Molecular Cancer Therapeutics 1659 Camptothecin- and etoposide-induced apoptosis in human leukemia cells is independent of cell death receptor-3 and -4 aggregation but accelerates tumor necrosis factor–related apoptosis-inducing ligand–mediated cell death Stephane Bergeron,1 Myriam Beauchemin,1 expression level of SODD, FADD-DN, TRADD-ND, and and Richard Bertrand1,2 DAP3-DN. However, camptothecin or VP-16 treatment in combination with tumor necrosis factor–related apopto- 1 Centre de recherche, Centre hospitalier de l’Universite´ de sis-inducing ligand (TRAIL) substantially accelerated Montre´al-Hoˆpital Notre-Dame and Institut du cancer de Montre´al and 2Departement de me´decine, Universite´ de Montre´al, kinetics of apoptosis than treatment with camptothecin, Montre´al, Quebec, Canada 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 Abstract that DR4 aggregation mediated by camptothecin or VP-16 During camptothecin- and etoposide (VP-16)-induced could represent a mean that accelerates TRAIL-induced apoptosis in HL-60 cells, the expression level of cell death apoptosis. [Mol Cancer Ther 2004;3(12):1659–69] receptor-3 (DR3), cell death receptor-4 (DR4), and FAS remained mostly unchanged, whereas the expression of Introduction silencers of death domain (SODD) and FLICE inhibitory Chemotherapeutic agents, including DNA topoisomerase proteins, inhibitors of the cell death receptor signaling I and II inhibitors commonly used in the treatment of pathways, decreased substantially. By indirect immuno- fluorescence and immunoperoxidase imaging and with gel hematopoietic tumor cells, could eliminate these cells by rapid activation of apoptosis (1, 2). Apoptosis is triggered filtration column chromatography, we observed rapid by different entry sites, including the best-studied mito- aggregation at the cell surface and the appearance of high molecular weight protein complexes primarily involving chondrial and cell death receptor pathways, the well- described granzyme B pathway in apoptosis by CTL and DR3, and DR3 and DR4 after camptothecin and VP-16 the less-characterized routes via apical procaspase-2 acti- treatment, respectively. Both drugs failed to rapidly promote FAS aggregation in these cells. The high vation, lysoapoptase, and cathepsin activation through lysosomal leakage and calpain activation driven by Ca2+ expression level of SODD or of dominant negative forms release from the endoplasmic reticulum (3–7). These path- of FADD (FADD-DN) and DAP3 (DAP3-DN), or of NH - 2 ways lead to sequential activation of a series of cysteine terminal deletion mutant of TRADD (TRADD-ND) achieved aspartate-specific proteases, called the proteolytic caspase by transient transfection experiments, did not impair the cascade, which results in the orderly death of cells (8). kinetics of apoptosis after camptothecin and VP-16 Several studies have shown that the transcriptional treatment in HL-60 and U937 cells. Taken together, these observations suggested that camptothecin and VP-16 activity of p53 and activation of downstream target genes in response to genotoxic stress, including those provoked induced rapid aggregation of DR4 and DR3, but paradoxi- by DNA topoisomerase I and II inhibitors, contribute to cally, the importance of these events in signaling apoptosis is uncertain, because the kinetics of apoptosis evoking apoptosis. Among the p53-mediated apoptotic effector genes activated by DNA damage, several proa- were unaffected, even in the presence of a high poptotic Bcl-2 family members, including Bax, Noxa, Puma and Bid (9–12), and Peg/Pw1 (13), a group of genes named PIGs (14) and the cell death receptor family Received 3/12/03; revised 10/7/04; accepted 10/15/04. members, including FAS, cell death receptor-4 (DR4), and Grant support: Canadian Institutes of Health Research (R. Bertrand). DR5 (15–17), have been recognized as p53-dependent R. Bertrand is a scholar of the Fonds de la recherche en sante´ target genes associated with the activation of apoptosis in du Que´bec. S. Bergeron obtained studentships from the Faculte´ des ´etudes supe´rieures de l’Universite´ de Montre´al and the Institut du response to genotoxic stress. Although some of these genes cancer de Montre´al. could also be regulated in a p53-independent manner, The costs of publication of this article were defrayed in part by the much less attention has been paid to the p53-independent payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to apoptotic pathways induced by cancer radiotherapy and indicate this fact. chemotherapy. Requests for reprints: Richard Bertrand, Centre de recherche, Centre The importance of the cell death receptor family in hospitalier de l’Universite´ de Montre´al-Hoˆpital Notre-Dame, 1560 signaling programmed cell death after genotoxic stress Sherbrooke Street East, Room Y-5634, Montreal, Quebec, Canada H2L 4M1. Phone: 514-890-8000, ext. 26615; Fax: 514-412-7591. has been the subject of several studies. This family so far E-mail: [email protected] includes the membrane death receptors and their respec- Copyright C 2004 American Association for Cancer Research. tive ligands tumor necrosis factor (TNF)-R1 and TNF-a Mol Cancer Ther 2004;3(12). December 2004 Downloaded from mct.aacrjournals.org on September 28, 2021. © 2004 American Association for Cancer Research. 