title: Inhibitor of apoptosis protein hILP undergoes caspase-mediated cleavage during T lymphocyte apoptosis.
abstract: Several endogenous or viral inhibitors of apoptosis, including Bcl-2, Bcl-xL, FLIP, p35, and CrmA, have been shown to be cleaved by caspases during apoptosis. In this study, we demonstrate that the endogenous inhibitor of apoptosis, hILP/XIAP, is also cleaved in apoptotic T lymphocytes, generating at least one prominent fragment of 29 kDa. This p29 cleaved fragment was detected in Jurkat cells induced to apoptose by anti-Fas antibody, staurosporin, or VP-16. The cleavage of hILP appears to be caspase mediated because the production of the p29 protein was inhibited by the pan-caspase peptide inhibitor, Z-VAD.FMK. In Jurkat cells engineered to overexpress CrmA, cleavage of hILP in response to anti-Fas antibody or staurosporin was inhibited, whereas overexpression of Bcl-2 abrogated the cleavage in response to VP-16. Cleavage of hILP was also observed in cell-free reactions using in vitro translated hILP and recombinant caspase-3 or -7. Moreover, we found that the p29 hILP fragment retained the ability to bind caspase-3 and -7, as shown previously for full-length or BIR-2 hILP. The p29 cleavage product was also detected during T-cell receptor-mediated apoptosis in peripheral blood lymphocytes from normal donors. Furthermore, tumor-associated T lymphocytes purified from ascites of patients with ovarian cancer expressed fragmented hILP, which was not detected in control T cells purified from peripheral blood of normal donors. Our results suggest that the cleavage of hILP represents an important event in apoptosis of T lymphocytes in both normal and pathological in vivo settings.
authors.affiliation: Department of Pharmacology, University of Pittsburgh School of Medicine, and University of Pittsburgh Cancer Institute, Pennsylvania 15123, USA.
authors.forename: D E
authors.initials: DE
authors.lastname: Johnson
authors.forename: B R
authors.initials: BR
authors.lastname: Gastman
authors.forename: E
authors.initials: E
authors.lastname: Wieckowski
authors.forename: G Q
authors.initials: GQ
authors.lastname: Wang
authors.forename: A
authors.initials: A
authors.lastname: Amoscato
authors.forename: S M
authors.initials: SM
authors.lastname: Delach
authors.forename: H
authors.initials: H
authors.lastname: Rabinowich
mesh_terms: D000590:Amino Acid Chloromethyl Ketones / Q000494:pharmacology; D000906:Antibodies / Q000494:pharmacology; D017209:Apoptosis / Q000187:drug effects; D020169:Caspases / Q000378:metabolism*; D015853:Cysteine Proteinase Inhibitors / Q000494:pharmacology; D004791:Enzyme Inhibitors / Q000378:metabolism; D005047:Etoposide / Q000494:pharmacology; D006801:Humans; D019169:Jurkat Cells; D011506:Proteins / Q000378:metabolism*; D019311:Staurosporine / Q000494:pharmacology; D013601:T-Lymphocytes / Q000166:cytology* / Q000502:physiology*; D051636:X-Linked Inhibitor of Apoptosis Protein; D019014:fas Receptor / Q000502:physiology
chemical_list: D000590:Amino Acid Chloromethyl Ketones; D000906:Antibodies; D015853:Cysteine Proteinase Inhibitors; D004791:Enzyme Inhibitors; D011506:Proteins; D051636:X-Linked Inhibitor of Apoptosis Protein; C496866:XIAP protein, human; C096713:benzyloxycarbonylvalyl-alanyl-aspartyl fluoromethyl ketone; D019014:fas Receptor; D005047:Etoposide; D020169:Caspases; D019311:Staurosporine