Apoptosis-resistant Mitochondria in T Cells Selected for Resistance to Fas Signaling*

Gui-Qiang WangDagger §, Brian R. Gastman§, Eva WieckowskiDagger , Leslie A. GoldsteinDagger , Asaf RabinovitzDagger , Xiao-Ming YinDagger , and Hannah RabinowichDagger ||**

From the Departments of Dagger  Pathology and  Otolaryngology, University of Pittsburgh School of Medicine and || University of Pittsburgh Cancer Institute, Pittsburgh, Pennsylvania 15213

Received for publication, July 13, 2000, and in revised form, November 2, 2000



    ABSTRACT
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Jurkat leukemic T cells are highly sensitive to the extrinsic pathways of apoptosis induced via the death receptor Fas or tumor necrosis factor-related apoptosis-inducing ligand as well as to the intrinsic/mitochondrial pathways of death induced by VP-16 or staurosporin. We report here that clonal Jurkat cell lines selected for resistance to Fas-induced apoptosis were cross-resistant to VP-16 or staurosporin. Each of the apoptotic pathways was blocked at an apical phase, where common regulators of apoptosis have not yet been defined. The Fas pathway was blocked at the level of caspase-8, whereas the intrinsic pathway was blocked at the mitochondria. No processing or activity of caspases was detected in resistant cells in response to either Fas-cross-linking or VP-16 treatment. Also, no apoptosis-associated alterations in the mitochondrial inner membrane, outer membrane, or matrix were detected in resistant Jurkat cells treated with VP-16. Thus, no changes in permeability transition, loss in inner membrane cardiolipin, generation of reactive oxygen species, or release of cytochrome c were observed in resistant cells treated with VP-16. Further, unlike purified mitochondria from wild type cells, those obtained from resistant cells did not release cytochrome c or apoptosis-inducing factor in response to recombinant Bax or truncated Bid. These results identify a defect in mitochondria ability to release intermembrane proteins in response to Bid or Bax as a mechanism of resistance to chemotherapeuetic drugs. Further, the selection of VP-16-resistant mitochondria via elimination of Fas-susceptible cells may suggest the existence of a shared regulatory component between the extrinsic and intrinsic pathways of apoptosis.



    INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Susceptibility to apoptosis is an essential prerequisite for successful treatment of tumor cells by cytotoxic T lymphocytes, natural killer cells, radiation, or chemotherapy. However, resistance to apoptosis has been established as one of the mechanisms responsible for the failure of therapeutic approaches in many types of cancer, including hematopoietic malignancies. At least two pathways of caspase activation have been delineated, including extrinsically and intrinsically stimulated cascades (1-3). The extrinsic pathway involves apoptosis mediated by cell surface death receptors, such as Fas, tumor necrosis factor receptor, or TRAIL1 receptor. Fas stimulation results in oligomerization of the receptors and recruitment of the adapter protein Fas-associated death domain (FADD) and caspase-8, forming a death-inducing signaling complex (DISC) (4). Autoactivation of caspase-8 at the DISC is followed by activation of effector caspases, including caspase-3, -6, and -7, which function as downstream effectors of the cell death program. The intrinsic pathway is mediated by diverse apoptotic stimuli, which converge at the mitochondria. Release of cytochrome c from the mitochondria to the cytoplasm initiates a caspase cascade. Cytosolic cytochrome c binds to apoptosis protease-activating factor 1 (Apaf-1) and procaspase-9, generating a DISC-like complex, "apoptosome" (5, 6). Within the apoptosome, caspase-9 is activated, leading to processing of caspase-3. The two pathways of apoptosis, extrinsic/death receptor and intrinsic/mitochondrial, converge on caspase-3 and subsequently on other proteases and nucleases that drive the terminal events of programmed cell death. The significance of Apaf-1, caspase-9, and caspase-3 for the execution of apoptosis has been confirmed by genetic studies in mice (7-9). Cells derived from Apaf-1, caspase-9, and caspase-3 knockout mice demonstrated defects in response to a variety of apoptotic stimuli. However, T lymphocytes from mice lacking Apaf-1 or caspase-9 were susceptible to apoptosis signaled via Fas or tumor necrosis factor receptors (10, 11). Also, cytochrome c-deficient cells were susceptible to death receptor-mediated apoptosis (12). These findings support the concept that the two apoptotic pathways are discrete and function in parallel. However, each of the pathways is amplified by activated components of the "other" pathway. In cases of low initial caspase-8 activation, the direct effect of caspase-8 on caspase-3 is amplified by a mitochondrial loop (1). In this scenario, caspase-8 cleaves off an N-terminal fragment of Bid, allowing the tBid to translocate to the mitochondria and induce cytochrome c release (13-15). Also, the intrinsic pathway is amplified by components of the death receptor cascade, as cytostatic drugs have been reported to activate caspase-8 secondarily to mitochondrial damage (16, 17). Currently, cross-talk between the extrinsic and intrinsic pathways has been observed mainly as an amplification loop at the level of execution, but not initiation, of each of the cascades. Also, tumor cell lines studied for resistance to apoptosis were resistant to either the death receptor or the mitochondrial pathway but not cross-resistant to both pathways (18). Cross-resistance has been observed in cells overexpressing an inhibitor, such as X-linked inhibitor of apoptosis (XIAP/hILP), which targets downstream effector molecules, such as caspase-3, shared by both pathways (19-22). Also, members of the Bcl-2 family, which play a major role in control of the mitochondria-dependent apoptotic pathway (23), were reported to protect some cell lines and tissues from Fas-induced apoptosis but not others (24, 25). However, attenuation of Fas-mediated apoptosis by Bcl-2 might be mediated by inhibition of the mitochondrial amplification loop (1).

