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

1998; Elsevier BV; Volume: 273; Issue: 8 Linguagem: Inglês

10.1074/jbc.273.8.4523

1083-351X

Anu Srinivasan, Feng Li, Angela Wong, Lalitha Kodandapani, Robert Smidt, Joseph Krebs, Lawrence C. Fritz, Joseph C. Wu, Kevin J. Tomaselli,

NF-κB Signaling Pathways

Stimulation of the Fas or tumor necrosis factor receptor 1 (TNFR1) cell surface receptors leads to the activation of the death effector protease, caspase-8, and subsequent apoptosis. In some cells, Bcl-xL overexpression can inhibit anti-Fas- and tumor necrosis factor (TNF)-α-induced apoptosis. To address the effect of Bcl-xL on caspase-8 processing, Fas- and TNFR1-mediated apoptosis were studied in the MCF7 breast carcinoma cell line stably transfected with human Fas cDNA (MCF7/F) or double transfected with Fas and human Bcl-xL cDNAs (MCF7/FB). Bcl-xL strongly inhibited apoptosis induced by either anti-Fas or TNF-α. In addition, Bcl-xL prevented the change in cytochrome c immunolocalization induced by anti-Fas or TNF-α treatment. Using antibodies that recognize the p20 and p10 subunits of active caspase-8, proteolytic processing of caspase-8 was detected in MCF7/F cells following anti-Fas or TNF-α, but not during UV-induced apoptosis. In MCF7/FB cells, caspase-8 was processed normally while processing of the downstream caspase-7 was markedly attenuated. Moreover, apoptosis induced by direct microinjection of recombinant, active caspase-8 was completely inhibited by Bcl-xL. These data demonstrate that Bcl-xL can exert an anti-apoptotic function in cells in which caspase-8 is activated. Thus, at least in some cells, caspase-8 signaling in response to Fas or TNFR1 stimulation is regulated by a Bcl-xL-inhibitable step. Stimulation of the Fas or tumor necrosis factor receptor 1 (TNFR1) cell surface receptors leads to the activation of the death effector protease, caspase-8, and subsequent apoptosis. In some cells, Bcl-xL overexpression can inhibit anti-Fas- and tumor necrosis factor (TNF)-α-induced apoptosis. To address the effect of Bcl-xL on caspase-8 processing, Fas- and TNFR1-mediated apoptosis were studied in the MCF7 breast carcinoma cell line stably transfected with human Fas cDNA (MCF7/F) or double transfected with Fas and human Bcl-xL cDNAs (MCF7/FB). Bcl-xL strongly inhibited apoptosis induced by either anti-Fas or TNF-α. In addition, Bcl-xL prevented the change in cytochrome c immunolocalization induced by anti-Fas or TNF-α treatment. Using antibodies that recognize the p20 and p10 subunits of active caspase-8, proteolytic processing of caspase-8 was detected in MCF7/F cells following anti-Fas or TNF-α, but not during UV-induced apoptosis. In MCF7/FB cells, caspase-8 was processed normally while processing of the downstream caspase-7 was markedly attenuated. Moreover, apoptosis induced by direct microinjection of recombinant, active caspase-8 was completely inhibited by Bcl-xL. These data demonstrate that Bcl-xL can exert an anti-apoptotic function in cells in which caspase-8 is activated. Thus, at least in some cells, caspase-8 signaling in response to Fas or TNFR1 stimulation is regulated by a Bcl-xL-inhibitable step. Apoptosis is a genetically controlled form of cell death that is conserved from worms to humans (1Steller H. Science. 1995; 267: 1445-1449Crossref PubMed Scopus (2425) Google Scholar). A diverse set of stimuli can trigger the apoptotic process in virtually all nucleated cells (1Steller H. Science. 1995; 267: 1445-1449Crossref PubMed Scopus (2425) Google Scholar, 2Thompson C.B. Science. 1995; 267: 1456-1462Crossref PubMed Scopus (6177) Google Scholar). 