And Etoposide-Induced Apoptosis in Human Leukemia Cells Is

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 Montreál-Hoˆpital Notre-Dame and Institut du cancer de Montreál and 2Departement de me´decine, Universite´ de Montreál, kinetics of apoptosis than treatment with camptothecin, Montreál, 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 immunofluorescence 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 études supe´rieures de l’Universite´ de Montreál and the Institut du response to genotoxic stress. Although some of these genes cancer de Montreál. 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 Montreál-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

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 adapter proteins (22). of TRADD (TRADD-ND) achieved in transient transfection In nonstimulated cells, SODD is believed to be associated experiments, did not slow the kinetics of apoptosis in with these receptors, inhibiting DISC formation. Upon camptothecin- or VP-16-treated HL60 and U937 cells. stimulation by their respective ligands, the dissociation of Camptothecin or VP-16 treatment in combination with SODD from the receptors leads to the formation of active TRAIL substantially accelerated the kinetics of apoptosis DISC (22). than treatment with camptothecin, VP-16 or TRAIL alone. Participation of the cell death receptor family in signal- In contrast, cotreatment of camptothecin or VP-16 with ing programmed cell death after anticancer drug treatment TWEAK or TL1A did not accelerate apoptosis in HL60 had been suggested first by observations in patients with cells. Our results suggest that the importance of DR3 and acute myeloid leukemia, where increased FAS expression DR4 in signaling apoptosis in HL60 and U937 cells after in blast cells was associated with a better initial response to camptothecin or VP-16 treatment is uncertain, but campto- chemotherapy (23). In some cells, cross-resistance between thecin or VP-16 could accelerate TRAIL-, but not TWEAK- anticancer drugs and FAS-mediated apoptosis was also or TL1A-induced apoptosis in HL60 cells. noted (24). Together, these findings indicated that drug- induced apoptosis could be mediated by the death receptor FAS signaling pathway, or reflected alterations in common downstream pathways of apoptosis. Several studies have Materials and Methods reported that FAS, with or without the activation of its Reagents specific ligand, participates in anticancer radiotherapy- and The radioactive precursors [2-14C]-thymidine (59 mCi/ chemotherapy-induced apoptosis in a variety of tumor cell mmol) was obtained from ICN BioMedicals (Costa Mesa, models. Conversely, others proposed that drug-induced CA). Camptothecin and VP-16 were purchased from Sigma apoptosis was independent of the FAS signaling pathway Chemical Co. (St. Louis, MO). Recombinant hTRAIL was

