Bcl-xL Functions Downstream of Caspase-8 to Inhibit Fas- and Tumor Necrosis Factor Receptor 1-induced Apoptosis of MCF7 Breast Carcinoma Cells

• DOI: 10.1074/jbc.273.8.4523
• Publication Date: 2002-07-26T14:47:21Z
• Authors: Anu Srinivasan; Feng Li; Angela Wong; Lalitha Kodandapani; Robert Smidt; Joseph F. Krebs; Lawrence C. Fritz; Joe C. Wu; Kevin J. Tomaselli
• Keywords: Fas ligand; Caspase 8; Bcl-xL

Stimulation of the Fas or tumor necrosis factor receptor 1 (TNFR1) cell surface receptors leads to activation death effector protease, caspase-8, and subsequent apoptosis. In some cells, Bcl-xL overexpression can inhibit anti-Fas- (TNF)-α-induced To address effect on caspase-8 processing, Fas- TNFR1-mediated apoptosis were studied in MCF7 breast carcinoma line stably transfected with human cDNA (MCF7/F) double cDNAs (MCF7/FB). strongly inhibited induced by either anti-Fas TNF-α. addition, prevented change cytochrome c immunolocalization TNF-α treatment. Using antibodies that recognize p20 p10 subunits active proteolytic processing was detected MCF7/F cells following TNF-α, but not during UV-induced MCF7/FB processed normally while downstream caspase-7 markedly attenuated. Moreover, direct microinjection recombinant, completely Bcl-xL. These data demonstrate exert an anti-apoptotic function which is activated. 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Thompson 3759-3764Crossref (207) Biochemical studies have functions upstream caspase-3 inhibiting Fas-induced Jurkat also shown (28Boise localize cytoplasmic membranes mitochondria, endoplasmic reticulum, nuclei (29Hockenbery D.M. Nunez Milliman Screiber R.D. 1990; 348: 334-336Crossref (3530) 30Krajewski Tanaka Takayama Schibler Fenton Reed J.C. Cancer Res. 1993; 53: 4701-4714PubMed 31Lithgow van Driel Bertram Cell Growth Differ. 3: 411-417Google 32González-Garcia Pérez-Ballestero Boise Nùñez Development. 120: 3033-3042Crossref Given localization plasma membrane-localized, DISC-associated we interested determining whether stimulation. We chose study express doubly Since endogenously, afforded opportunity same cell. study, that, although overexpressing protected apoptosis, normal kinetics. blocks caspase-8. Bcl-xLcan block (MCF7/FB) (kind gifts Dr. V. Dixit, Genentech, Inc., San Francisco, CA) grown RPMI 1640 medium, supplemented 10% fetal bovine serum, 200 units/ml penicillin, μg/ml streptomycin, neomycin, 150 hygromycin. Cells 60–75% confluence co-treated 50 ng/ml (MBL, PanVera Labs, Madison, WI) plus cycloheximide 40 TNF (R&D Systems, Minneapolis, MN) cycloheximide. For UV treatment, irradiated 100 mJ/cm2 short wavelength (UV Crosslinker, Fisher) then incubated 37 °C. At various times, harvested scraping rubber policeman centrifuging 700 × g. (∼20 million/plate) lysed 300 μl lysis buffer (10 mm Hepes, pH 7.4, 42 KCl, 5 MgCl2, phenylmethylsulfonyl fluoride, 0.1 EDTA, EGTA, dithiothreitol, pepstatin A, leupeptin, aprotinin, 0.5% CHAPS). Following 30-min incubation ice, lysates centrifuged 14,000 g, clear supernatants for Western analysis. Protein concentrations measured using Bio-Rad DC determination kit (Bio-Rad). Caspase-8 most prodomain sequence (corresponding amino acids 1–212) deleted cloned into pET21b (Novagen, WI), transformed Escherichia coli BL21 (DE3), expressed as COOH-terminal 6-His fusion protein. Bacterial cultures LB/ampicillin °C mmisopropyl-1-thio-β-d-galactopyranoside 4 h 25 °C, pellets collected centrifugation 10 min 2000 g prepared sonicating Tris, 20 NaCl, 0.1% Triton X-100, mg/ml lysozyme, (30,000 min). His-tagged purified bacterial lysate nickel chromatography Hi-trap column (Pharmacia Biotech Inc.) eluted imidazole gradient (60 1m). found enzymatically (specific activity = 0.111 μmol AMC/mg protein/min). Unit enzyme defined amount