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In this study, we demonstrate that, although MCF7 cells overexpressing Bcl-xL are protected from anti-Fas- or TNF-induced apoptosis, caspase-8 is processed with normal kinetics. In addition, Bcl-xL blocks apoptosis induced by microinjection of active caspase-8. Thus, Bcl-xLcan block apoptosis in cells in which caspase-8 has been activated. MCF7 cells stably transfected with human Fas cDNA (MCF7/F) or both human Fas and human Bcl-xL cDNAs (MCF7/FB) (kind gifts of Dr. V. Dixit, Genentech, Inc., San Francisco, CA) were grown in RPMI 1640 medium, supplemented with 10% fetal bovine serum, 200 units/ml penicillin, 200 μg/ml streptomycin, 200 μg/ml neomycin, and 150 μg/ml hygromycin. Cells at 60–75% confluence were co-treated with either 50 ng/ml anti-Fas (MBL, PanVera Labs, Madison, WI) plus 1 μg/ml cycloheximide or 40 ng/ml TNF (R&D Systems, Minneapolis, MN) plus 1 μg/ml cycloheximide. For UV treatment, cells were irradiated with 100 mJ/cm2 short wavelength UV (UV Crosslinker, Fisher) and then incubated at 37 °C. At various times, cells were harvested by scraping with a rubber policeman and centrifuging at 700 × g. Cells (∼20 million/plate) were lysed in 300 μl of lysis buffer (10 mm Hepes, pH 7.4, 42 mm KCl, 5 mm MgCl2, 1 mm phenylmethylsulfonyl fluoride, 0.1 mm EDTA, 0.1 mm EGTA, 1 mm dithiothreitol, 1 μg/ml pepstatin A, 1 μg/ml leupeptin, 5 μg/ml aprotinin, and 0.5% CHAPS). Following a 30-min incubation on ice, cell lysates were centrifuged at 14,000 × g, and the clear supernatants were used for Western analysis. Protein concentrations of the lysates were measured using the Bio-Rad DC protein determination kit (Bio-Rad). Caspase-8 cDNA with most of the prodomain sequence (corresponding to amino acids 1–212) deleted was cloned into pET21b (Novagen, Madison, WI), transformed into Escherichia coli BL21 (DE3), and expressed as a COOH-terminal 6-His fusion protein. Bacterial cultures grown in LB/ampicillin at 37 °C were induced with 1 mmisopropyl-1-thio-β-d-galactopyranoside for 4 h at 25 °C, and cell pellets were collected by centrifugation for 10 min at 2000 × g at 4 °C. Bacterial lysates were prepared by sonicating the pellets in 25 mm Tris, 20 mm NaCl, 0.1% Triton X-100, 0.1 mg/ml lysozyme, and centrifuging at 4 °C (30,000 × g for 40 min). His-tagged caspase-8 was purified from bacterial lysate by nickel chromatography using a Hi-trap column (Pharmacia Biotech Inc.) and eluted with an imidazole gradient buffer (60 mm to 1m). The protein eluted from the column was found to be processed to p20 and p10 subunits and was enzymatically active (specific activity = 0.111 μmol of AMC/mg of protein/min). Unit activity of enzyme is defined as the amount of enzyme required to generate 1 μmol of AMC/h from 10 μm AcDEVD-AMC substrate in a 250 μl of assay mixture. To generate antibodies to caspase-8, rabbits were immunized with recombinant caspase-8 prepared as described above. Affinity purification columns were generated by binding denatured caspase-8 to cross-linked 6% beaded agarose through sulfhydryl groups (Sulfolink Kit, Pierce). Columns were incubated with the immune serum overnight and followed by washing with 10 mm Tris-HCl, pH 7.4, and a high salt buffer (500 mm NaCl in 10 mm Tris-HCl, pH 7.4). Caspase-8 antibodies were eluted using 100 mm glycine, pH 2.5 (33Harlow E. Lane D. Antibodies: A Laboratory Manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1988: 285-317Google Scholar). Specificity of the affinity purified caspase-8 antibody was confirmed by Western blotting against a panel of recombinant human caspases (caspase-2, -3, -6, -7, -8, -9, and -10). The caspase-8 antibody strongly recognized the p20 and p10 subunits of caspase-8 and cross-reacted only with p17, but not the p10, subunit of caspase-3. To generate the monoclonal antibody 1E8, mice were immunized with the peptide CRGTELDCGIETD (corresponding to the COOH terminus of the p20 subunit of human CPP32) conjugated to keyhole limpet hemocyanin through the amino terminus. B cells