Mol Cancer Ther 2004;3(12). December 2004

from Calbiochem-Novabiochem Co. (San Diego, CA), and or VP-16 (50 mol/L) treatment. After 30 minutes incubation recombinant hTWEAK and hTL1A were obtained from with the drugs, the cells were centrifuged and resuspended R&D Systems (Minneapolis, MN). The fluorogenic peptide in drug-free medium. TRAIL (250 ng/mL), TWEAK (500 substrates acetyl-Asp-Glu-Val-Asp-7-amino-4- methyl-cou- ng/mL), and TL1A (500 ng/mL) treatments were done marin (Ac-DEVD-AMC), benzyloxycarbonyl-Ile-Asp-Thr- during and after camptothecin or VP-16 exposure. Asp-7-amino-4-trifluoro methylcoumarin (z-IETD-AFC), Caspase Activity Determinations and acetyl-Leu-Glu-His-Asp-7-amino-4-trifluoromethyl- Control and drug-treated cells were homogenized at 4jC coumarin (Ac-LEHD-AFC) were provided from Bachem for 30 minutes in lysis buffer containing 100 mmol/L Bioscience, Inc. (King of Prussia, PA) or Calbiochem- HEPES (pH 7.5), 5 mmol/L EDTA, 5 mmol/L DTT, 20% Novabiochem. Rabbit polyclonal anti-human caspase- glycerol, and 0.1% NP40. The samples were centrifuged 8 (559932) and DR4 (66891N), and mouse monoclonal (10,000 Â g for 10 minutes at 4jC), and supernatants col- anti-human TRADD (B36-2) and FADD (A66-2) antibodies lected as cytosolic extracts. Caspase activity was measured were purchased from PharMingen, Inc. (San Diego, CA). by monitoring fluorescence continuously in a dual lumi- Rabbit polyclonal anti-human FAS (sc-715) and SODD (sc- nescence LS 50B fluorometer (Perkin-Elmer, Buckingham- 8980) antibodies were obtained from Santa Cruz Biotech- shire, UK) at an excitation wavelength of 380 nm and an nology (Santa Cruz, CA). Rabbit polyclonal anti-human emission wavelength of 460 nm for the substrate Ac-DEVD- DR3 (06-839) and mouse monoclonal anti-FLAG (M2) were AMC and at an excitation wavelength of 400 nm and an from Upstate Biotechnology (Lake Placid, NY), rabbit emission wavelength of 505 nm for the substrates z-IETD- polyclonal anti-human FLIP-g/y (343006) from Calbio- AFC and Ac-LEHD-AFC. Reactions were conducted in chem-Novabiochem and mouse monoclonal anti-HA (clone cuvettes, and temperature was maintained at 37jC with a 12CA5) from Roche Diagnostics (Laval, Que). All other water-jacketed sample compartment. DEVDase and IET- chemicals and reagents were acquired from Sigma Chem- Dase activities were studied in an optimal reaction buffer ical, ICN, or local sources. (31) containing 100 mmol/L HEPES (pH 7.5), 100 mmol/L Cell Culture, cDNA Cloning, Transfection, DNA Label- NaCl, 10% glycerol, 0.1% CHAPS, 10 mmol/L DTT, 1 ing, and DrugTreatments mmol/L EDTA, and 200 Amol/L fluorogenic peptide The human HL60 and U937 cell lines, from the substrates. LEHDase activity was measured in a reaction American Type Culture Collection (Manassas, VA), were buffer containing 100 mmol/L MES (pH 6.5), 10% PEG8000, j grown in suspension culture at 37 C under 5% CO2 in a 0.1% CHAPS, 10 mmol/L DTT, 1 mmol/L EDTA, and 500 humidified atmosphere in RPMI 1640 supplemented with Amol/L Ac-LEHD-AFC (31). Enzyme activities were 10% heat-inactivated fetal bovine serum, 100 units/mL determined as initial velocities (relative intensity penicillin and 100 Ag/mL streptomycin. The expression minuteÀ1 mgÀ1) and the results are expressed as fold- vectors pRK-FLAG-SODD and pcDNA3-GFP-FADD (80– increase relative to control, untreated cells. 