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¹ Centre de recherche, Centre hospitalier de l'Université de Montréal-Hôpital Notre-Dame and Institut du cancer de Montréal and ² Departement de médecine, Université de Montréal, Montréal, Quebec, Canada

Requests for reprints: Richard Bertrand, Centre de recherche, Centre hospitalier de l'Université de Montréal-Hôpital Notre-Dame, 1560 Sherbrooke Street East, Room Y-5634, Montreal, Quebec, Canada H2L 4M1. Phone: 514-890-8000, ext. 26615; Fax: 514-412-7591. E-mail: richard.bertrand{at}umontreal.ca

During camptothecin- and etoposide (VP-16)-induced apoptosis in HL-60 cells, the expression level of cell death receptor-3 (DR3), cell death receptor-4 (DR4), and FAS remained mostly unchanged, whereas the expression of silencers of death domain (SODD) and FLICE inhibitory proteins, inhibitors of the cell death receptor signaling pathways, decreased substantially. By indirect immunofluorescence and immunoperoxidase imaging and with gel filtration column chromatography, we observed rapid aggregation at the cell surface and the appearance of high molecular weight protein complexes primarily involving DR3, and DR3 and DR4 after camptothecin and VP-16 treatment, respectively. Both drugs failed to rapidly promote FAS aggregation in these cells. The high expression level of SODD or of dominant negative forms of FADD (FADD-DN) and DAP3 (DAP3-DN), or of NH ₂-terminal deletion mutant of TRADD (TRADD-ND) achieved by transient transfection experiments, did not impair the kinetics of apoptosis after camptothecin and VP-16 treatment in HL-60 and U937 cells. Taken together, these observations suggested that camptothecin and VP-16 induced rapid aggregation of DR4 and DR3, but paradoxically, the importance of these events in signaling apoptosis is uncertain, because the kinetics of apoptosis were unaffected, even in the presence of a high expression level of SODD, FADD-DN, TRADD-ND, and DAP3-DN. However, camptothecin or VP-16 treatment in combination with tumor necrosis factor–related apoptosis-inducing ligand (TRAIL) substantially accelerated kinetics of apoptosis than treatment with camptothecin, VP-16, or TRAIL alone. In contrast, cotreatment of camptothecin or VP-16 with TWEAK or TL1A did not facilitate apoptosis in HL60 cells. These findings suggest that DR4 aggregation mediated by camptothecin or VP-16 could represent a mean that accelerates TRAIL-induced apoptosis.

Key Words: Apoptosis • DR3 • DR4 • Fas • DNA topoisomerase • CPT • VP16

Grant support: Canadian Institutes of Health Research (R. Bertrand). R. Bertrand is a scholar of the Fonds de la recherche en santé du Québec. S. Bergeron obtained studentships from the Faculté des études supérieures de l'Université de Montréal and the Institut du cancer de Montréal.

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.

Received 3/12/03; revised 10/ 7/04; accepted 10/15/04.