from excised spleens were fused with Sp2/0 myeloma cells. Hybridomas were screened by enzyme-linked immunosorbent assay and Western analysis. Single cell cloning was done by limiting dilution, and IgG was purified from monoclonal supernatants of large scale cultures using a protein G-Sepharose column (33Harlow E. Lane D. Antibodies: A Laboratory Manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1988: 285-317Google Scholar). The 1E8 monoclonal was tested against the panel of recombinant caspases (see above) and found to recognize the p20 subunits of caspase-3 and -7. The monoclonal antibody to procaspase-3 was purchased from Transduction Laboratories (Lexington, KY). Cell lysates (50 μg of protein/lane) were resolved by SDS-polyacrylamide gel electrophoresis on 16% gels (Novex, La Jolla, CA) and transferred to Immobilon polyvinylidene difluoride membranes (Millipore, Bedford, MA). Membranes were blocked in PBS, 0.1% Tween 20 (PBST) + 0.4% casein (I-block, Tropix, Bedford, MA). Blots were incubated in 1 μg/ml primary antibody diluted in PBST/casein for 1 h. Following three washes in PBST, blots were incubated for 1 h in 1:15,000 dilutions of alkaline phosphatase conjugated goat anti-rabbit IgG or goat anti-mouse IgG (Tropix) in PBST/casein. Blots were then washed twice with PBST, twice in assay buffer (10 mm diethanolamine, pH 10.0, 1 mm MgCl2), and then incubated in 250 μm chemiluminescent substrate CSPD (Tropix) in assay buffer and exposed to Biomax film (Eastman Kodak Co.) overnight. MCF7/F and MCF7/FB cells were plated at 25,000 cells per chamber in 8-well chamber slides (Nunc, Naperville, IL). Cells were treated with 40 ng/ml TNF plus 1 μg/ml cycloheximide for various times. Prior to immunostaining, cells were fixed in 10% formalin in PBS for 15 min, washed with PBS, and stored at 4 °C for up to 24 h. Fixed cells were blocked for 1 h in a buffer containing PBS, 10% normal goat serum, and 0.4% Triton X-100. The cells were then incubated for 1 h in 0.5 μg/ml anti-cytochrome c antibody (clone 6H2.B4, Pharmingen, La Jolla, CA) in a buffer containing PBS, 2% normal goat serum, and 0.4% Triton X-100. Following three washes with PBST, cells were incubated for 1 h with 4 μg/ml Texas Red conjugated goat anti-mouse IgG (Molecular Probes, Portland, OR). Finally, cells were washed three times with PBST and mounted. Phase contrast and fluorescent images were captured on a Sony CatsEye digital camera using appropriate filters. MCF7 cells were scored for apoptosis (phase bright, condensed cells) and for cytochrome c immunolocalization. For each time point, at least 150–250 cells were counted; each time course experiment constituted duplicate or triplicate slides. The data presented are from a single experiment but are representative of results obtained in three experiments. Cell microinjection was performed using a Nikon Diaphot 300 inverted microscope fitted with an Eppendorf pressure injector (model 5246) and micromanipulator (model 5171). Microinjection needles (about 0.1-μm inner diameter) were made from glass capillaries using a horizontal electrode puller (Sutter Instrument, model P-97) and loaded using Eppendorf microloaders. MCF7/F or MCF7/FB cells were plated on glass cellocate coverslips (Eppendorf) 24 h prior to injection. To identify injected cells, the injectate contained 0.3% solution of dextran conjugated to Texas Red (Molecular Probes) in water. Dye alone or dye plus active caspase-8 were injected into the cytoplasm of MCF7 cells (pressure, 80 to 100 hPa; time, 0.3 s). Cells were switched into fresh medium immediately after injection. The concentration of caspase-8 in the pipette was 60 ng/ml, equivalent to 3 units/μl of active enzyme. Based on the approximate volume delivered per cell (0.05 pl) (34Minaschek G. Bereiter-Hahn J. Berthold G. Exp. Cell Res. 1989; 183: 434-442Crossref PubMed Scopus (53) Google Scholar), the concentration of caspase-8 delivered per cell is estimated to be 3 fg or 1.5 × 10−7 units of activity. 