208) were kind gifts from Drs. W. Liu (Tularik, Inc., San DNA Fragmentation Assays Francisco, CA; ref. 22) and H. Wajant (Institut fuer To visualize oligonucleosome-sized DNA fragmentation, Zelbiologie und Immunologie, Stuttgart, Germany; ref. cellular DNA was extracted and DNA electrophoresis was 26), respectively. From the expression vectors pRK-myc- done on 1.6% agarose gel in Tris-acetate buffer (pH 8.0) TRADD obtained from Dr. Goeddel (Tularik; ref. 27), we prior to ethidium bromide staining (2). A DNA filter generated a NH2-terminal deletion mutant of TRADD elution assay was used to quantitate DNA fragmentation, (TRADD-ND; ref. 28) lacking the NH2-terminal 1 to 108 as described previously (2). The results were expressed as amino acids, by PCR using specific adapter primers the percentage of DNA fragmented in treated cells containing BamHI sequences. The DAP3-DN cDNA that compared with DNA fragmented in untreated cells lacks the COOH-terminal 231 to 398 amino acids (29), was (background), using the formula (F À F0 /1 À F0) Â 100, generated by reverse transcription-PCR from polyA(+) where F and F0 represent DNA fragmentation in treated RNA extracted from HL60 cells, using specific adapter and control cells, respectively. primers containing NotI sequences. The amplified fragment Indirect Immunofluorescence and Immunoperoxidase was first cloned in pCR2.1 TOPO vector (TA cloning sys- Staining tem; Invitrogen, San Diego, CA) and then subcloned at the Approximately 1.0 Â 105 control and drug-treated cells BamHI or NotI restriction sites in the eukaryotic expression were spread by cytocentrifugation on glass slides and fixed vector pCEP4 (Invitrogen, Carlsbad, CA) that has been in ice-cold ethanol for 10 minutes and air-dried for 10 modified to include hemagglutinin epitote Tag sequences minutes. For immunofluorescence microscopy, nonspecific (HA-tag) and Kozak consensus sequences (30). All trans- binding sites were blocked with 5% bovine serum albumin fections were done by electroporation at 0.27 kV (Gene in the presence of irrelevant antibodies (antimouse immu- Pulser, Bio-Rad, Hercules, CA) with 10 Ag of each plasmid noglubulin G, 15 Ag/mL; Vector Laboratories, Birmingham; and 10 Â 106 cells. For DNA-labeling, cells were grown CA); then the slides were washed in PBS and incubated with [2-14C]-thymidine (0.02 Ci/mL) for 24 hours and with the primary antibody (10 Ag/mL) for 1 hour at chased overnight in isotope-free medium prior to electro- room temperature. After several washes in PBS, the slides poration or drug treatment. Exponentially growing cells were probed with anti-rabbit FITC-conjugated secondary (5 Â 105 cells/mL) were used for camptothecin (1.0 mol/L) antibodies (10 Ag/mL; Vector Laboratories) for 1 hour at

