Death Induction by Recombinant Native TRAIL and Its Prevention by a Caspase 9 Inhibitor in Primary Human Esophageal Epithelial Cells
2004; Elsevier BV; Volume: 279; Issue: 38 Linguagem: Inglês
10.1074/jbc.m404541200
ISSN1083-351X
AutoresSeok‐Hyun Kim, Kunhong Kim, Jae Kwagh, David T. Dicker, Meenhard Herlyn, Anil K. Rustgi, Youhai Chen, Wafik S. El‐Deiry,
Tópico(s)NF-κB Signaling Pathways
ResumoThe cytotoxic death ligand TRAIL (tumor necrosis factor-related apoptosis-inducing ligand) is a tumor-specific agent under development as a novel anticancer therapeutic agent. However, some reports have demonstrated toxicity of certain TRAIL preparations toward human hepatocytes and keratinocytes through a caspase-dependent mechanism that involves activation of the extrinsic death pathway and Type II signaling through the mitochondria. We have isolated and purified both His-tagged protein and three versions of native recombinant human TRAIL protein from Escherichia coli. We found that 5 mm dithiothreitol in the purification process enhanced oligomerization of TRAIL and resulted in the formation of hyper-oligomerized TRAILs, including hexamers and nonomers with an extremely high potency in apoptosis induction. Although death-inducing signaling complex formation was much more efficient in cells treated with hyper-oligomerized TRAILs, this did not convert TRAIL-sensitive Type II HCT116 colon tumor cells to a Type I death pattern as judged by their continued sensitivity to a caspase 9 inhibitor. Moreover, TRAIL-resistant Type II Bax-null colon carcinoma cells were not converted to a TRAIL-sensitive Type I state by hyper-oligomerized TRAIL. Primary human esophageal epithelial 2 cells were found to be sensitive to all TRAIL preparations used, including trimer TRAIL. TRAIL-induced death in esophageal epithelial 2 cells was prevented by caspase 9 inhibition for up to 4 h after TRAIL exposure. This result suggests a possible therapeutic application of caspase 9 inhibition as a strategy to reverse TRAIL toxicity. Hyper-oligomerized TRAIL may be considered as an alternative agent for testing in clinical trials. The cytotoxic death ligand TRAIL (tumor necrosis factor-related apoptosis-inducing ligand) is a tumor-specific agent under development as a novel anticancer therapeutic agent. However, some reports have demonstrated toxicity of certain TRAIL preparations toward human hepatocytes and keratinocytes through a caspase-dependent mechanism that involves activation of the extrinsic death pathway and Type II signaling through the mitochondria. We have isolated and purified both His-tagged protein and three versions of native recombinant human TRAIL protein from Escherichia coli. We found that 5 mm dithiothreitol in the purification process enhanced oligomerization of TRAIL and resulted in the formation of hyper-oligomerized TRAILs, including hexamers and nonomers with an extremely high potency in apoptosis induction. Although death-inducing signaling complex formation was much more efficient in cells treated with hyper-oligomerized TRAILs, this did not convert TRAIL-sensitive Type II HCT116 colon tumor cells to a Type I death pattern as judged by their continued sensitivity to a caspase 9 inhibitor. Moreover, TRAIL-resistant Type II Bax-null colon carcinoma cells were not converted to a TRAIL-sensitive Type I state by hyper-oligomerized TRAIL. Primary human esophageal epithelial 2 cells were found to be sensitive to all TRAIL preparations used, including trimer TRAIL. TRAIL-induced death in esophageal epithelial 2 cells was prevented by caspase 9 inhibition for up to 4 h after TRAIL exposure. This result suggests a possible therapeutic application of caspase 9 inhibition as a strategy to reverse TRAIL toxicity. Hyper-oligomerized TRAIL may be considered as an alternative agent for testing in clinical trials. Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) 1The abbreviations used are: TRAIL, tumor necrosis factor-related apoptosis-inducing ligand; DISC, death-inducing signaling complex; hTR, His6-tagged TRAIL; Ni-NTA, nickel-nitrilotriacetic acid; FADD, Fas-associating death domain-containing protein; nTR, native TRAIL preparation; GFP, green fluorescent protein; DTT, dithiothreitol; c-FLIP, cellular FLICE (FADD-like interleukin-1β-converting enzyme)-inhibitory protein; Bax, BCL-2-associated X protein. is a Type II transmembrane protein that binds to one of four receptors that have been identified in humans, including TRAIL-R1/DR4 (1Pan G. O'Rourke K. Chinnaiyan A.M. Gentz R. Ebner R. Ni J. Dixit V.M. Science. 1997; 276: 111-113Crossref PubMed Scopus (1562) Google Scholar), TRAIL-R2/KILLER/DR5 (2Walczak H. Degli-Esposti M.A. Johnson R.S. Smolak P.J. Waugh J.Y. Boiani N. Timour M.S. Gerhart M.J. Schooley K.A. Smith C.A. Goodwin R.G. Rauch C.T. 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Both DR4 and DR5 are pro-apoptotic receptors, which contain a cytoplasmic death domain and mediate apoptosis on binding to TRAIL. By contrast, TRID and TRUNDD do not contain a cytoplasmic death domain and block the function of TRAIL by competing with TRAIL-R1 and TRAIL-R2 for binding of TRAIL (8Sheridan J.P. Marsters S.A. Pitti R.M. Gurney A. Skubatch M. Baldwin D. Ramakrishnan L. Gray C.L. Baker K. Wood W.I. Goddard A.D. Godowski P. Ashkenazi A. Science. 1997; 277: 818-821Crossref PubMed Scopus (1533) Google Scholar). Apoptosis is an essential process for the regulation of home-ostasis and development. The apoptotic process can be induced by two different pathways (9Stennicke H.R. Salvesen G.S. Biochim. Biophys. Acta. 2000; 1477: 299-306Crossref PubMed Scopus (296) Google Scholar). The intrinsic pathway, also called the mitochondrial pathway, is induced by intracellular signals such as oncogene activation, DNA-damaging agents, and growth factor deprivation. 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Miller R.E. Ariail K. Gliniak B. Griffith T.S. Kubin M. Chin W. Jones J. Woodward A. Le T. Smith C. Smolak P. Goodwin R.G. Rauch C.T. Schuh J.C. Lynch D.H. Nat. Med. 1999; 5: 157-163Crossref PubMed Scopus (2236) Google Scholar, 17Ashkenazi A. Pai R.C. Fong S. Leung S. Lawrence D.A. Marsters S.A. Blackie C. Chang L. McMurtrey A.E. Hebert A. DeForge L. Koumenis I.L. Lewis D. Harris L. Bussiere J. Koeppen H. Shahrokh Z. Schwall R.H. J. Clin. Investig. 1999; 104: 155-162Crossref PubMed Scopus (2008) Google Scholar). Even in tumor cell lines that show resistance to TRAIL, combined treatment with chemotherapeutic drugs or ionizing radiation has revealed induction of tumor cell death (18Griffith T.S. Fialkov J.M. Scott D.L. Azuhata T. Williams R.D. Wall N.R. Altieri D.C. Sandler A.D. Cancer Res. 2002; 62: 3093-3099PubMed Google Scholar, 19Jazirehi A.R. Ng C.P. Gan X.H. Schiller G. Bonavida B. Clin. Cancer Res. 2001; 7: 3874-3883PubMed Google Scholar, 20Kim M.R. Lee J.Y. Park M.T. Chun Y.J. Jang Y.J. Kang C.M. Kim H.S. Cho C.K. Lee Y.S. Jeong H.Y. Lee S.J. FEBS Lett. 2001; 505: 179-184Crossref PubMed Scopus (47) Google Scholar, 21Kim Y. Suh N. Sporn M. Reed J.C. J. Biol. Chem. 2002; 277: 22320-22329Abstract Full Text Full Text PDF PubMed Scopus (292) Google Scholar, 22Lacour S. Hammann A. Wotawa A. Corcos L. Solary E. Dimanche-Boitrel M.T. Cancer Res. 2001; 61: 1645-1651PubMed Google Scholar, 23Nagane M. Pan G. Weddle J.J. Dixit V.M. Cavenee W.K. Huang H.J. Cancer Res. 2000; 60: 847-853PubMed Google Scholar, 24Nimmanapalli R. Perkins C.L. Orlando M. O'Bryan E. Nguyen D. Bhalla K.N. Cancer Res. 2001; 61: 759-763PubMed Google Scholar). However, some reports have demonstrated a toxicity of certain TRAIL preparations against primary human hepatocytes through a caspase-dependent mechanism that involves activation of the extrinsic death pathway (25Jo M. Kim T.H. Seol D.W. Esplen J.E. Dorko K. Billiar T.R. Strom S.C. Nat. Med. 2000; 6: 564-567Crossref PubMed Scopus (766) Google