100–150 cells were injected per condition and cells were examined at various time points by phase contrast or fluorescence microscopy. Apoptotic cells were identified morphologically as round, condensed, phase bright cells. Photomicrographs were prepared from digital images obtained using a Sony CatsEye digital camera. MCF7 breast carcinoma cells stably expressing Fas alone (MCF7/F) or Fas plus Bcl-xL (MCF7/FB) (35Duan H. Chinnaiyan A.M. Hudson P.L. Wing J.P. He W.-W. Dixit V.M. J. Biol. Chem. 1996; 271: 1621-1625Abstract Full Text Full Text PDF PubMed Scopus (325) Google Scholar) were treated with anti-Fas/cycloheximide or TNF/cycloheximide or exposed to UV irradiation. As determined by cell rounding and nuclear condensation (Fig. 1 A), all three stimuli induced apoptosis in 70–90% of MCF7/F cells within 24 h (Fig. 1 B). In contrast, about 90% of the cells expressing Bcl-xL were resistant to Fas, TNF, or UV, remaining flat with nuclei that appeared normal by phase contrast microscopy (Fig. 1,A and B) or after staining with Hoechst 33258 (data not shown). Bcl-2 has been shown to inhibit cytochrome c release from mitochondria in pre-apoptotic cells (36Yang J. Liu X. Bhalla K. Kim C.N. Ibrado A.M. Cai J. Peng T.-I. Jones D.P. Wang X. Science. 1997; 275: 1129-1132Crossref PubMed Scopus (4394) Google Scholar, 37Kluck R.M. Bossey-Wetzel E. Green D.R. Newmeyer D.D. Science. 1997; 275: 1132-1136Crossref PubMed Scopus (4265) Google Scholar, 38Kharbanda S. Pandey P. Schofield L. Isreals S. Roncinske R. Yoshida K. Bharti A. Yuan Z.-M. Saxena S. Weichselbaum R. Nalin C. Kufe D. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 6939-6942Crossref PubMed Scopus (368) Google Scholar). Cytochrome crelease from mitochondria of Bcl-xL-transfected MCF7 cells treated with anti-Fas or TNF was studied by immunocytochemistry using a cytochrome c monoclonal antibody. In untreated cells, cytochrome c was localized to mitochondria in a perinuclear, punctate staining pattern (Fig. 2 A). Consistent with previous observations (37Kluck R.M. Bossey-Wetzel E. Green D.R. Newmeyer D.D. Science. 1997; 275: 1132-1136Crossref PubMed Scopus (4265) Google Scholar), cytochrome c immunolocalization changed markedly following treatment with the apoptotic stimuli (Fig. 2 A). Within 5 h of treatment with TNF, more than 10% of the MCF7/F cells had an altered cytochrome c staining pattern, increasing to >90% of the cells by 24 h (Fig. 2 B). The altered cytochrome c staining pattern observed was either one in which the immunoreactive cytochromec was no longer confined to mitochondria, but had become diffusely localized in the cytoplasm, or in which the cells became immunonegative for cytochrome c. In contrast, in the MCF7/FB cells TNF treatment did not lead to a significant change in the cytochrome c immunolocalization pattern (Fig. 2 A). Instead, 90% of the Bcl-xL-expressing cells at 24 h post TNF treatment had a normal staining pattern (Fig. 2 B). Similar observations were made on MCF7/FB cells treated with anti-Fas or UV radiation (data not shown). Thus, as observed in other apoptotic paradigms (37Kluck R.M. Bossey-Wetzel E. Green D.R. Newmeyer D.D. Science. 