Mol Cancer Ther 2004;3(12). December 2004

room temperature. For immunoperoxidase assay, endoge- rapidly induced caspase activation, including DEVDase nous peroxidase activities were blocked with 3% H2O2 (caspase-3 like), IETDase (caspase-8 like), and LEHDase solution for 15 minutes. The slides were then probed and (caspase-9 like) activities (Fig. 1A), associated by DNA stained with Vectastain ABC Elite Kit reagents, according fragmentation. This DNA fragmentation was quantitated to the manufacturer’s protocols (Vector Laboratories). by DNA filter elution assay and visualized as oligonucleo- Images were generated with a Nikon Optiphot-2 micro- some-sized DNA ladders on ethidium bromide-stained scope equipped with a thermoelectrically cooled CCD agarose gels (Fig. 1B). Rapid activation of IETDase camera (Model DC330E, DageMTI, Inc., Michigan City, IN) (caspase-8 like) and LEHDase (caspase-9 like) activities hooked up to a PC computer. All images were analyzed could suggest the participation of both the cell death with Clemex Vision software (version 3.0.036, Clemex, receptor and mitochondrial pathways, to signal apoptosis Longueuil, QC, Canada). after camptothecin and VP-16 treatment in HL60 cells. Protein Expression by Western Blot Analysis However, several studies have revealed the existence of cell For Western blot analysis, cells were collected and death receptor-independent mechanisms for procaspase- homogenized in lysis buffer containing 5 mmol/L HEPES 8 activation (32, 33). (pH 7.4), 160 mmol/L KCl, 40 mmol/L NaCl, 10 mmol/L To more directly evaluate the participation of DR3, MgCl2, 1 mmol/L phenylmethylsulfonyl fluoride, 1 mmol/ DR4 and FAS in signaling caspase activation after L DTT, 0.5% NP40, and a cocktail of protease inhibitors camptothecin or VP-16 treatment, we first looked for (Complete, Roche Molecular Biochemicals, Montreal, QC, death receptor aggregation at the cell surface by indirect Canada) at 4jC for 30 minutes. After centrifugation (10,000 immunofluorescence and immunoperoxidase staining Â g, 10 minutes), the supernatants were collected as protein (Fig. 2). Found primarily as monomeric proteins in resting extracts. Proteins were loaded for SDS-PAGE and electro- cells, these receptors, upon activation, aggregate into high blotted on nitrocellulose membranes. Immunoblots were molecular weight complexes (34) that can be visualized as revealed by enhanced chemiluminescence reagent (Amer- dense, patchy clusters, and aggregates by indirect immu- sham Life Science, Piscataway, NJ). Relative densitometry nofluorescence staining (35). Exposure of HL60 cells to analysis of film exposure was based on integrated density camptothecin or VP-16 failed to induce large and dense values using an Alpha Imager 2000 digital imaging system staining aggregates of FAS after indirect immunofluores- (Alpha Innotech Co., San Jose, CA). cence (Fig. 2, top) or immunoperoxidase staining (Fig. 2, Gel Filtration Column Chromatography bottom). In contrast, DR3 immunoreactivity was detected as Gel filtration chromatography was done on a Superose dense and brightly stained clusters after camptothecin or 6HR 16/50 column connected to a fast protein liquid VP-16 treatment (Fig. 2). Similarly, DR4 was visualized as chromatography system (Amersham Life Science). The dense patchy staining after VP-16 treatment, and slightly equilibration, lysis and elution buffer contained PBS, 1% weaker after camptothecin treatment (Fig. 2). NP40, and a cocktail of protease inhibitors (Complete, To evaluate if DR3 and DR4 aggregation resulted from Roche Molecular Biochemicals). Cellular extracts were an increase in their expression after camptothecin or VP- prepared from 5 Â 108 control or drug-treated cells, 16 treatment, the kinetics of their expression were pelleted by centrifugation, and lysed in a 500-AL volume. analyzed by Western blotting and immunocytofluorom- After centrifugation, soluble extracts (500 AL) were injected etry. As shown in Fig. 3, the expression level of DR3, DR4, onto a column for elution at a flow rate of 500 AL/min, and and FAS remained mostly unchanged for at least 8 hours 2.5-mL fractions were collected at 5-minute intervals. The after camptothecin or VP-16 treatment in HL60 cells. The corresponding molecular weights and Stokes’ radius for variations observed in some experiments were not signifi- each standard (Amersham Life Science) were RNase A cantly different (Student’s t test: P > 0.05). In contrast, the (13.7 kDa, 16 A˚ ), chymotrypsinogen A (25 kDa, 20.9 A˚ ), FLIPS expression level decreased after camptothecin or VP- ovalbumin (43 kDa, 30.5 A˚ ), albumin (67 kDa, 35.5 A˚ ), al- 16 treatment, with the most rapid and pronounced effect dolase (158 kDa, 48.1 A˚ ), catalase (232 kDa, 52.2 A˚ ), occurring after VP-16 (Student’s t test: P < 0.008 for apoferritin (440 kDa, 61.0 A˚ ), and thyroglobulin (669 kDa, camptothecin at 8 hours and P < 0.001 for VP-16 at 4, 6, and 85 A˚ ). Void volume corresponded to the elution of Blue 8 hours). Similarly, the SODD expression level also Dextran 2000. After column chromatography, 80-AL ali- declined significantly after camptothecin or VP-16 treat- quots from each fraction were analyzed by SDS-PAGE and ment (Student’s t test: P < 0.001 for camptothecin at 4, 6, Western blotting. and 8 hours; P < 0.008 for VP-16 at 4 hours; and P < 0.001 at 6 and 8 hours; Fig. 3). To confirm some of these observations, the total protein amount of DR3, DR4, and FAS was also measured by indirect immunocytofluorom- Results etry in permeable cells. The fluorescence intensity distri- Aggregration at the Cell Surface of DR3 and DR4 bution detected for DR3, DR4, and FAS remained mostly after Camptothecin and VP-16 Treatment in HL60 Cells unchanged in the absence of and after camptothecin or The kinetics of camptothecin- and VP-16-induced apo- VP-16 treatment (data not shown). Altogether, these ptosis in HL60 cells were reported previously (1, 2). Short observations indicated that DR3 and DR4 aggregation on drug treatments (30 minutes) with camptothecin or VP-16 the cell surface did not result from an increased expression