In the current study, we demonstrate the selection of cells resistant to a mitochondrial pathway (intrinsic) by elimination of Fas-sensitive cells (extrinsic pathway). The selection of clonal cell lines completely cross-resistant to inducers of the two apoptotic pathways may suggest the existence of an upstream regulatory component shared by the extrinsic and the intrinsic pathways.


    EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Reagents-- Agonistic anti-Fas Ab (CH-11, IgM) was purchased from Upstate Biotechnology Inc. (Lake Placid, NY). Staurosporin and VP-16 were purchased from Sigma. Recombinant caspase-3 and caspase-8, anti-cytochrome c mAb, rabbit anti-caspase-3 Ab, and anti-CD3 zeta -chain mAb (clone 610B10) were purchased from Pharmingen. Anti-hILP/XIAP mAb (clone 28), anti-caspase-7 mAb (clone 51), and anti-caspase-2 mAb (clone 47) were from Transduction Laboratories (Lexington, KY); rabbit anti-caspase-8 Ab was from StressGen Biotechnology (Victoria BC, Canada); caspase-8-specific mAb (clone 5F7) was from Upstate Biotechnology, Inc.; anti-caspase-9 was from Oncogene (Cambridge, MA); anti-Bcl-2 mAb (clone 100), rabbit anti-Bcl-xL, and goat anti-apoptosis-inducing factor (AIF) Ab were from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA); anti-poly(ADP-ribose) polymerase (PARP) mAb (clone C2-10) was from Enzyme Systems (Livermore, CA); and anti-voltage-dependent anion channel (VDAC) mAb (clone 31HC) was from Calbiochem. Rabbit anti-Bid Ab was generated as described previously (26). Chloromethyl X-rosamine (CMXRos), nonyl acridine orange (NAO), and hydroethidine (HE) were from Molecular Probes, Inc. (Eugene, OR); phosphatidylethanolamine-conjugated anti-APO2.7, fluorescein isothiocyanate-annexin V and propidium iodide were from CLONTECH (Palo Alto, CA); anti-beta -actin mAb (clone AC-15), horse heart cytochrome c, and dATP were from Sigma; and the TRAIL kit was from Alexis (San Diego, CA).

Preparation of GST-Bax and His-tagged tBid-- Mouse Bax-Delta TM (amino acids 1-173) was cloned into pGEX-2T (Amersham Pharmacia Biotech) in fusion with GST protein at its N terminus. Bacterial Escherichia coli strain DH5alpha (Life Technologies, Inc.) was transformed and cultured in LB medium. When A600 reached 0.7-1.0, isopropyl beta -D-thiogalactoside was added at 0.1 mM to induce the expression of fusion protein. Bacteria were harvested after 2-3 h of incubation at 37 °C and lysed by sonication in lysis buffer (1% Triton X-100, 1 mM EDTA in PBS). After centrifugation, the supernatant was incubated with preswollen glutathione beads for 30 min at 4 °C. The beads were then precipitated and washed by centrifugation. GST-BAX-Delta TM was eluted with 5 mM glutathione in 50 mM Tris-HCl, pH 8.0.

