levels of the membrane forms of DR5 proteins (Fig. 1F) . In contrast, Fas was highly expressed on human hepatocytes (Fig. 1E) , and DR4 and/or DR5 was highly expressed in human tumor lines WM793 and H460 (Fig. 1E and F) . These studies indicate that the lack of DR4 and low levels of DR5 expression in human hepatocytes may contribute to the cell resistance to TRAIL.

FasL but not TRAIL Induces Apoptosis in Human Hepatocytes in Chimeric Mice.
Next we tested TRAIL and FasL hepatotoxicity in SCID/Alb-uPA chimeric mice whose livers are largely composed of human hepatocytes (8) . The chimeric mice were generated through crossing hemizygous Alb-uPA transgenic mice with homozygous SCID/bg mice as described in the Materials and Methods. Two-month-old chimeric mice were injected intravenously with 500 µg of TRAIL in 100 µL normal saline, 30 µg of the antibody cross-linked Flag-FasL (30 µg of Flag-FasL mixed with 12 mg antibody in 100 µL normal saline), or 100 µL normal saline. All of the chimeric mice that received cross-linked Flag-FasL injection succumbed within 90 minutes, whereas the chimeric mice injected with 500 µg TRAIL remained alive up to 4 and 16 hours when the mice were sacrificed for necropsy. Serum tests showed no significant difference in hAAT and ALT concentrations before and after TRAIL injection in chimeric mice (Fig. 2A) .

View larger version (72K): [in this window] [in a new window] [Download PPT slide]   Fig. 2. TRAIL does not trigger apoptosis in human hepatocytes in the chimeric mice. A, serum tests for hAAT and ALT in the chimeric mice at 0 and 16 hours after intravenous injection of 500 µg TRAIL. B, H&E (top), antihuman hepatocyte (middle), and anticleaved caspase-3 antibody-stained sections (bottom) of the livers obtained from the chimeric mice injected intravenously with saline (left), 500 µg TRAIL (middle), or 30 µg FasL (right). The nodules of human hepatocytes (H) and surrounding mouse hepatocytes (M) were observed in the chimeric livers. C, Western blot detection of cleavage products of caspase-8 (Casp-8), caspase-3 (Casp-3), and DFF45 in the homogenized liver tissues from two chimeric mice intravenously injected with 500 µg TRAIL for 16 hours and two chimeric mice intravenously injected with 30 µg FasL for 1.5 hours. Extracellular signal-regulated kinase (ERK) 1/2 was used as a loading control.

TRAIL Inhibits Tumor Growth but Causes No Injury to Human Hepatocytes in Chimeric Mice.
To show TRAIL selective antitumor activity in the chimeric mice, we injected 2-month-old chimeric mice intraperitoneally with 8 x 10⁶ WM793 cells on the left side. On the next day, three chimeric mice received intraperitoneal injections of 100 µg TRAIL on the right side with 100 µL normal saline in the control group of three mice. Animals were treated twice daily for 10 days. All of the mice treated with TRAIL remained healthy, whereas in the control group, one mouse died on the 39th day, and the remaining two mice appeared sick. All of the remaining mice were sacrificed on the 40th day for necropsy. On histologic examination, the hepatocytes within nodules appeared normal and stained positively for a human hepatocyte marker but negatively for cleaved caspase-3 antibody in TRAIL- and saline-treated mice (Fig. 3A) . Serum tests showed no significant difference in the hAAT concentrations before and after TRAIL injection in the chimeric mice (Fig. 3B) . Necropsy showed large tumor masses limited to peritoneal space in the saline-treated control group, with an average tumor weight of 1.6 g (Fig. 3C) . Of three TRAIL-treated mice, however, one remained free of tumor, whereas the other two mice harbored small tumor masses within the peritoneal space. The mean tumor weight of the TRAIL-treated mice was 0.25 g (Fig. 3C) .

View larger version (107K): [in this window] [in a new window] [Download PPT slide]   Fig. 3. Intraperitoneal injection of TRAIL inhibits tumor growth in chimeric mice. A, H&E, antihuman hepatocyte, and anticleaved caspase-3 antibody-stained sections of the livers from chimeric mice that received intraperitoneal injections twice daily of 100 µg of TRAIL or 100 µL of normal saline for 10 days, followed by sacrifice on the 40th day. The tumors (T) invaded chimeric livers in a control mouse. B, serum test for hAAT concentrations in the chimeric mice on the day of tumor inoculation (week 0) and the following weeks up to the 4th week after tumor inoculation. C, mean intraperitoneal W793 tumor weights in the chimeric mice that received intraperitoneal injection of TRAIL or normal saline as described in A. All of the intraperitoneal tumor masses were grouped and weighed in each mouse for calculation of mean tumor weight. The data were compared using Student’s t test (*P < 0.01).

View larger version (43K): [in this window] [in a new window] [Download PPT slide]   Fig. 4. Intraperitoneal injection of TRAIL inhibits subcutaneous tumor growth without triggering apoptosis of the chimeric livers. A, Western blot detection of caspase-8, caspase-3, and DFF45 cleavage in the liver tissues from chimeric mice injected intraperitoneally with 100 µg normal saline or 100 µg TRAIL twice per day for 10 days. The mice were sacrificed, and each of four liver lysates in TRAIL-treated mice and four liver lysates in saline-treated mice were subjected to Western blot analysis. H460 cells were treated with medium or 300 ng/mL TRAIL for 6 hours, and the cell lysates were included in the Western blot analysis. B, cell viability assay for cell death in H460 cells after exposure to 300 ng/mL TRAIL for the times indicated. C, subcutaneous tumor sizes measured from the day of tumor inoculation (day 0) and followed for 10 days in the chimeric mice injected intraperitoneally with normal saline or TRAIL as described in A. Data are mean ± SE (n = 4). D, serum hAAT and ALT concentrations on the day of tumor inoculation (day 0) and day 10 in the chimeric mice injected with normal saline or TRAIL as described in A. Data are mean ± SE (n = 4).

	ACKNOWLEDGMENTS

 
We thank Ms. Ida Hoffman for her donation in support of this project.

	FOOTNOTES

 
Grant support: Ida Hoffman Cancer Research Fund and CIHR grants to C. Hao, D. L. J. Tyrrell, and N. M. Kneteman. C. Hao is the Alberta Heritage Foundation for Medical Research Clinical Investigator, and N. M. Kneteman is the Wyeth-CIHR Chair of Transplantation.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Requests for reprints: Chunhai Hao, Department of Pathology and Laboratory Medicine, Oncology, and Hematology, Emory University School Medicine, 1365-C Clifton Road, Atlanta, GA 30322, or Norman M. Kneteman, Department of Surgery, University of Alberta, Edmonton, Alberta, Canada T6G 2B7. E-mail: chao{at}emory.edu or nkneteman{at}cha.ab.ca

Received 7/20/04. Revised 9/13/04. Accepted 10/10/04.

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