Mol Cancer Ther 2004;3(12). December 2004

Figure 1. Kinetics of caspase activation and DNA fragmentation in camptothecin- and VP-16-treated HL60 cells. Cells were treated with camptothecin (1.0 Amol/L, left) or VP- 16 (50 Amol/L, right) for 30 minutes followed by incubation in drug-free medium. A, at the indicated times (x axis, h) after camptothecin or VP- 16 treatment, DEVDase (.), IETDase (n), and LEHDase (E) activities were measured as initial velocities and data were expressed as fold increase (y axis) relative to control cells. Points, mean of three independent experiments; bars, FSE. B, [2-14C]-thymidine-labeled cells treated with camptothecin or VP-16. At various time points (x axis, h) after drug treatment, DNA fragmentation was determined by DNA filter elution assay. Points, mean % DNA fragmentation of three independent experiments; bars, FSE. Inset, Total DNA extracted from control and drug- treated cells at 3 hours after camptothecin or VP-16 and visualized by ethidium bromide staining after agarose gel electrophoresis.

of these receptors, because a comparable level of proteins DR3 and DR4 revealed a large distribution of these was detected in untreated and treated HL60 cells. In proteins from low to very high molecular weight masses, addition, the decreased expression of two inhibitors of the whereas the expression profile of FAS showed a death receptor signaling pathways after camptothecin or narrower distribution (Fig. 4A). In camptothecin- or VP- VP-16 treatment suggests the participation of these cell 16-treated cells, at 2 and 3.5 hours after treatment, the death receptor signaling pathways in apoptosis activation elution profiles of DR3 shifted and concentrated toward and/or amplification in the cells. fractions 26 to 28 corresponding to molecular weight Formation of High Molecular Weight Complexes masses ranging approximately from 500 to 300 kDa. The Involving DR3 and DR4 after Camptothecin and VP-16 elution profiles of DR4, at 2 and 3.5 hours after VP-16 Tr e a t m e n t treatment, exhibited similar behaviour, shifting and con- Initial attempts failed to immunoprecipitate the DR3 centrating in a few fractions (24–26) corresponding to high and DR4 death-inducing signaling complexes after molecular weight masses ranging from f700 to 500 kDa. In camptothecin or VP-16 treatment in HL60 cells, in the contrast, the elution profiles of DR4 after camptothecin absence of added ligands. To explore the formation of treatment did not show striking differences compared with these complexes, a gel filtration column chromatography control cells. Similarly, the elution profiles of FAS after approach, followed by Western blot analysis of the camptothecin or VP-16 treatment remained mostly multiple collected fractions, was chosen. This approach unchanged compared with control cells (Fig. 4A). allowed us to separate and monitor proteins of interest We next monitored the elution profiles of two adapter within a large range of molecular masses, revealing proteins, TRADD and FADD, associated with DR3 or DR4 multioligomer and complex formation. death-inducing signaling complexes. Neither TRADD nor In untreated control HL60 cells, the elution profiles of FADD coeluted in fractions with DR3 (fraction 26–28) or

Mol Cancer Ther 2004;3(12). December 2004

Figure 2. Effect of camptothecin and VP-16 treatment on FAS, DR3, and DR4 aggregation at the cell surface of HL60 cells. Cells were untreated (CONTROL, left) or treated with camptothecin (1.0 Amol/L, middle) or VP-16 (50 Amol/L, right) for 30 minutes followed by incubation in drug-free medium. At 4 hours after treatment, FAS, DR3, and DR4 aggregation was visualized by indirect immunofluorescence (top) and immunoperoxidase (bottom) staining. Images were generated with a Nikon Optiphot-2 microscope equipped with a thermoelectrically cooled CCD camera (model DC330E).

DR4 (fraction 24–26) after camptothecin or VP-16 treatment immunofluorescence and immunoperoxidase staining (Fig. 4B). Similarly, we monitored the elution profiles of experiments and indicated that DR3, and DR3 and DR4 SODD, a specific inhibitory protein of DR3, and FLIPS, an formed oligomers and aggregates of high molecular inhibitor of both DR3 and DR4 death-inducing signaling weight after camptothecin and VP-16 treatment, respec- complexes. Whereas FLIPS did not coelute in fractions tively. The effect of camptothecin on DR4 aggregation with DR3 or DR4, SODD showed strong coelution profiles remains enigmatic. Whereas some patchy staining after with DR3 at 2 and 3.5 hours after camptothecin treatment, camptothecin treatment was observed by indirect immu- and in some fractions 3.5 hours after VP-16 treatment (Fig. nofluorescence and immunoperoxidase imaging, no shift 4C). Finally, the elution profiles of procaspase-8 were and cluster formation of high molecular weight involving distinct than those of DR3 and DR4 after camptothecin DR4 were detected by gel filtration column chromatog- treatment, but a few amount of procaspase-8 coeluted with raphy after camptothecin. Under the conditions of these DR3 and/or DR4 at 3.5 hours after VP-16 treatment. The experiments, we were also unable to observe coelution cleaved fragments of active caspase-8 were also detected of the adapter proteins TRADD and FADD within the after camptothecin and VP-16 treatment (Fig. 4D). DR3 and DR4 complexes, suggesting that the aggregates Altogether, these observations were consistent with the were not formed by typical active DISC components.

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Conversely, some procaspase-8 coeluted with DR3 and/ or DR4 after VP-16 treatment, suggesting the formation of weak DR3-procaspase-8- and/or DR4/procaspase-8- containing complexes. Interestingly, we found a strong coelution association between SODD and DR3, suggesting the presence of this death inhibitory protein within the DR3 complexes predominantly after camptothecin treatment.