Mouse tBid (amino acids 60-195) was cloned into a pET23dwHis vector modified from the pET23d(+) vector (Novagen, WI), in fusion with the 6-histidine tag at its N terminus. Bacteria E. coli strain BL21(DE3) was transformed and cultured at 37 °C in Terrific Broth. The induction of expression was started at 0.8-1.0 A600 by the addition of 0.4 mM isopropyl beta -D-thiogalactoside at 37 °C for 2-3 h. The bacterial pellets were resuspended and sonicated in the buffer containing 5 mM imidazole, 500 mM NaCl, and 20 mM Tris-HCl, pH 7.9. After centrifugation, the supernatants were passed through a His-Bind nickel-agarose affinity chromatographic column precharged with 50 mM NiSO4 (Novagen, WI). The columns were washed with the washing buffer containing 60 mM imidazole, 500 mM NaCl, and 20 mM Tris-HCl, pH 7.9. The proteins were eluted with the elution buffer containing 400 mM imidazole, 500 mM NaCl, and 20 mM Tris-Cl, pH 7.9, and were further purified using a Sephadex G-50 column balanced with phosphate-buffered saline.

Cell Lines and Clones-- Jurkat T leukemic cell line was obtained from the American Type Culture Collection (ATCC; Manassas, VA). Jurkat cells were grown in RPMI 1640 medium containing 10% fetal calf serum, 2 mM L-glutamine, and 100 units/ml each penicillin and streptomycin (complete medium). The generation of stable cell lines expressing epitope-tagged CrmA or Bcl-2 proteins has been described previously (27-30). Transfected cell lines were maintained in complete medium supplemented with 0.5 mg/ml G418 (Life Technologies).

To select for cells resistant to Fas-mediated apoptosis, wild-type Jurkat T cell line was repeatedly exposed to the agonistic anti-Fas mAb, CH-11 (200 ng/ml). Initial treatment induced apoptosis in 99% of the cells. Surviving cells were expanded in culture, and following several consecutive selections with anti-Fas Ab, the cultured cells were further selected by flow cytometry cell sorting to include only Fas-positive cells. Anti-Fas Ab was routinely added to the resistant cell line approximately once a month. Clones of Fas-resistant Jurkat cells were obtained by limiting dilution, and seven of the clonal cell lines were subjected to analysis of mechanisms of resistance. Similar features of resistance to apoptosis were found in all tested clones (as described in this study).

Induction of Apoptosis-- Jurkat cells plated at 0.5-1 × 106 cells/ml in complete medium were treated with VP-16 (20-40 µM), agonistic anti-Fas Ab (200-500 ng/ml), staurosporin (0.5-1 µM), or TRAIL (100 nM; enhancer, 2 µg/ml) at 37 °C for varying lengths of time, as indicated for each experiment. In several experiments, Jurkat cells were treated with cycloheximide (10 µg/ml) for 4 h prior to the addition of anti-Fas-Ab, CH-11.

Measurements of Apoptosis-- DNA fragmentation was assessed by the JAM assay, in which loss of [3H]TdR-labeled DNA was measured (31). DNA labeling of Jurkat target cells was performed by incubation of the cells in the presence of 5 µCi/ml [3H]TdR for 18-24 h at 37 °C. The cells were treated with anti-Fas Ab, VP-16, or staurosporin for various lengths of time, as indicated, and then harvested (Mach IIM, TOMTEC) onto glass fiber filters. The radioactivity of unfragmented DNA, retained on the glass fiber filters, was measured by liquid scintillation counting. Specific DNA fragmentation was calculated according to the following formula: percentage of specific DNA fragmentation = 100 × (S - E)/S, where S represents retained DNA in the absence of effector cells (spontaneous) and E represents experimentally retained DNA in the presence of tumor (effector) cells.

Cytofluorometric analyses of apoptosis were performed by costaining with propidium iodide and fluorescein isothiocyanate-annexin V conjugate (CLONTECH). Propidium iodide was used to identify breaks in DNA as a feature of late apoptosis, and annexin V was used to assess aberrant phosphatidylserine exposure (32). The staining was performed according to the manufacturer's directions.