Scholar, 26Nagata S. Nat. Med. 2000; 6: 502-503Crossref PubMed Scopus (51) Google Scholar). Further studies suggested that this toxicity could be avoided by use of native TRAIL but not tagged TRAIL (27Lawrence D. Shahrokh Z. Marsters S. Achilles K. Shih D. Mounho B. Hillan K. Totpal K. DeForge L. Schow P. Hooley J. Sherwood S. Pai R. Leung S. Khan L. Gliniak B. Bussiere J. Smith C.A. Strom S.S. Kelley S. Fox J.A. Thomas D. Ashkenazi A. Nat. Med. 2001; 7: 383-385Crossref PubMed Scopus (636) Google Scholar). In this study, we generated His-tagged and native TRAIL using several biochemical purification methods. In the case of native TRAIL produced in the presence of dithiothreitol, we observed and purified hyper-oligomerized forms that were found to be highly potent in death induction assays. We investigated the toxicity of the various recombinant TRAIL preparations toward normal human esophageal epithelial cells and found evidence of significant toxicity. We further developed a strategy for using caspase 9 blockade to inhibit toxicity and now report that administration of a caspase 9 inhibitor is effective for a period of up to 4 h following TRAIL exposure. Our studies have implications for the use of TRAIL as an anticancer agent during its utilization in clinical trials. Expression Plasmids for Tagged and Untagged Soluble Human TRAIL—The extracellular portion (aa 95∼281) of the human TRAIL cDNA was amplified and cloned into pQE80L (Qiagen) after digestion with BamHI and HindIII to generate the His6-tagged TRAIL expression plasmid (pQE-hTR). The primer sequences used were 5′-AAGGCGGATCCACCTCTGAGGAAACCATTTC-3′ (hTR-S) and 5′-CCCAAGCTTTTAGCCAACTAAAAAGGCCCCGA-3′ (hTR-AS). To make a non-tagged native human TRAIL expression plasmid, the His6 tag and amino acids 95∼113 were removed from pQE-hTR by a two-step PCR-mediated ligation. The first PCR step was performed to amplify the vector and TRAIL sequences using primers for the vector sequence 5′-CGTATCACGAGGCCCTTTCGTC-3′ (Head-S) and 5′-TTTCTCTCACCATAGTTAATTTCTCCTCTTTAA (Head-AS)-3′ and for TRAIL aa 114∼281, 5′-GAAATTAACTATGGTGAGAGAAAGAGGTCCTCA-3′ (TR114) and 5′-CCCAAGCTTTTAGCCAACTAAAAAGGCCCCGA-3′ (hTR-AS), respectively. Pfu Turbo DNA polymerase was used to prevent 3′-nucleotide addition. The amplified products from the first step were mixed and reamplified with primers Head-S and hTR-AS. The amplified product was cloned into pQE80L after digestion with EcoRI and HindIII to generate pQE-nTR. Purification of Soluble Recombinant Human TRAIL—Recombinant TRAILs were expressed in DH10B bacteria as the expression host. The overnight seed culture was diluted 100-fold into 1.0 liter of LB broth and incubated for 3 h at 37 °C. Isopropyl-1-thio-β-d-galactopyranoside (0.5 mm) was added to induce recombinant protein expression, and bacterial cells were incubated overnight at 30 °C. Bacteria were harvested and homogenized in lysis buffer (50 mm sodium phosphate, pH 8.0, 300 mm NaCl, 10 mm imidazole, and 10 mm β-mercaptoethanol or 5 mm dithiothreitol). Recombinant TRAILs were isolated from the soluble fraction using Ni-NTA agarose beads (Qiagen) after washing with lysis buffer containing 20 mm imidazole. Isolated proteins were dialyzed against phosphate-buffered saline with 10 mm β-mercaptoethanol. The purity of the recombinant TRAIL proteins was confirmed by SDS-PAGE and Coomassie Blue gel staining. Separation of Oligomerized Forms of Soluble Recombinant Human TRAIL—The recombinant human TRAIL purified from the Ni-NTA-agarose column application was subjected to gel filtration chromatography to isolate oligomerized forms of TRAIL. The HiLoad 60/120 Superdex 200 prep grade column was used in the AKTA fast protein liquid chromatography system (Amersham Biosciences). Cell Lines and Culture—Human primary esophageal epithelial cells (EPC2) were described previously (28Andl C.D. Mizushima T. Nakagawa H. Oyama K. Harada H. Chruma K. Herlyn M. Rustgi A.K. J. Biol. Chem. 2003; 278: 1824-1830Abstract Full Text Full Text PDF PubMed Scopus (222) Google Scholar). EPC2 cells were cultured in keratinocyte-SFM (Invitrogen) containing bovine pituitary extract (40 μg/ml) and epidermal growth factor (1 ng/ml). Human foreskin fibroblasts were described previously (29Meier F. Nesbit M. Hsu M.Y. Martin B. Van Belle P. Elder D.E. Schaumburg-Lever G. Garbe C. Walz T.M. Donatien P. Crombleholme T.M. Herlyn M. Am. J. Pathol. 