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Fas-Mediated Apoptosis in Human Prostatic Carcinoma Cell Lines Occurs Via Activation of Caspase-8 and Caspase-71

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Fas-Mediated Apoptosis in Human Prostatic Carcinoma Cell Lines Occurs Via Activation of Caspase-8 and Caspase-71 [CANCER RESEARCH 58. 5870-5875. December 15. 1998] Fas-mediated Apoptosis in Human Prostatic Carcinoma Cell Lines Occurs via Activation of Caspase-8 and Caspase-71 Oskar W. Rokhlin, Rebecca A. Glover, and Michael B. Cohen2 From the Departments of Pathology ¡O.W. R., R. A. C., M. B. C.¡and Urolog\ ¡M.B. C.¡,The University of Iowa, and Veterans Affairs Medical Center ¡M.B. C.¡,Iowa City, Iowa 52242 ABSTRACT and -10 may be dependent on autocatalysis, which is triggered by interaction with FADD or FADD-like death-effector proteins that We previously demonstrated that treatment with cycloheximide (CHX) receive the apoptotic signal from Fas- or TNF-a-receptors (5). The converted the phenotype of Fas-resistant human prostatic carcinoma cell mode of activation of caspase-8 remained unclear because caspase-8 lines to Fas-sensitive and that resistance to Fas-mediated apoptosis was represents the most proximal caspase in the Fas- and TNF-a-mediated due to a dominant-negative protein(s). In this study, we investigated the sequential activation of caspase family members, to gain insight into the apoptosis. However, it has recently been shown that clustering of likely site of action of the suppressor protein(s). We did not find Tyr-Val- procaspase-8 to Fas-FADD complex causes activation of caspase-8 Ala-Aspase activity in any of the cell lines examined. Time-dependent due to an intrinsic proteolytic activity of the zymogen (6). Asp-Glu-Val-Aspase activity was detected during Fas-mediated apoptosis We have previously shown that of six human prostatic carcinoma in Fas-sensitive cell lines PC3 and ALVA31. Asp-Glu-Val-Aspase activity cell lines investigated (ALVA31, DU 145, JCA1, LNCaP, ND1, and in Fas-resistant cell lines 1)1 145 and .1CVI. was detected only under PC3), agonistic anti-Fas antibody induced apoptosis in only two, PC3 combined treatment with CHX and anti-Fas agonistic mAb. In experi and ALVA31. However, treatment with CHX converted the pheno ments with caspase inhibitors we show that Fas-mediated apoptosis in type of Fas-resistant cell lines DU 145 and JCA1 to Fas sensitive (7). PC3 is mainly executed by the caspase-3 subfamily, but another mem- Subsequently, we generated somatic hybrids between Fas-resistant ber(s) of the caspase family may be involved in Fas-mediated apoptosis in and Fas-sensitive cell lines and found that hybrid cells were resistant ALVA31, DU145, and JCA1. Western blot analysis revealed that Fas- ligation activated caspase-7, but not caspase-3. The activated form of to apoptosis (8). Thus, resistance to Fas-mediated apoptosis is thought caspase-8 was detected in DU 145 only after 4 h of simultaneous treatment to be an active process mediated by a labile protein(s), and dominates with CHX and anti-Fas mAb, whereas in PC3 caspase-8 was found to be over sensitivity. However, the individual caspases responsible for activated after l h of Fas-ligation. We have also found that treatment with Fas-mediated apoptosis in prostatic carcinoma cell lines have not been staurosporin did not activate caspase-8, whereas staurosporin induced identified. In addition, the hierarchy of caspase activation is unknown. apoptosis at the same levels in both Fas-resistant and Fas-sensitive cell In this study, we investigated the possible involvement of caspase lines. These results suggest that an inhibitory protein(s), which suppresses subfamilies in Fas- and TNF-a-mediated apoptosis in human prostatic apoptosis in Fas-resistant cell lines, presumably acts at the apex of apop- carcinoma cell lines. To perform these experiments, we used ICE- totic cascade by preventing the activation of caspase-8. like- (YVAD) and CPP32-like (DEVD)-specific substrates, five dif ferent cell-permeable inhibitors of caspases and mAbs specific to both INTRODUCTION proenzyme and the large subunits of caspase-8, -3, and -7. Our data indicate that the CPP32-like subfamily is involved in Fas-mediated Execution of apoptosis in eukaryotic cells is an active biochemical apoptosis in PC3 and ALVA31 and in TNF-a-mediated apoptosis in process that depends upon activation of proenzymes of the aspartate- specific cysteine protease family (caspases; reviewed in Refs. 1-3). PC3. However, none of the inhibitors used completely prevented TNF-a-mediated apoptosis in ALVA31. These results, therefore, in Comparative analysis of the caspases reveals the existence of three subfamilies: an ICE3 (3) subfamily (comprising caspases-1, -4, and dicate that in addition to caspases another protease(s) may participate in the execution of TNF-a-mediated apoptosis in ALVA31. We have -5), a CPP32 subfamily (comprising caspases-3, -6, -7, -8, -9, and