Apoptosis-associated alterations in mitochondria were assessed by flow cytometry analysis using special dyes designed to evaluate mitochondrial events. Disruption of the mitochondrial inner transmembrane potential (Delta Psi m) was measured by the cationic lipophilic fluorochrome CMXRos. Cells were incubated at 37 °C for 15 min in the presence of CMXRos (0.1 µM, fluorescence at 600 nm) followed by immediate analysis of fluorochrome incorporation (33).

Generation of superoxide anions was measured by HE (Molecular Probes). Cells were incubated at 37 °C for 15 min in the presence of HE (2 µM, fluorescence at 600 nm) followed by immediate analysis of fluorochrome incorporation (34).

Decrease in mitochondrial mass was assessed by NAO (Molecular Probes), reported to measure the inner mitochondrial cardiolipin content (35). Cells were stained with 0.1 µM NAO in RPMI medium for 15 min at 37 °C. Cells were washed and immediately analyzed by flow cytometry.

Staining of cells with anti-APO2.7 served as an additional marker for apoptotic mitochondria. This Ab was raised against apoptotic mitochondria and stains a p38 antigen exposed on apoptotic mitochondria (36).

Preparation of Cytosolic Extracts-- Cytosolic extracts were prepared from clones of wild-type or resistant Jurkat cells, as described previously (37). Briefly, cultured Jurkat cells were washed twice with phosphate-buffered saline and then resuspended in ice-cold buffer (20 mM HEPES, pH 7.0, 10 mM KCl, 1.5 mM MgCl2, 1 mM sodium EDTA, 1 mM sodium EGTA, 1 mM dithiothreitol, 250 mM sucrose, and protease inhibitors). After incubation on ice for 20 min, cells (2.5 × 106/0.5 ml) were disrupted by Dounce homogenization (20 strokes). Nuclei were removed by centrifugation at 650 × g for 10 min at 4 °C. Cellular extracts were obtained as the supernatants resulting from centrifugation at 14,000 × g at 4 °C for 30 min.

Caspase-3 activation was initiated by the addition of 10 µM horse heart cytochrome c and 1 mM dATP to 10 µl of cellular extract at 30 °C for 1 h. To enforce processing of caspase-8, recombinant caspase-3 was added to the cell extracts. The extracts were boiled in sample buffer, resolved by SDS-PAGE, and immunoblotted for processing of caspases.

Western Blot Analysis-- To generate whole cell extracts, cells were lysed in 1% Nonidet P-40, 20 mM Tris-base, pH 7.4, 137 mM NaCl, 10% glycerol, 1 mM phenylmethylsulfonyl fluoride, 10 µg/ml leupeptin, and 10 µg/ml aprotinin. Proteins were resolved by SDS-PAGE and transferred to polyvinylidene difluoride membranes, as described previously (28, 30). Following probing with a specific primary Ab and horseradish peroxidase-conjugated secondary Ab, the protein bands were detected by enhanced chemiluminescence (Pierce).

Subcellular Fractionation-- After induction of apoptosis, Jurkat cells were harvested in isotonic mitochondrial buffer (20 mM sucrose, 20 mM HEPES, 10 mM KCl, 1.5 mM MgCl2, 1 mM EDTA, 1 mM EGTA, 1 mM dithiothreitol, 10 µg/ml leupeptin, 1 mM phenylmethylsulfonyl fluoride, 10 µg/ml aprotinin) and Dounce-homogenized by 15-20 strokes (38). Samples were transferred to Eppendorf centrifuge tubes and centrifuged at 650 × g for 5 min at 4 °C to eliminate nuclei and unbroken cells. The resulting supernatant was centrifuged at 10,000 × g for 30 min at 4 °C to obtain the heavy membrane pellet (HM). The supernatant was further centrifuged at 100,000 × g for 1 h at 4 °C to yield the final soluble cytosolic fraction, S-100. HM and S-100 subcellular fractions were assessed for the presence of cytochrome c by Western blot analysis.