1997; 275: 1132-1136Crossref PubMed Scopus (4265) Google Scholar, 39Kim C.N. Wang X. Huang Y. Ibrado A.M. Liu L. Fang G. Bhalla K. Cancer Res. 1997; 57: 3115-3120PubMed Google Scholar), Bcl-xLexpression prevents the changes in cytochrome cimmunolocalization that accompany apoptosis induced by either Fas or TNFR1 stimulation. In summary, by several criteria (Figs. 1 and 2), heterologous expression of Bcl-xL protects MCF7 cells from Fas- and TNFR1-induced apoptosis. Caspase-8 processing following treatment with anti-Fas or TNF was studied by immunoblotting using affinity-purified caspase-8 antibodies that recognize the p20 and p10 subunits, but not the proform, of caspase-8. The specificity of the caspase-8 antibody was determined by immunoblotting of purified recombinant human caspase-2, -3, -6, -7, -8, -9, and -10. The caspase-8 antibody was highly selective for both the p20 and p10 subunits of caspase-8, cross-reacting only with the caspase-3 p17, but not p10, subunit (data not shown). To rule out the possibility that the p20 subunit observed in the MCF7 cells following anti-Fas or TNF treatment (see below) was not caspase-3 p20, caspase-3 expression in MCF7/F cells was determined by immunoblotting with a caspase-3-specific monoclonal antibody. While caspase-3 was readily detected in extracts of Jurkat cells, there was no detectable caspase-3 in extracts of MCF7/F cells (Fig. 3 G). Thus, the p20 subunit observed following anti-Fas or TNF treatment corresponds to caspase-8 p20 and not caspase-3 p17. The p20 and p10 subunits of active caspase-8 were first detectable in lysates of anti-Fas and TNF treated MCF7/F cells at 2 and 4 h post-treatment, respectively (Fig. 3, A and B). Caspase-8 p20 and p10 subunits were detectable out to 8 h following anti-Fas or TNF treatment but declined rapidly thereafter, becoming undetectable by 24 h. Caspase-8 processing previously described in other cell types (17Medema J.P. Scaffidi C. Kischkel F.C. Shevchenko A. Mann M. Krammer P.H. Peter M.E. EMBO J. 1997; 16: 2794-2804Crossref PubMed Scopus (1038) Google Scholar) was similarly transitory but occurred with more rapid kinetics than observed here. Interestingly, caspase-8 processing was not observed in the UV treated MCF7/F cells at any time point following UV irradiation (Fig. 3 C). Caspase-8 processing was also not observed following treatment with 1 μm staurosporine (data not shown). Processing of caspase-8 was also studied in MCF7/FB cells following anti-Fas or TNF stimulation. The appearance of caspase-8 p20 and p10 subunits appeared with the same kinetics and to the same degree in Bcl-xL-expressing MCF7/FB cells as in the control MCF7/F cells (Fig. 3, A and B). Thus, overexpression of Bcl-xL in MCF7 cells does not block processing of caspase-8, even though it blocks subsequent apoptosis. Caspase-7 processing was also studied in the MCF7/F and MCF7/FB cells using a monoclonal antibody, 1E8, that recognizes the p20 subunits of both caspase-3 and -7. In MCF7 cells, however, 1E8 recognizes only caspase-7 p20 (Fig. 3, D and E), since caspase-3 is not expressed at detectable levels (Fig. 3 G). As described previously for TNF (35Duan H. Chinnaiyan A.M. Hudson P.L. Wing J.P. He W.-W. Dixit V.M. J. Biol. Chem. 1996; 271: 1621-1625Abstract Full Text Full Text PDF PubMed Scopus (325) Google Scholar), caspase-7 was processed in MCF7 cells in response to anti-Fas, TNF treatment, or UV radiation (Fig. 3,D–F). Caspase-7 p20 was first detected at 4 h and peaked at 16 h following anti-Fas, TNF, or UV (Fig. 3,D–F). In contrast to MCF7/F cells, the appearance of the caspase-7 p20 subunit was substantially attenuated in the MCF7/FB cells following anti-Fas or TNF treatment and completely inhibited following UV irradiation (Fig. 3, D–F). Thus, although caspase-8 is processed normally in MCF7/FB cells, caspase-7 processing is partially inhibited by Bcl-xL. Caspase-7 processing in response to UV irradiation was completely suppressed in the MCF7/FB cells, whereas in response to anti-Fas or TNF, caspase-7 processing was not completely suppressed (Fig. 3). This suggested that the processed caspase-8 observed in anti-Fas or TNF treated MCF7/FB cells might be enzymatically active and could contribute to the initial processing of some caspase-7 directly, as observed in vitro (16Srinivasula S.M. Ahmad M. Fernandes-Alnemri T. Litwack G. Alnemri E.S. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 14486-14491Crossref PubMed Scopus (481) Google Scholar, 22Muzio M. Salvesen G.S. Dixit V.M. J. Biol. Chem. 1997; 272: 2952-2956Abstract Full Text Full Text PDF PubMed Scopus (312) Google Scholar). To determine if the processed caspase-8 in MCF7/FB cells was indeed active, we attempted to measure caspase-8 protease activity in extracts of anti-Fas and TNF-treated cells at various time points. The tetrapeptide Ac-DEVD-AMC is a substrate for caspase-8 (16Srinivasula S.M. Ahmad M. Fernandes-Alnemri T. Litwack G. Alnemri E.S. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 14486-14491Crossref PubMed Scopus (481) Google Scholar) as well as other caspases. However, no significant DEVD-cleaving activity was observed in MCF7 cell lysates at any time point following anti-Fas or TNF treatment (data not shown). Our inability to measure caspase-8 enzymatic activity in extracts of MCF7/F cells is probably due to the >100-fold lower rate of catalysis of DEVD-AMC substrate by caspase-8 as compared with caspase-3. 2J. Krebs, unpublished observations. Although we were unable to measure caspase-8 activity directly even in control MCF7/F cells, we hypothesized that if the processed caspase-8 in MCF7/FB cells was active, the MCF7/FB cells should be resistant to apoptosis induced by intracellular delivery of active caspase-8. Therefore, we compared the ability of caspase-8 to induce apoptosis in MCF7/F and MCF7/FB cells following microinjection of active, recombinant caspase-8. Microinjection of a solution containing 3 units/μl human caspase-8 led to apoptosis in 70% of MCF7/F cells by 6 h post-injection (Fig. 4). Caspase-8-injected cells displayed rounded morphology with condensed nuclei similar in appearance to either anti-Fas- or TNF-treated cells (Fig. 4 A). However, in MCF7/FB cells, apoptosis induced by microinjection of caspase-8 was nearly completely inhibited (Fig. 4 B). Caspase-8-injected MCF7/FB cells remained normal in appearance with a smooth, flattened morphology and normal nuclei (Fig. 4 A). Thus, Bcl-xL was capable of inhibiting apoptosis induced by delivery of active caspase-8 into the cell cyotosol. This study demonstrates that caspase-8 is processed with normal kinetics in MCF7 cells rendered resistant to Fas- and TNFR1-induced apoptosis by overexpression of Bcl-xL. Thus, Bcl-xL can function to block apoptosis in cells in which the membrane proximal caspase-8 has been activated. Consistent with this observation, Bcl-xL inhibited MCF7 cell apoptosis induced by microinjection of recombinant, active caspase-8. In several Fas-expressing cell types studied thus far (17Medema J.P. Scaffidi C. Kischkel F.C. Shevchenko A. Mann M. Krammer P.H. Peter M.E. EMBO J. 1997; 16: 2794-2804Crossref PubMed Scopus (1038) Google Scholar), caspase-8 is recruited to the membrane DISC following Fas ligation and is proteolytically processed from its 54-kDa zymogen form to an active protease containing p20 and p10 subunits (17Medema J.P. Scaffidi C. Kischkel F.C. Shevchenko A. Mann M. Krammer P.H. Peter M.E. EMBO J. 1997; 16: 2794-2804Crossref PubMed Scopus (1038) Google Scholar). That caspase-8 functions as a component of a similar DISC comprising TNFR1, TRADD, and FADD has been suggested by the ability of a dominant negative mutation of procaspase-8 to inhibit TNF-induced apoptosis (15Boldin M.P. Goncharov T.M. Goltsev Y.V. Wallach D. Cell. 1996; 85: 803-815Abstract Full Text Full Text PDF PubMed Scopus (2102) Google Scholar). The present study demonstrating activation of caspase-8 in response to TNF treatment provides the first direct evidence that this caspase also functions in the TNFR1 signaling pathway. Interestingly, apoptosis induced by UV irradiation or staurosporine did not lead to caspase-8 processing in MCF7 cells, suggesting that these stimuli engage