2000; 156: 193-200Abstract Full Text Full Text PDF PubMed Scopus (185) Google Scholar) and grown in Dulbecco's modified Eagle's medium with 10% fetal bovine serum and antibiotics. The recombinant human soluble DR5 was produced using the Pichia pastoris system as we described previously (30Song K. Chen Y. Goke R. Wilmen A. Seidel C. Goke A. Hilliard B. J. Exp. Med. 2000; 191: 1095-1104Crossref PubMed Scopus (324) Google Scholar). The purified soluble DR5 contains 1-2 ng of lipopolysaccharide/mg of protein as determined by Limulus amebocyte lysate assay. This is comparable with human serum albumin purchased from Sigma, which contains 1-4 ng of lipopolysaccharide/mg of protein. Assessment of TRAIL-mediated Apoptosis—For detecting apoptosis mediated by TRAIL, the active caspase 3 assay was performed using a Cytofix/Cytoperm kit (BD Biosciences) as described previously (31Kim K. Takimoto R. Dicker D.T. Chen Y. Gazitt Y. El-Deiry W.S. Int. J. Oncol. 2001; 18: 241-247PubMed Google Scholar). Briefly, 5 × 105 cells were seeded into a 6-well plate. After 24 h, the cells were treated with TRAIL. After treatment, the cells were harvested, fixed, and incubated with 0.125 μg/μl rabbit anti-active caspase 3 antibody (clone C92-605, Pharmingen) for 20 min. After washing, the cells were probed with 0.125 μg/μl phycoerythrin-conjugated goat anti-rabbit secondary antibody (CALTAG Laboratories) for 20 min. The intensity of phycoerythrin was analyzed by flow cytometry using a Beckman Coulter Epics Elite analyzer. For the analysis of the sub-G1 fraction, cells were fixed in 70% ethanol, stained with 50 μg/ml propidium iodide and RNase A for 30 min at room temperature, and then analyzed by flow cytometry. DISC Immunoprecipitation—H460 cells (4 × 107) were harvested and suspended in 2 ml of Dulbecco's modified Eagle's medium with 100 ng/ml trimer or hexamer TRAIL. After incubation with TRAIL preparations for 5, 10, or 20 min, cells were collected and lysed in 1 ml of DISC immunoprecipitation buffer (10 mm Tris, pH 7.5, 150 mm NaCl, 10% glycerol, 1 mm EDTA, 1% Triton X-100) with protease inhibitor mixture. Cell lysates (400 μl) were incubated overnight with 5 μl of rabbit anti-FADD antibody (Cell Signaling) at 4 °C. Complexes were precipitated by protein A-agarose (Invitrogen) and suspended in 50 μl of SDS sample buffer after three washes with DISC immunoprecipitation buffer. Immunoprecipitates were subjected to SDS-PAGE and Western blotting. Generation of Recombinant TRAILs and Their Toxicity against Human Cancer Cell Lines—hTR, which has an N-terminal histidine tag, was purified from the DH10B Escherichia coli strain after overnight incubation in 0.5 mm isopropyl-1-thio-β-d-galactopyranoside for protein induction. We used three different expression and purification schemes for the generation of native TRAILs (Table I). All TRAILs were purified using an Ni-NTA-agarose column application, and TRAIL protein purity was confirmed by SDS-PAGE and Coomassie Blue staining (Fig. 1A). Although the native TRAIL preparations (nTRs) did not contain a His6 tag in their sequence, they still bound to the Ni-NTA-agarose column, but the interaction was relatively weak, allowing separation from the beads at 40 mm imidazole, whereas the His-tagged hTR required 250 mm imidazole to be released (data not shown).Table IConditions for generation of recombinant TRAILsTagAmino acids residuesGrowth mediumPurification bufferhTRHis695-281, 23 kDaLBPhosphate buffer with 10 mm