also demonstrated that caspase-8 is activated first during Fas-medi -10), and an ICH-l/Nedd2 (caspase-2) subfamily. Although members ated apoptosis, which in turn induced activity of caspase-7. Impor of the ICE-like and CPP32-like subfamilies have different substrate tantly, we observed the same hierarchy of caspase activation in specificity, all caspases are cleaved at specific Asp residues and, Fas-sensitive and Fas-resistant cell lines under treatment with CHX based on that observation, a model of hierarchiecal activation of and Fas-ligation. Taken together, these results indicate that an inhib caspases has been proposed (4). In this model, caspase-8 has been itor protein(s) in Fas-resistant cell lines act upstream possibly inhib termed an initiator protease, which activates executioner proteases iting the activation of caspase-8. such as caspase-3 or caspase-7. Once procaspase-3 is activated by upstream caspases, it could activate procaspase-6, which in turn may feed back on procaspase-3, resulting in a protease amplification cycle. MATERIALS AND METHODS It has been also suggested that activation of the upstream caspase-8 Cell Culture. The various human prostatic carcinoma cell lines were described previously (7). Cells were cultured in RPMI1640 supplemented with Received 7/10/98; accepted 10/16/98. 100 units/ml penicillin, 100 /ng/ml srteptomycin, 10 rriMHEPES, 1 mM sodium The costs of publication of this article were defrayed in part by the payment of page pyruvate, 10% heat-inactivated PCS (Hyclone Laboratories), 0.1 mM 2-mer- charges. This article must therefore be hereby marked advertisement in accordance with 18 I'.S.C Section 1734 solely to indicate this fact. captoethanol, and 2 mM L-glutamine. Cells were subcultured at 1:10 dilution by 1Supported in part by NIH Grant CA 76673 (to M. B. C.). trypsinization for 2-4 min at room temperature. 2 To whom requests for reprints should be addressed, at Department of Pathology. The Quantitative DNA Fragmentation Assay. The method was described University of Iowa. 200 Hawkins Drive. 5216 RCP. Iowa City, IA 52242-1009. Voice: (319) 356-4510; Fax: (319) 356-8470; E-mail: [email protected]. previously (7). Briefly, to quantitate DNA lost during apoptosis, cells were 'The abbreviations used are: ICE. interleukin-1ß-converting enzyme; AMC, amino- prelabeled overnight with [3H]thymidine and then cultured for various times in methylcoumarin: Ac, acetyl; CHO. aldehyde: CMK. chloromethylketone; FMK, fluoro- 96-well flat-bottomed plates (5-10,000 cells/well) in the presence of different melhylkelone: Z. benzyloxycarbonyl; VAD, Val-Ala-Asp; YVAD, Tyr-Val-Ala-Asp; concentrations of TNF-a and anti-Fas mAb (IPO-4). In separate experiments, DEVD, Asp-Glu-Val-Asp; VDVAD. Val-Asp-Val-Ala-Asp; IETD. Ile-GIu-Thr-Asp; PARP, poly (ADP-ribose) polymerase; Rb, retinoblastoma protein: CHX, cycloheximide; cells were treated with kinase and phosphatase inhibitors: okadaic acid, sodium mAb, monoclonal antibody; CPP32, 32 kDa cysteine protease; FADD, Fas-associating orthovanadate, staurosporin, and genistein (Sigma Chemical Co.). The radio protein with death domain; TNF. tumor necrosis factor. activity was measured by liquid scincillation counting in triplicate or sextu- 5870 Downloaded from cancerres.aacrjournals.org on October 2, 2021. © 1998 American Association for Cancer Research. CASPASE ACTIVATION IN PROSTATIC CARCINOMA plicate samples. The percentage of DNA fragmentation was calculated as 40 60 follows: PC3 ALVA31 50- A 30 cpm in untreated group-cpm in treated group o x 100 40- cpm in untreated group 30- Inhibition of Apoptosis. To investigate the role of different inhibitors in 20- apoptosis, the DNA fragmentation assay was performed in the presence of five o different cell-permeable inhibitors of caspases: Z-VAD-FMK. Ac-YVAD- S 10 „-O—O O—O—•O*""" YVADase CMK. Z-DEVD-FMK, and Z-VDVAD-FMK (all from Calbiochem), and 10- Ac-IETD-CHO (BIOMOL). Inhibitors and apoptosis inducers (anti-Fas mAb x -x x ~_L'XX o or TNF-a) were added at the time of plating, and apoptosis was measured by 60 DNA fragmentation assay. JCA1 DU 145 Assay of Caspase Activity. To measure caspase activity, cytosolic extracts were prepared as described (9) by repeated freezing and thawing of cells in is 2 40H KPM buffer [50 mM PIPES-NaOH (pH 7.0), 50 mM KC1, 10 mM EGTA, 1.92 o. mM MgCli, 1 mM DTT, 10 jag/ml cytochalasin B, and 2 jug/ml pepstatin. |ÃŽ30- leupeptin. and antipain]. Protein lysate (25-50 jig) was incubated at room le E temperature for 30. 60. and 90 min in assay buffer [50 mM PIPES-KOH (pH S 20- 7.0), 0.1 mM EDTA, and 10% glycerol] with 20 JJ.Mfluorescent substrates: Ac-DEVD-AMC as a CPP32-like caspase substrate, and Ac-YVAD-AMC as < 10-1 an ICE-like caspase substrate. Fluorescence at 360/460 nm was measured with CHX a FL500 fluoremeter (Bio-Tek Instruments, Ine). Measurements were cali o brated against a standard curve of AMC (Sigma Chemical Co.).
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