Figure 4. High molecular weight complex formation in camptothecin- and VP-16-treated HL60 cells. Cells were treated with camptothecin (1.0 Figure 3. Kinetics of FAS, DR3, DR4, FLIPs, and SODD expression in Amol/L) or VP-16 (50 Amol/L) for 30 minutes followed by incubation in drug- camptothecin- and VP-16-treated HL60 cells. Cells were treated with free medium. Cell extracts prepared from control and drug-treated cells at 2 camptothecin (1.0 Amol/L) or VP-16 (50 Amol/L) for 30 minutes followed and 3.5 hours after treatment were subjected to gel filtration chromatog- by incubation in drug-free medium. At the indicated times after raphy on a Superose 6HR 16/50 column (flow rate, 500 AL/min). Fractions camptothecin (left) or VP-16 (right) treatment, Western blot analysis of 2.5 mL were collected (number above), and 80-AL aliquots of each was done. Typical film exposures and relative densitometry analysis done fraction were analyzed by SDS-PAGE and Western blotting. Arrows, elution in triplicate of n experiments. ., data distribution; open columns, means; profiles of the molecular weight standard proteins. A, elution profiles of bars, FSD; filled columns, means; bars, FSE; P values, Student’s t test. DR3, DR4, and FAS. B, elution profiles of TRADD and FADD. C, elution Differences with P < 0.05 were considered significant. CRK-L expression profiles of SODD and FLIPs. D, elution profile of procaspase-8 and caspase- as loading control. 8 fragments.

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Figure 5. Effect of FADD-DN, TRADD-ND, DAP3-DN, and SODD overexpression on camptothecin- and VP-16-induced apoptosis in HL60 and U937 cells. HL-60 (A and B) and U937 (C and D) cells were transfected by electroporation with control vector, GFP-FADD-DN, HA-TRADD- ND, DAP3-DN, or FLAG-SODD. Transfection efficiency was determined by measuring GFP by flow cytometry (f70%). After 24 hours, cells were treated with camptothecin (1.0 Amol/L) or VP-16 (50 Amol/L) for 30 minutes followed by incubation in drug-free medium. A and C, representative expression level of GFP-FADD-DN, HA-TRADD-ND, DAP3-DN, or FLAG-SODD in HL60 (A) and U937 (C) cells 24 hours after transfection. B and D, at the indicated times (x axis, h) after camptothecin or VP-16 treatment, kinetics of DNA fragmentation were determined by DNA filter elution assays. B, points, mean % DNA fragmentation of three independent experiments; bars, FSE. D, points, mean F range of two independent experiments. n, control vector; E, GFP-FADD-DN; !, HA-TRADD-ND; x, DAP3-DN; ., FLAG-SODD.

SODD, FADD-DN, TRADD-ND, and DAP3-DN Overex- VP-16 treatment. Similar results were obtained in U937 pression Did Not Impair Camptothecin and VP-16- cells (Fig. 5C and D). These results revealed that the im- Induced Apoptosis in HL60 and U937 Cells portance of DR3 and DR4 aggregation to signal apoptosis From the above experiments, it was difficult to in these cells after camptothecin or VP-16 treatment is associate or not a functional role for the DR3 and DR4 uncertain, or at least not linked with the death-inducing high molecular weight aggregates in signaling caspase signaling pathways inhibited by a high expression level of activation and apoptosis after camptothecin or VP-16 SODD, FADD-DN, TRADD-ND, and DAP3-DN. treatment. Whereas all or part of the DR3 complexes Combination Treatment of Camptothecin or VP-16 could be inhibited by the presence of SODD, no adapter withTRAIL,TWEAK, orTL1A or inhibitory proteins were found to be associated with Several reports have shown that chemotherapeutic the DR3 or DR4 clusters, but a low amount of agents increase FAS- or TRAIL-induced apoptosis, asso- procaspase-8 was detected in high molecular weight ciated or not with up-regulation of FAS, DR4 or DR5 fractions with DR3 and/or DR4 after VP-16 treatment. To (reviewed in ref. 18). Thus, to investigate a functional further elucidate these questions, we examined the significance for camptothecin- and VP-16-mediated DR3 consequences of overexpressing SODD, FADD-DN, and DR4 aggregation, we examined the kinetics of TRADD-ND, and DAP3-DN on camptothecin- and VP- apoptosis in HL60 cells treated with TRAIL, the DR4 16-induced apoptosis. Figure 5A shows representative ligand, and TWEAK or TL1A, described as putative DR3 expression level of these proteins achieved in transient ligands, alone or in combination with camptothecin or transfection experiments on HL60 cells. The high expres- VP-16. Figure 6 shows that camptothecin (1.0 Amol/L) or sion level of SODD, FADD-DN, TRADD-ND, and DAP3- VP-16 (50 Amol/L) treatment in combination with TRAIL DN did not impair the kinetics of DNA fragmentation (Fig. (250 ng/mL), substantially accelerated kinetics of apoptosis 5B) and caspase activation, including IETDase (caspase- than treatment with camptothecin, VP-16, or TRAIL alone. 8 like), LEHDase (caspase-9 like), and DEVDase (caspase-3 In contrast, cotreatment of camptothecin (1.0 Amol/L) or like) activities (data not shown), after camptothecin or VP-16 (50 Amol/L) with TWEAK (500 ng/mL), or TL1A