BMEaBME, β-mercaptoethanol.nTR1—114-281, 19 kDaLBPhosphate buffer with 10 mm BMEaBME, β-mercaptoethanol.nTR2—114-281, 19 kDaLBPhosphate buffer with 5 mm DTTbDTT, dithiothreitol.nTR3—114-281, 19 kDaLB with 100 μm ZnSO4Phosphate buffer with 5 mm DTTbDTT, dithiothreitol.a BME, β-mercaptoethanol.b DTT, dithiothreitol. Open table in a new tab The death-inducing activity of each TRAIL was tested in H460, a human lung adenocarcinoma cell line, which is sensitive to TRAIL. All the purified recombinant TRAIL proteins could induce apoptosis in H460 cells (Fig. 1B). The hTR and nTR1 proteins induced less than 15% cell death at 10 ng/ml. The hTR protein induced more than 40% cell death at 100 ng/ml, whereas nTR1 was only slightly less toxic than hTR, resulting in 30% cell death at 100 ng/ml. Incubation of TRAIL proteins in the presence of soluble DR5 protein resulted in a blockade of apoptosis, thereby confirming the specificity of hTR and nTR1 for the TRAIL-mediated death receptor pathway. We also used a decoy receptor-expressing SW480 human colorectal carcinoma cell line to further confirm the specificity of hTR and nTR1 for TRAIL receptor-mediated apoptosis (Fig. 1, C and D). We introduced a cytosolic death domain-deleted mouse KILLER/DR5 gene, which was fused with GFP into the SW480 cells (SWMK), and found that this artificial decoy receptor could protect from cell death induced by hTR or nTR1. The death-inducing activity of hTR was comparable with that of commercially available preparations (32Kim K. Fisher M.J. Xu S.Q. El-Deiry W.S. Clin. Cancer Res. 2000; 6: 335-346PubMed Google Scholar, 33Burns T.F. El-Deiry W.S. J. Biol. Chem. 2001; 276: 37879-37886Abstract Full Text Full Text PDF PubMed Google Scholar). Interestingly, the death-inducing activity of nTR2 and nTR3 appeared to be higher than that observed with nTR1, although all three were expressed from the same expression plasmid and host. Death induction following exposure to nTR2 and nTR3 resulted in a cell death rate greater than 40%, even at 10 ng/ml TRAILs, and death occurred rapidly by 1 h after treatment with higher doses of TRAIL. This hyperactivity of nTR2 and nTR3 was mediated specifically by the cell surface TRAIL death receptors because soluble DR5 protein almost completely blocked their cytotoxicity. These results suggested that nTR2 and nTR3 were somehow different from hTR and nTR1, although they were composed of a homogeneous TRAIL component. These results prompted us to examine the multimeric state of the TRAIL molecules. Hyper-oligomerization Correlates with High Cytotoxicity of nTR2 and nTR3—Gel filtration chromatography revealed that, depending on the method of purification, TRAIL molecules were heterogeneous in their oligomeric state (Fig. 2). In the case of hTR, one peak corresponding to the trimer was observed, whereas nTR1 appeared to be a mixture of monomer, dimer, and trimer molecules. The existence of an N-terminal His tag appeared to facilitate the trimerization of TRAIL molecules. Surprisingly, nTR2 and nTR3, which were purified in a 5 mm DTT-containing buffer, appeared to consist of timer, hexamer, and a small amount of nonomer. Each peak corresponding to a trimer, hexamer, or nonomer was composed of TRAIL molecules as verified by reducing SDS-PAGE. There was no apparent difference in the molecular composition of nTR2 versus nTR3, which suggested that the addition of zinc ions in the culture medium did not influence the oligomerization state of TRAIL molecules. However, there was a highly significant difference in the molecular conformation in nTR1 versus nTR2, although the same procedure was used in the overexpression and purification of the TRAIL molecules, except that the reducing agent was changed from 10 mm β-mercaptoethanol to 5 mm DTT. The use of DTT during purification greatly enhanced the oligomerization of native TRAIL molecules, which resulted in the formation of hexamers and nonomers. DTT