the apoptotic pathway independently of caspase-8. Stable expression of Bcl-xL in MCF7 cells greatly attenuated apoptotic changes observed following anti-Fas, TNF or UV treatment. A similar inhibition by Bcl-xL of anti-Fas- and TNF-induced apoptosis has also been observed in MCF7 cells transiently transfected with Bcl-xL and in cells microinjected with purified recombinant Bcl-xL. 3R. Armstrong and F. Li, unpublished observations. Our observations place Bcl-xL downstream of activated caspase-8 processing in the Fas- and TNFR1-induced apoptotic cascade in MCF7 cells. Consistent with this conclusion, Bcl-xL was capable of inhibiting apoptosis induced by microinjection of active caspase-8. Biochemical studies have shown that processed, active caspase-8 is released from the DISC following receptor stimulation (17Medema J.P. Scaffidi C. Kischkel F.C. Shevchenko A. Mann M. Krammer P.H. Peter M.E. EMBO J. 1997; 16: 2794-2804Crossref PubMed Scopus (1038) Google Scholar). Although we did not study Fas-induced DISC formation directly, we would predict that it occurs normally in the Bcl-xL overexpressing cells. Although the subcellular localization and targets of activated caspase-8 are unknown, caspase-8 microinjected into the cytosol may have access to the targets of endogenous caspase-8. If true, then the caspase-8-injected cells are a good model of the anti-Fas- and TNF-treated cells. In both paradigms, Bcl-xL overexpression curtailed the apoptotic signaling cascade initiated by active caspase-8. A previous study demonstrated that Bcl-xL could inhibit apoptosis in Jurkat cells in which caspase-like activity was not completely suppressed (28Boise L.H. Thompson C.B. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 3759-3764Crossref PubMed Scopus (207) Google Scholar). Our data suggest that caspase-8 could have been activated in that Bcl-xL paradigm as well. How does Bcl-xL prevent apoptosis after the activation of the receptor-associated caspase-8 protease? The target substrates of caspase-8 in cells are unknown. Based on in vitro data, caspase-3, -6, and -7 could be downstream targets of caspase-8 (16Srinivasula S.M. Ahmad M. Fernandes-Alnemri T. Litwack G. Alnemri E.S. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 14486-14491Crossref PubMed Scopus (481) Google Scholar,22Muzio M. Salvesen G.S. Dixit V.M. J. Biol. Chem. 1997; 272: 2952-2956Abstract Full Text Full Text PDF PubMed Scopus (312) Google Scholar), thus propagating the caspase signal. Alternatively, caspase-8 could cleave noncaspase targets, thus altering their functions (40Rudel T. Bokoch G.M. Science. 1997; 276: 1571-1574Crossref PubMed Scopus (602) Google Scholar). Whatever proteins function as caspase-8 targets, overexpression of Bcl-xL must attenuate either their cleavage or the consequences of those cleavages. Bcl-xL, like Bcl-2, is thought to exert its antiapoptotic function in association with the intracellular membranes of the mitochondria, endoplasmic reticulum, and nuclei (29Hockenbery D.M. Nunez G. Milliman C. Screiber R.D. 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Alternatively, the key targets of caspase-8 could be physically associated with and regulated by Bcl-xL. Bcl-xLhas been shown to interact with several proteins, including Bax (41Sedlak T.W. Oltvai Z.N. Yang E. Wang K. Boise L.H. Thompson C.B. Korsmeyer S.J. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 7834-7838Crossref PubMed Scopus (783) Google Scholar) and the Caenorhabditis elegans cell death effector, CED4 (42Wu D. Wallen H.D. Nunez G. Science. 1997; 275: 1126-1129Crossref PubMed Scopus (285) Google Scholar, 43Ottilie S. Wang Y. Banks S. Chang J. Vigna N.J. Weeks S. Armstrong R.C. Fritz L.C. Oltersdorf T. Cell Death Differentiation. 