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J. Biol. Chem., Vol. 279, Issue 38, 40044-40052, September 17, 2004

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Death Induction by Recombinant Native TRAIL and Its Prevention by a Caspase 9 Inhibitor in Primary Human Esophageal Epithelial Cells^*

Seok-Hyun Kim, Kunhong Kim, Jae G. Kwagh¶, David T. Dicker, Meenhard Herlyn||, Anil K. Rustgi**, Youhai Chen, and Wafik S. El-Deiry¶¶

From the Laboratory of Molecular Oncology and Cell Cycle Regulation, Howard Hughes Medical Institute, Department of Medicine, Genetics, and Pharmacology, and the Abramson Cancer Center, School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania 19104, ^¶Endocrinology Department, The Joseph Stokes Jr. Research Institute, The Children's Hospital of Philadelphia, Philadelphia, Pennsylvania 19104, ^||Molecular and Cellular Oncogenesis Program, The Wistar Institute, Philadelphia, Pennsylvania 19104, ^**Gastroenterology Division, Department of Medicine, and the Abramson Cancer Center, School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania 19104, and Department of Pathology and Laboratory Medicine, School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania 19104

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.

Received for publication, April 23, 2004 , and in revised form, June 28, 2004.

^* This work was supported in part by funds from the Howard Hughes Medical Institute, National Institutes of Health Grant CA 098101, and National Institutes of Health Center for Molecular Studies in Digestive and Liver Diseases. This work was presented at the 94th annual meeting of the American Association for Cancer Research, July 11-14, 2003, Washington, D. C. 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.

Present address: Dept. of Biochemistry and Molecular Biology, Yonsei University College of Medicine, Seoul, Korea.

^¶¶ Assistant Investigator of the Howard Hughes Medical Institute. To whom correspondence should be addressed: 415 Curie Blvd., CRB 437, Philadelphia, PA 19104. Tel.: 215-898-9015; Fax: 215-573-9139; E-mail: wafik{at}mail.med.upenn.edu.

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