Mitochondria Purification-- Jurkat cells were suspended in mitochondria buffer (MIB) composed of 0.3 M sucrose, 10 mM MOPS, 1 mM EDTA, and 4 mM KH2PO4 and lysed by Dounce homogenization as described previously (33). Briefly, nuclei and debris were removed by 10 min of centrifugation at 650 × g, and a pellet containing mitochondria was obtained by two successive spins at 10,000 × g for 12 min. The washed mitochondria pellet was resuspended in MIB and layered on a Percoll gradient consisting of four layers of 10, 18, 30, and 70% Percoll in MIB. After centrifugation for 35 min at 13,500 × g, the purified mitochondria were collected at the 30/70 interface. Mitochondria were diluted in MIB containing 1 mg/ml bovine serum albumin (at least a 10-fold dilution required to remove Percoll). The mitochondrial pellet was obtained by a 30-min spin at 20,000 × g and used immediately in the cytochrome c release assay.

To assess insertion of Bax or tBid into the mitochondria membrane, purified mitochondria were treated with 0.1 M Na2CO3 (pH 11.5) for 20 min on ice to remove alkali-sensitive proteins attached to the membrane (39).

Cytochrome c Release Assay-- Purified mitochondria (100 µg of protein) were incubated with recombinant tBid or Bax at various doses as indicated in 20 µl of MIB at 30 °C for 30 min. Mitochondria were pelleted by centrifugation at 4000 × g for 5 min. The resulting supernatants or mitochondria were mixed with lysis buffer and analyzed by SDS-PAGE and immunoblotting for the presence of cytochrome c.


    RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Cross-resistance to Fas- and VP-16-induced Apoptosis-- Selected clonal T cell lines that survived in the presence of agonistic anti-Fas Ab were also found to be completely resistant to apoptotic signals of VP-16 known to initiate a mitochondrial cascade of apoptosis. The degree of resistance to apoptosis induced through the extrinsic or the intrinsic pathway was higher than that observed in Jurkat cells engineered to overexpress either CrmA or Bcl-2. While CrmA-overexpressing Jurkat cells were only partially resistant to Fas-mediated apoptosis, complete resistance was observed in the selected Jurkat cells (Fig. 1A). As expected, CrmA-Jurkat cells were highly susceptible to VP-16-induced cell death, whereas the Fas-resistant Jurkat cells were as resistant to VP-16 as Jurkat cells overexpressing Bcl-2 (Fig. 1A). These results demonstrate that the mechanism(s) responsible for resistance to apoptosis in the selected Jurkat cells regulate(s) the two known pathways of apoptosis. The cross-resistance to the two pathways of apoptosis was also demonstrated by flow cytometry. As shown in Fig. 1B, propidium iodide-positive and annexin-positive cells were detected in Jurkat-sensitive cells treated either with anti-Fas Ab, staurosporin, or VP-16 for 14 h. However, in resistant Jurkat cells treated similarly with either Fas cross-linking or VP-16, no significant level of apoptotic cells was observed. Few apoptotic cells were detected in the resistant clones treated with staurosporin. All clones obtained by limiting dilution of the parental resistant Jurkat cell line possessed cross-resistance to both Fas- and VP-16-apoptotic signals.



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Fig. 1.   Resistance of clonal Jurkat cells to Fas and VP-16 apoptotic signals. A, resistance of Jurkat cells was assessed by the JAM assay as compared with Jurkat cells overexpressing CrmA or Bcl-2. [3H]TdR-labeled clonal Jurkat cell lines were treated with agonistic anti-Fas Ab (CH-11; 200 ng/ml) or VP-16 (20 µM) for 14 h. DNA degradation was measured by loss of [3H]TdR-labeled DNA. The error bars represent the S.E. of eight replicates. B, resistance to Fas and VP-16 apoptotic signals was assessed by flow cytometry. Clonal Jurkat cell lines were treated with agonistic anti-Fas Ab or VP-16, as described above. The cells were then stained with annexin V (2 µg/ml) and propidium iodide (5 µg/ml), and the presence of apoptotic cells was assessed by flow cytometry.