1660 Cell Death Receptors, Apoptosis, Camptothecin, and VP-16 ligand, FAS and FAS ligand, cell death receptor-3 (DR3) (reviewed in ref. 18). Interestingly, Fulda et al. (25) found and APO-3L/TWEAK and TL1A ligands, DR4 and DR5 that in type I cells, both the FAS signaling system and the that share the TNF-related apoptosis-inducing ligand mitochondrial pathway participated in triggering apoptosis (TRAIL), and the orphan receptor DR6 (reviewed in ref. after drug treatment, whereas in type II cells, apoptosis was 18). Activation of these cell death receptors by their cognate predominantly controlled by mitochondria-driven events. ligands results in receptor trimerization and the formation More recently, DR4 and DR5 expression levels and their of high molecular weight death-inducing signaling com- shared ligand TRAIL have been shown to increase after a plexes (DISC). These DISC contain adapter proteins, variety of DNA-damaging agents, including DNA top- including FADD, TRADD, DAP3, and a combination of oisomerase I and II inhibitors, in a p53-dependent and p53- them (e.g., TRADD-FADD for TNF-R1 and DR3, DAP3- independent manner. In some of these studies, evidence FADD for DR4 and DR5, and FADD-only for FAS), which has been provided for the participation of both cell death bind to the aggregated receptor through death domain receptor– and mitochondria-mediated signaling events interactions (reviewed in ref. 18). FADD also contains a triggering apoptosis after drug treatment. In addition, death effector domain and recruits apical procaspase-8 (or several studies have revealed that combined treatment procaspase-10) via death effector domain interactions. Once with a cell death ligand, including FAS ligand and TRAIL, initiator procaspase-8 zymogen is recruited to the DISC, it and DNA-damaging agents, including DNA topoisomerase begins to cluster and undergoes autoprocessing to generate I and II inhibitors, significantly resulted in enhanced active caspase-8. In type I cells, procaspase-8 activation at synergistic cell death (reviewed in ref. 18). Altogether, the DISC directly promotes the activation of procaspase-3 these observations supported the idea that such treatment and apoptosis in a mitochondria-independent manner (19). combinations may be useful for clinical cancer therapy. However, in most cells, the type II cells, strong activation Despite these numerous studies, to the best of our knowl- of procaspase-8 and procaspase-3 requires amplification of edge, the participation of DR3 in drug-induced apoptosis is the initial signal through the mitochondrial cell death path- still unrevealed. way (19). In these type II cells, the BH3-only protein BID The present experiments were designed to examine the is a specific proximal substrate of caspase-8. Cleavage of participation of DR3 and DR4, compared with FAS, in Bid by caspase-8 yields a COOH-terminal BH3-containing p53-deficient leukemia cell lines well described to rapidly fragment that activates downstream mitochondrial apopto- undergo apoptosis after DNA topoisomerase I and II tic events (20). inhibitor treatment. We observed that camptothecin and The cell death receptor pathways are also regulated by etoposide (VP-16) did not induce an increase in DR3, DR4, negative control proteins, including FLICE inhibitory and FAS expression but provoked the down-regulation of proteins (FLIPS) and silencer of death domains (SODD). SODD and FLIPS. Camptothecin promoted strong DR3 FLIPS contains two death effector domains and competes aggregation at the cell surface and, to a lesser extent, DR4, with FADD for binding to procaspase-8 (or procaspase-10), whereas VP-16 significantly stimulated DR3 and DR4 preventing procaspase-8 (or procaspase-10) recruitment and aggregation. However, the high expression level of SODD activation at the DISC (21). SODD specifically binds to the or of dominant negative forms of FADD (FADD-DN) and death domains of TNF-R1 and DR3, preventing the DAP3 (DAP3-DN), or of NH2-terminal deletion mutant recruitment of their respective