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Figure 6. Cotreatment of camptothecin or VP-16 with TRAIL, TWEAK, or TL1A in HL60 cells. [2-14C]-Thymi- dine-labeled cells were treated with (A) 250 ng/mL TRAIL, (B)500ng/mL TWEAK, and (C) 500 ng/mL TL1A alone or in combination with 1.0 Amol/ L camptothecin or 50 Amol/L VP-16. Kinetics of DNA fragmentation, at various time points (xaxis, h), was determined by DNA filter elution assay. Points, mean % DNA fragmentation (n =4inA, n =3inB and C); bars, FSE. n, camptothecin or VP-16 alone; E, TRAIL, TWEAK, or TL1A alone; ., camptothecin or VP-16 in combination with TRAIL, TWEAK, or TL1A.

(500 ng/mL), did not sensitize or accelerate apoptosis than receptor family members, including FAS, DR4, or DR5, treatment with camptothecin, VP-16, TWEAK, or TL1A with or without the activation of their specific ligands, in alone in HL60 cells. The HL60 cells were resistant to apoptosis induced by radiotherapy and chemotherapy in a TWEAK- or TLA1-mediated apoptosis at concentrations variety of cancer cells. In contrast, others have reported that up to 10 Ag/mL (data not shown). genotoxic stress-induced apoptosis could be independent of the cell death receptor signaling pathways (reviewed in ref. 18). From these studies, it seemed that in some cells, Discussion both a cell death signaling system involving FAS, DR4, or In the present study, we examined the participation of DR3 DR5 and the mitochondrial pathway participated in and DR4 in signaling apoptosis after DNA topoisomerase I triggering apoptosis after drug treatment, whereas in other and II inhibitor treatment in HL60 and U937 cells. Several cells, apoptosis was predominantly controlled by mito- studies have suggested the participation of cell death chondria-driven events.