Abrogation of Caspase Processing and Activation in Resistant Jurkat Cells-- To determine the mechanism involved in resistance to both Fas and VP-16, we tested the processing of endogenous caspases in sensitive and resistant clonal Jurkat cells in response to stimuli by anti-Fas Ab, VP-16, or staurosporin. As shown in Fig. 2, cross-linking of Fas on sensitive Jurkat cells induced processing of caspase-8, -3, -2, -7, and -9. No such processing was observed in resistant Jurkat cells stimulated for periods as long as 14, 24, or 48 h. Likewise, stimulation by VP-16 induced processing of these caspases in sensitive cells but not in the resistant Jurkat cells treated for as long as 48 h. The observed Fas-mediated activation of caspase-9 at 14 h poststimulation of sensitive Jurkat cells results possibly from a mitochondrial amplification loop mediated by caspase-8-cleaved Bid (13-15, 40). Activation of caspase-8 in a late phase of the mitochondrial apoptotic cascade, as seen for VP-16-treated sensitive Jurkat cells, has been previously documented (17). These results demonstrate that caspases known to be involved in either Fas-induced or VP-16-induced cascades are not processed in resistant Jurkat cells. Caspase activation by staurosporin was also significantly blocked in resistant cells as compared with their sensitive counterpart. However, staurosporin-induced processing of caspase-2 was detected in resistant cells following 14 h of treatment. As shown in Fig. 1B and corroborated by this analysis of caspase processing, apoptosis induced by staurosporin in resistant Jurkat cells was significantly, but not completely, blocked.



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Fig. 2.   Abrogation of caspase processing in resistant clonal Jurkat cells. Wild-type and resistant Jurkat cells were treated with anti-Fas Ab (200 ng/ml), staurosporin (0.5 µM), or VP-16 (20 µM) for 14, 24, or 48 h. At the end of the treatment period, whole cell extracts were separated by SDS-polyacrylamide gels, and resolved proteins were transferred to a polyvinylidene difluoride membrane. The processing of the indicated caspases was assessed by immunoblotting with specific Abs as detailed under "Experimental Procedures." Equal amounts of protein were loaded after quantification, and immunoblotting for beta -actin served as an additional loading control. Each panel represents the results of at least three experiments.

To further assess activity of endogenous caspases, we compared sensitive and resistant Jurkat cells for caspase activity in cleaving known endogenous substrates. The substrates tested included the following endogenous proteins: PARP, known to serve as substrate for caspase-3 and -7 (41); XIAP, demonstrated recently by us (30) and others (42) to be cleaved by caspase-3 and -7; TcR zeta -chain, reported by us to serve as substrate for caspase-3 and -7 (28); and the anti-apoptosis proteins Bcl-2 and Bcl-xl, reported to convert to proapoptosis regulators following cleavage by caspase-3 (43, 44). As demonstrated in Fig. 3, no cleavage of any of the substrates tested was detected in resistant Jurkat cells treated with either anti-Fas Ab, VP-16, or staurosporin for 14 h.



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Fig. 3.   Abrogation of caspase cleaving activity of endogenous substrates in resistant clonal Jurkat cells. Wild-type and resistant Jurkat cells were treated with anti-Fas Ab (200 ng/ml), staurosporin (0.5 µM), or VP-16 (20 µM) for 14 h. At the end of the treatment period, whole cell extracts were separated by SDS-polyacrylamide gels, and resolved proteins were transferred to a polyvinylidene difluoride membrane. The cleavage of endogenous substrates was assessed by immunoblotting using specific antibodies to PARP, XIAP, TcR zeta -chain, Bcl-XL, or Bcl-2.

Since caspase-8, the apical caspase of the Fas-apoptotic cascade, was not processed in resistant Jurkat clones in response to Fas cross-linking, we examined whether it would be processed by exogenously added caspase-3. Caspase-8 has been shown to be cleaved by caspase-3 downstream of mitochondrial damage induced by gamma -radiation (17). As shown in Fig. 4, processing of prodomain caspase-8 as well as the detection of the p20 subunit was similarly observed in extracts of either sensitive or resistant Jurkat cell lines treated with recombinant caspase-3.



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Fig. 4.   Processing of endogenous caspase-8 in extracts of resistant Jurkat cells by recombinant caspase-3. Recombinant caspase-3 was added to extracts of sensitive or resistant Jurkat cells at 0.3 or 1.5 µM for 30 min at 37 °C. The proteins were resolved by SDS-PAGE and probed with anti-caspase-8 mAb (5F7; UBI) (top). The membrane was stripped and reprobed with polyclonal anti-caspase-8 (Ab3; StressGen), which detects the p20 subunit (bottom).

Caspase-3 has been identified as an executioner caspase in both the Fas- and VP-16 apoptotic cascades. Therefore, we examined the processing of endogenous caspase-3 in resistant Jurkat cells in the presence of either exogenous recombinant caspase-8 or exogenous cytochrome c and dATP. As shown in Fig. 5, caspase-3 subunits were detected in either sensitive or resistant Jurkat cells treated with recombinant caspase-8 or cytochrome c and dATP to enforce processing of prodomain caspase-3.