also led to the disappearance of monomers and dimers observed with nTR1. We next investigated the apoptosis-inducing capacity of various TRAIL preparations and found that hexamers and nonomers were the highly cytotoxic component of nTR2 and nTR3. Hexamer and nonomer TRAIL proteins could kill the majority of H460 cells within 1 h of treatment at 100 ng/ml, and no cells survived after 4 h of treatment. In contrast, trimer TRAIL exposure resulted in more modest cell killing under the same conditions. Cell death induced by hyper-oligomerized TRAIL proteins was blocked by the soluble DR5 protein (Fig. 3A). We also checked the cleavage of caspases following TRAIL exposure. Trimer TRAIL treatment resulted in cleavage of procaspases 8, 9, and 3, but significant amounts of pro-caspases remained after 4 h of treatment. In the case of hexamer TRAIL, we observed cleavage of most of the cellular caspases 8 and 3 within 1 h, accompanied by complete disappearance of the pro-form of caspases 8 and 3 within 2 h. Significant amounts of pro-caspase 9 remained at 1 h but disappeared by 2 h on treatment with hexamer TRAIL. The extent of cleavage of the caspases was proportional to the potency of the TRAILs (Fig. 3B). Hyper-oligomerized TRAIL Induced More Efficient DISC Formation—Because hexamer TRAIL induced apoptosis more efficiently than trimer TRAIL, we wondered whether DISC formation was more efficient following binding of hexamer versus trimer TRAIL. We performed DISC immunoprecipitation using anti-FADD antibody to examine the recruitment and cleavage of DISC components following exposure of H460 cells to either trimer or hexamer TRAIL. Using 100 ng/ml trimer or hexamer TRAIL, we observed a time-dependent increase in the recruitment and cleavage of caspase 8 and c-FLIP to the DISC (Fig. 3C). We also observed increased recruitment of DR4 to the DISC. This increase was much greater in hexamer TRAIL-treated cells showing a significantly greater amount of recruitment and cleavage even at a 5-min exposure, which was comparable with the amount observed following 20 min of exposure to the trimer TRAIL. We also investigated whether the more efficient DISC formation and death induction by hexamer TRAIL might influence whether cell death occurred by a Type I versus a Type II mechanism. To determine whether cell death became less sensitive to inhibition by the caspase 9 inhibitor LEHD-fluoromethyl ketone (34Ozoren N. Kim K. Burns T.F. Dicker D.T. Moscioni A.D. El-Deiry W.S. Cancer Res. 2000; 60: 6259-6265PubMed Google Scholar), we treated either H460 (Type I) or HCT116 (Type II) cells with trimer or hexamer TRAIL and examined cell death induction at 6 h (Fig. 4A). H460 cells showed marginal death protection by the caspase 9 inhibitor (C9I) treatment, whereas complete death protection was conferred by the caspase 8 inhibitor (C8I), consistent with a Type I cell death protection pattern. There was no difference in the degree of observed death protection by the caspase inhibitors whether trimer or hexamer TRAIL was used to treat the H460 cells. In HCT116 cells, the C9I completely blocked cell death induced by either trimer or hexamer TRAIL. Despite more efficient formation of DISC by hexamer TRAIL, this did not alleviate the need to amplify the death signal through mitochondrial activation of caspase 9 as occurs in the Type II cell death pattern. We further confirmed these observations by examining the sub-G1 fraction and colony viability (Fig. 4, B and C, respectively) of Bax-null HCT116 cells, which are TRAIL-resistant because the Bax deficiency prevents their death by a Type II mechanism (33Burns T.F. El-Deiry W.S. J. Biol. Chem. 2001; 276: 37879-37886Abstract Full Text Full Text PDF PubMed Google Scholar, 35Wang S. El-Deiry W.S. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 15095-15100Crossref PubMed Scopus (131) Google Scholar). Bax-null cells remained resistant regardless of the oligomeric state of TRAIL used. Native TRAIL, Including Trimer TRAIL, Induced Death of Human Primary Esophageal Epithelial Cells—TRAIL is believed to selectively induce cell death of tumor cells but not most normal cells. We tested our TRAIL preparations for potential cytotoxic effects toward normal human cell lines. Human foreskin fibroblasts appeared completely refractory to TRAIL-induced death regardless of the method of TRAIL preparation (Fig. 5A). In contrast, EPC2 cells displayed significant sensitivity to the various TRAIL preparations (Fig. 5B). We recently demonstrated that these cells are sensitive to His-tagged recombinant TRAIL (36Kim K. Nakagawa H. Fei P. Rustgi A.K. El-Deiry W.S. Cell Death Differ. 2004; 11: 583-587Crossref PubMed Scopus (20) Google Scholar). We treated the EPC2 cells with 50 ng/ml of each TRAIL preparation and, using the active caspase 3 fluorescence-activated cell sorter assay, found that EPC2 were sensitive to all four TRAIL preparations (Fig. 5B); however, the hyper-oligomerized TRAIL preparations were clearly more toxic toward the EPC2 cells. There have been some reports that His-tagged or leucine zipper TRAIL preparations induced cell death in human primary keratinocytes or hepatocytes (25Jo M. Kim T.H. Seol D.W. Esplen J.E. Dorko K. Billiar T.R. Strom S.C. Nat. Med. 2000; 6: 564-567Crossref PubMed Scopus (766) Google Scholar, 27Lawrence D. Shahrokh Z. Marsters S. Achilles K. Shih D. Mounho B. Hillan K. Totpal K. DeForge L. Schow P. Hooley J. Sherwood S. Pai R. Leung S. Khan L. Gliniak B. Bussiere J. Smith C.A. Strom S.S. Kelley S. Fox J.A. Thomas D. Ashkenazi A. Nat. Med. 2001; 7: 383-385Crossref PubMed Scopus (636) Google Scholar, 37Qin J.Z. Bacon P.E. Chaturvedi V. Bonish B. Nickoloff B.J. Exp. Dermatol. 2002; 11: 573-583Crossref PubMed Scopus (25) Google Scholar). There was also a report that optimally trimerized soluble TRAIL did not induce apoptosis in human hepatocytes (27Lawrence D. Shahrokh Z. Marsters S. Achilles K. Shih D. Mounho B. Hillan K. Totpal K. DeForge L. Schow P. Hooley J. Sherwood S. Pai R. Leung S. Khan L. Gliniak B. Bussiere J. Smith C.A. Strom S.S. Kelley S. Fox J.A. Thomas D. Ashkenazi A. Nat. Med. 2001; 7: 383-385Crossref PubMed Scopus (636) Google Scholar). When we compared the dose dependence of cell death induced by trimer TRAIL in EPC2 and H460 cells, EPC2 cells showed a sensitivity to TRAIL similar to that of the H460 lung cancer cells (Fig. 5C), although our trimer TRAIL has optimally trimerized conformation according to our gel filtration results (Fig. 2). TRAIL-induced Cell Death in Human Primary Esophageal Epithelial Cells Was Prevented by Treatment with a Caspase 9 Inhibitor Even after Initiation of Apoptosis—We previously reported that human primary hepatocytes were sensitive to His-tagged TRAIL treatment, but this death was prevented by co-treatment with the C9I (34Ozoren N. Kim K. Burns T.F. Dicker D.T. Moscioni A.D. El-Deiry W.S. Cancer Res. 2000; 60: 6259-6265PubMed Google Scholar). We also showed that the C9I could not prevent apoptosis induced by TRAIL in H460 and SW480 cell lines because these tumor cell lines were killed by a Type I mechanism that was not sensitive to blockade of caspase 9 activated by the mitochondrial signaling pathway. In the present study, we evaluated the death-protective effect of C9I especially after the death signal had been initiated by TRAIL. First, we added the C9I or the soluble DR5 protein at 0-, 1-, 2-, 4-, and 6-h time points during a 6-h culture of EPC2 cells exposed to 100 ng/ml trimer TRAIL at time 0 (Fig. 6A). At the zero time point, when each of the death blockers, C9I or soluble DR5, was added with trimer TRAIL, the death was prevented completely. At the 1-h time point, in the case when the death blockers were added at 1 h after trimer TRAIL, the C9I prevented the death completely, whereas the soluble DR5 only pr
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