required generate AMC/h μm AcDEVD-AMC substrate 250 assay mixture. rabbits immunized recombinant described above. Affinity purification columns generated binding denatured cross-linked 6% beaded agarose through sulfhydryl groups (Sulfolink Kit, Pierce). Columns immune serum overnight followed washing Tris-HCl, high salt (500 NaCl 7.4). glycine, 2.5 (33Harlow Lane Antibodies: Laboratory Manual. Cold Spring Harbor Laboratory, Harbor, NY1988: 285-317Google Specificity affinity confirmed blotting against panel (caspase-2, -3, -6, -7, -8, -9, -10). recognized cross-reacted only p17, p10, subunit caspase-3. monoclonal 1E8, mice CRGTELDCGIETD COOH terminus CPP32) conjugated keyhole limpet hemocyanin terminus. B excised spleens fused Sp2/0 myeloma cells. Hybridomas screened enzyme-linked immunosorbent Single cloning done limiting dilution, IgG large scale G-Sepharose 1E8 tested (see above) -7. procaspase-3 purchased Transduction Laboratories (Lexington, KY). (50 μg protein/lane) resolved SDS-polyacrylamide gel electrophoresis 16% gels (Novex, La Jolla, transferred Immobilon polyvinylidene difluoride (Millipore, Bedford, MA). Membranes blocked Tween (PBST) + 0.4% casein (I-block, Tropix, Blots primary diluted PBST/casein h. three washes PBST, blots 1:15,000 dilutions alkaline phosphatase goat anti-rabbit anti-mouse (Tropix) PBST/casein. washed twice diethanolamine, 10.0, MgCl2), chemiluminescent CSPD exposed Biomax film (Eastman Kodak Co.) overnight. plated 25,000 per chamber 8-well slides (Nunc, Naperville, IL). treated times. Prior immunostaining, fixed formalin PBS 15 min, stored up 24 Fixed X-100. 0.5 anti-cytochrome (clone 6H2.B4, Pharmingen, 2% Texas Red (Molecular Probes, Portland, OR). Finally, times PBST mounted. Phase contrast fluorescent images captured Sony CatsEye digital camera appropriate filters. scored (phase bright, condensed cells) immunolocalization. each time point, 150–250 counted; course experiment constituted duplicate triplicate slides. presented single representative results obtained experiments. performed Nikon Diaphot inverted microscope fitted Eppendorf pressure injector (model 5246) micromanipulator 5171). Microinjection needles (about 0.1-μm inner diameter) made glass capillaries horizontal electrode puller (Sutter Instrument, P-97) loaded microloaders. cellocate coverslips (Eppendorf) prior injection. identify injected injectate contained 0.3% solution dextran Probes) water. Dye alone dye cytoplasm (pressure, 80 hPa; time, 0.3 s). switched fresh medium immediately concentration pipette 60 ng/ml, equivalent 3 units/μl enzyme. Based approximate volume delivered (0.05 pl) (34Minaschek Bereiter-Hahn Berthold Exp. 1989; 183: 434-442Crossref (53) estimated fg 1.5 10−7 units activity. 100–150 condition examined points fluorescence microscopy. Apoptotic identified morphologically round, condensed, bright Photomicrographs camera. expressing (35Duan Hudson P.L. Wing He W.-W. 1621-1625Abstract (325) Scholar) anti-Fas/cycloheximide TNF/cycloheximide irradiation. As determined rounding nuclear condensation (Fig. A), 70–90% within B). contrast, about 90% resistant UV, remaining flat appeared microscopy 1,A B) staining Hoechst 33258 (data shown). release mitochondria pre-apoptotic (36Yang Liu Bhalla Kim C.N. Ibrado Cai Peng T.-I. Jones D.P. 275: 1129-1132Crossref (4394) 37Kluck R.M. Bossey-Wetzel Green Newmeyer D.D. 1132-1136Crossref (4265) 38Kharbanda Pandey Schofield Isreals Roncinske Yoshida Bharti Yuan Z.