1997; 4: 526-533Crossref PubMed Scopus (25) Google Scholar). Evidence for an indirect interaction between Bcl-xL and certain caspases via a mammalian CED4-like molecule has been presented (44Chinnaiyan A.M. O'Rourke K. Lane B.R. Dixit V.M. Science. 1997; 275: 1122-1126Crossref PubMed Scopus (553) Google Scholar). However, whether any Bcl-xL-interacting proteins are targets of caspase-8 is unknown. Bcl-xL might also regulate the availability of a cofactor necessary either for caspase-8 to cleave its substrates or for the consequences of those cleavages to ensue. Recent evidence has suggested that Bcl-2 and Bcl-xL regulate the release of cytochrome c in pre-apoptotic cells (36Yang J. Liu X. Bhalla K. Kim C.N. Ibrado A.M. Cai J. Peng T.-I. Jones D.P. Wang X. Science. 1997; 275: 1129-1132Crossref PubMed Scopus (4394) Google Scholar, 37Kluck R.M. Bossey-Wetzel E. Green D.R. Newmeyer D.D. Science. 1997; 275: 1132-1136Crossref PubMed Scopus (4265) Google Scholar, 39Kim C.N. Wang X. Huang Y. Ibrado A.M. Liu L. Fang G. Bhalla K. Cancer Res. 1997; 57: 3115-3120PubMed Google Scholar). In the presence of additional cofactors present in cytosol, cytochromec is capable of activating caspase-3 (45Liu X. Kim C.N. Yang J. Jemmerson R. Wang X. 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Nature. 1997; 385: 353-357Crossref PubMed Scopus (721) Google Scholar). In either case, Bcl-xL would function upstream of cytochromec release, as suggested by previous studies (36Yang J. Liu X. Bhalla K. Kim C.N. Ibrado A.M. Cai J. Peng T.-I. Jones D.P. Wang X. Science. 1997; 275: 1129-1132Crossref PubMed Scopus (4394) Google Scholar, 37Kluck R.M. Bossey-Wetzel E. Green D.R. Newmeyer D.D. Science. 1997; 275: 1132-1136Crossref PubMed Scopus (4265) Google Scholar). The inability of Bcl-xL to inhibit apoptosis induced by direct intracellular microinjection of cytochrome c (47Duckett C.S. Li F. Wang Y. Tomaselli K.J. Thompson C.B. Armstrong R.C. Mol. Cell. Biol. 1997; 18: 608-615Crossref Scopus (192) Google Scholar) is consistent with this model. Regardless of the mechanism of Bcl-xL, our data suggest that caspase-8 signaling in this paradigm is propagated through a Bcl-xL-inhibitable step. This is surprising given the possibility that caspase-8 could activate downstream caspases directly (16Srinivasula S.M. Ahmad M. Fernandes-Alnemri T. Litwack G. Alnemri E.S. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 14486-14491Crossref PubMed Scopus (481) Google Scholar, 22Muzio M. Salvesen G.S. Dixit V.M. J. Biol. Chem. 1997; 272: 2952-2956Abstract Full Text Full Text PDF PubMed Scopus (312) Google Scholar). Data in Fig. 3 indicate that, in the Bcl-xL-expressing cells, activation of caspase-8 by anti-Fas or TNF leads to the processing of a small amount of caspase-7, whereas in UV-treated cells, in which caspase-8 is not activated, Bcl-xL completely prevents caspase-7 processing. If caspase-8 directly processes some caspase-7, it appears that this level of processing is below the threshold for inducing marked apoptotic changes in the MCF7/FB cells. The need for a Bcl-xL-inhibitable amplification of the caspase-8 signal may vary from one cell type to another (23Huang D.C. Cory S. Strasser A. Oncogene. 1997; 14: 405-414Crossref PubMed Scopus (231) Google Scholar, 24Erhardt P. Cooper G.M. J. Biol. Chem. 1996; 271: 17601-17604Abstract Full Text Full Text PDF PubMed Scopus (136) Google Scholar) depending, in part, on the complement of downstream caspases that are present. In this respect, MCF7 cells may be unique in their lack of expression of caspase-3. The model described here provides an opportunity to address this question. We gratefully acknowledge Dr. Vishva Dixit for providing the Fas-transfected and Fas/Bcl-xL-transfected MCF7 cells, Dr. Robert Armstrong for helpful discussions, Teresa Aja for help with cell culture, Salma Salchi for help with antibody purifications, and Lisa Trout for administrative assistance.