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In this study, we observed, by indirect immunofluor- FADD-DN that has a similar but more dominant effect escence and immunoperoxidase staining, the rapid aggre- compared with FLIPS in preventing the formation of active gation and oligomerization of DR3 and DR4, but not FAS, DISC. Also, similar experiments were done with other in HL60 cells treated with camptothecin or VP-16. Using gel adaptor proteins, including DAP3-DN and TRADD-ND. filtration column chromatography, we confirmed the High expression levels of FADD-DN, DAP3-DN, and aggregation of DR3 after camptothecin and VP-16, and of TRADD-ND achieved in transient transfection experiments DR4-only after VP-16 treatment. The molecular mecha- did not inhibit the kinetics of DNA fragmentation after nisms underlying DR3 and DR4 aggregation after campto- camptothecin and VP-16 treatment in HL60 and U937 cells. thecin or VP-16 treatment are still unknown. It is generally Altogether, these results are in agreement with previous believed that the death domains of these receptors can self- observations indicating that the presence of a neutralizing associate upon stimulation by their respective ligands or anti-FAS immunoglubulin G antibody failed to inhibit both independently of ligands when highly expressed. No doxorubicin- and VP-16-induced apoptosis in HL60 cells increase in DR3 or DR4 and FAS expression was observed (36), and that HL60 cells may be like type II cells with after camptothecin or VP-16 treatment in HL60 cells. respect to the importance of cell death receptor-initiated Although activation and the expression level of their apoptotic signaling after chemotherapy (25, 37). respective ligands were not measured in this study, earlier Although, a recruitment and activation of cell death reports have revealed that neither FAS nor its ligand was receptor/ligand systems are not absolutely required for up-regulated in doxorubicin-treated HL60 cells (36), and radiotherapy- and chemotherapy-induced apoptosis in a that DR4 and TRAIL levels did not increase in VP-16- or variety of tumor cells, including the HL60 cell line (this doxorubicin-treated HL60 cells (37). study and refs. 36, 37), many reports have revealed that SODD, the silencer of death domain-containing recep- combined cell death ligand and drug treatment produces a tors, including TNF-R1 and DR3 (22), showed strong synergistic cytotoxic effect, which may prove useful in the coelution profiles in fractions with DR3, predominantly treatment of various human tumors, including leukemia after camptothecin, and slightly after VP-16 treatment, by and lymphoma (reviewed in ref. 18). These effects were gel filtration column chromatography. These elution also observed in CEM and HL60 cells, where low-dose profiles suggested that the association between DR3 agonistic anti-FAS IgM antibodies or TRAIL ligand in and SODD could prevent the recruitment and binding of combination with DNA topoisomerase I or II inhibitors, the adapter proteins TRADD and FADD, blocking the elicited a synergistic cytotoxic effects, although neither FAS formation of active death-inducing signaling complexes. and its ligand nor DR4 and TRAIL were found to be up- Moreover, the expression level of SODD decreased signifi- regulated in these cells (36, 37). Similar observations were cantly at 4 hours after camptothecin and VP-16 treatment in made in our study, where camptothecin or VP-16 treatment HL60 cells, raising the possibility that DR3 could be in combination with TRAIL, substantially accelerated released from the blocking effect of SODD. Such release kinetics of apoptosis than treatment with camptothecin, could eventually initiate the formation of active death- VP-16 or TRAIL alone. Taken together, our results and inducing signaling complexes that would participate in those of others (36, 37) suggest that, even in the absence of signaling apoptosis. To further elucidate if decreased increased expression levels, some of these cell death SODD expression could have an effect on the kinetics of receptors rapidly aggregate at the cell surface after caspase activation and apoptosis induced by camptothecin camptothecin or VP-16 treatment. In turn, such aggregation and VP-16, a high expression level of SODD was achieved may set or sensitize these cells to low-dose cell death in transient transfection experiments. In these experiments, ligand treatment. a high expression level of SODD in HL60 and U937 cells In this study, we also observed that the less-characterized did not impair or slow the kinetics of apoptosis after DR3 rapidly aggregated at the cell surface and that the camptothecin and VP-16 treatment. These results indicated SODD expression level decreased after drug treatment. that the aggregation of DR3 and down-regulation of SODD Two ligands have been proposed to bind DR3, including were neither absolutely required nor strongly linked with APO-3L/TWEAK and TL1A, although some controversial the kinetics of apoptosis in these cells after camptothecin or data have emerged in the past concerning their specificity VP-16 treatment. (38–43). In our study, treatment with TWEAK or TL1A Similarly, the FLIPS expression level decreased signifi- alone did not induce apoptosis in HL60 cells, and cotreat- cantly after VP-16 treatment in HL60 cells. Although ment with camptothecin or VP-16 did not accelerate FLIPS did not coelute by gel filtration column chroma- kinetics of apoptosis than treatment with camptothecin or tography in fractions with DR4 or DR3, it was found in VP-16 alone. Thus, our data support those previous similar fractions with the adapter protein FADD and observations suggesting that TWEAK or TL1A are poor TRADD and with procaspase-8. FLIPS contains two death death ligand of DR3. effector domains and competes between adapter proteins In conclusion, camptothecin- and VP-16-induced apopto- and procaspase-8, preventing the formation of active sis in human leukemia HL60 and U937 cells is independent DISC (21). To further elucidate if decreased FLIPS of DR3 and DR4 signaling pathways. However, camptothe- expression could have an effect on camptothecin- or VP- cin or VP-16 substantially accelerate TRAIL-, but not 16-induced caspase activation and apoptosis, we used TWEAK- or TL1A-induced apoptosis in HL60 cells.

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Downloaded from mct.aacrjournals.org on September 28, 2021. © 2004 American Association for Cancer Research. 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, Myriam Beauchemin and Richard Bertrand

Mol Cancer Ther 2004;3:1659-1669.

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