-M. Saxena Weichselbaum Nalin Kufe 6939-6942Crossref (368) Cytochrome crelease Bcl-xL-transfected immunocytochemistry antibody. untreated localized perinuclear, punctate pattern 2 A). Consistent previous observations (37Kluck changed treatment Within more than had altered pattern, increasing >90% observed one immunoreactive cytochromec no longer confined become diffusely cytoplasm, became immunonegative c. did significant Instead, Bcl-xL-expressing post Similar radiation paradigms 39Kim Huang Fang 57: 3115-3120PubMed Bcl-xLexpression prevents changes cimmunolocalization accompany summary, several criteria (Figs. 2), heterologous expression protects immunoblotting affinity-purified proform, specificity caspase-2, -10. highly selective cross-reacting rule out possibility below) p20, caspase-3-specific While readily extracts there detectable G). corresponds p17. first post-treatment, respectively 3, 8 declined rapidly thereafter, becoming undetectable previously similarly transitory occurred rapid kinetics here. Interestingly, any point irradiation C). staurosporine Processing appearance degree control does even though Caspase-7 antibody, recognizes however, D E), since levels anti-Fas, 3,D–F). peaked 16 substantially attenuated D–F). partially suppressed whereas 3). This suggested might could contribute initial directly, vitro determine if indeed active, attempted measure protease TNF-treated points. tetrapeptide Ac-DEVD-AMC well caspases. However, DEVD-cleaving Our inability enzymatic probably due >100-fold lower rate catalysis DEVD-AMC compared 2J. Krebs, unpublished observations. unable directly hypothesized should delivery Therefore, led 70% 6 post-injection 4). Caspase-8-injected displayed rounded morphology nearly remained smooth, flattened capable cyotosol. demonstrates rendered membrane proximal observation, Fas-expressing far recruited ligation its 54-kDa That component FADD dominant negative mutation present demonstrating provides evidence pathway. suggesting these engage pathway independently Stable greatly inhibition transiently microinjected 3R. Li, place cascade conclusion, processed, would predict occurs subcellular targets unknown, cytosol may access endogenous If true, caspase-8-injected good paradigms, curtailed initiated caspase-like suggest paradigm well. How receptor-associated protease? target unknown. data, caspase-3, -7 Scholar,22Muzio propagating Alternatively, cleave noncaspase targets, altering (40Rudel Bokoch 276: 1571-1574Crossref Whatever must attenuate consequences those cleavages. Bcl-xL, like Bcl-2, association consider possible models system herein. cytosolic directly. require co-localization proteins, perhaps translocation internal membranes. date, neither nor inhibitory key physically associated Bcl-xLhas interact including Bax (41Sedlak T.W. Oltvai Z.N. Yang 92: 7834-7838Crossref (783) Caenorhabditis elegans effector, CED4 (42Wu Wallen H.D. 1126-1129Crossref (285) 43Ottilie Chang Vigna Weeks Oltersdorf Death Differentiation. 4: 526-533Crossref (25) Evidence indirect between via CED4-like (44Chinnaiyan B.R. 1122-1126Crossref (553) Bcl-xL-interacting regulate availability cofactor necessary cleavages ensue. Recent presence additional cofactors cytosol, activating (45Liu Jemmerson 86: 147-157Abstract (4447) profound TNF. stimulate propagation cascade, right compartment event. accomplish sequestering (38Kharbanda indirectly consequence channel-forming (46Minn Velez Schendel S.L. Liang Muchmore S.W. Fesik Fill 385: 353-357Crossref (721) case, release, (47Duckett C.S. Li Mol. 18: 608-615Crossref (192) model. Regardless mechanism our propagated surprising given Data Fig. indicate caspase-7, UV-treated processing. processes appears level below threshold inducing marked need amplification signal vary type another depending, part, complement present. respect, unique lack here question. gratefully acknowledge Vishva providing Fas-transfected Fas/Bcl-xL-transfected Robert helpful discussions, Teresa help culture, Salma Salchi purifications, Lisa Trout administrative assistance.