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Fig. 5.   Activation of endogenous caspase-3 in extracts of resistant Jurkat cells by exogenous caspase-8 or cytochrome c. A, wild-type and resistant Jurkat cells were treated with agonistic anti-Fas Ab (200 ng/ml) for 12 h and immunoblotted by anti-caspase-3 Ab. B, cytochrome c (10 µg/ml) together with dATP (1 mM; B) were added to cytosolic extracts of wild-type or resistant Jurkat cells. C, recombinant caspase-8 was added at the indicated doses to extracts of sensitive or resistant Jurkat cells. The extracts (B and C) were incubated at 30 °C for 1 h and then assessed by immunoblot analysis for the presence of the zymogen and active subunits of caspase-3.

Fas-induced apoptosis in various cells is enhanced in the presence of metabolic inhibitors such as cycloheximide (45). To investigate whether an endogenous protein inhibitor was involved in the observed resistance to the Fas-apoptotic cascade, wild-type and resistant Jurkat cells were treated with cycloheximide prior to the addition of anti-Fas Ab. While an increased percentage of apoptotic cells was detected in sensitive Jurkat cells, this combined treatment did not induce apoptosis in resistant Jurkat cells (data not shown). These observations were further confirmed by assessment of caspase-8 processing. Combined treatment of cycloheximide and anti-Fas Ab did not result in any processing of caspase-8 in resistant cells, while it significantly enhanced the processing seen in wild-type Jurkat cells (data not shown). These findings suggest that the observed block in caspase-8 processing was not mediated by an endogenously metabolized inhibitor.

To further identify the site of blockage, we investigated whether caspase-8 would be processed in response to signals via other death receptors known to use caspase-8 activation as an apical event in their cascade (46). To this end, sensitive and resistant Jurkat cells were cross-linked by TRAIL (100 ng/ml; enhancer, 2 µg/ml) for 14 h at 37 °C. No processing of caspase-8 was detected in resistant cells, whereas similar TRAIL cross-linking induced marked processing in sensitive cells (data not shown). The arrest in caspase-8 activation in both Fas and TRAIL receptor cascades, suggests that the block in the apoptotic cascade in resistant cells is at the level of caspase-8 and not at the receptor level. The block in caspase-8 activation was not mediated by up-regulated expression of FLICE/caspase-8 Inhibitory Protein (FLIP), since similar levels of FLIP-short or FLIP-long proteins were detected in sensitive and resistant Jurkat cells (data not shown). Also, similar levels of FADD expression were detected in sensitive and resistant Jurkat cells (data not shown).

Lack of Apoptosis-associated Alterations in Mitochondria of Resistant Jurkat Cells-- To determine the mechanism(s) of the observed resistance of Jurkat cells to VP-16, we assessed apoptosis-associated alterations within mitochondria using fluorescent dyes, which specifically target mitochondrial components or events. Sensitive and resistant Jurkat clones were treated for 8 h with either anti-Fas Ab or VP-16 and assessed for incorporation of the cationic lipophilic dye, CMXRos. Fas cross-linking in sensitive, but not in resistant, Jurkat cells caused significant reduction in incorporation of this dye (Fig. 6A). These results suggest that alterations in permeability transition in response to VP-16 are blocked in resistant Jurkat cells.



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Fig. 6.   Lack of apoptosis-associated alterations in mitochondria of resistant Jurkat cells treated with anti-Fas Ab or VP-16. Wild-type and resistant Jurkat cells treated with anti-Fas Ab (200 ng/ml) or VP-16 (20 µM) for 8 h were assessed by flow cytometry for mitochondrial changes. Staining with CMXRos (100 nM) served to assess changes in mitochondria permeability transition (A); NAO staining (100 nM) served to assess loss in mitochondria cardiolipin (B); HE staining (2 µM) served to assess the presence of reactive oxygen species (C); and anti-APO.27 Ab (2.5 µg/ml) served to detect a p38 antigen specific for apoptotic mitochondria (D).

We also assessed mitochondrial changes by NAO, a mitochondria-specific dye, which is independent of permeability transition. NAO is a probe that interacts specifically with nonoxidized cardiolipin, a lipid that is exclusively localized in the inner mitochondrial membrane (35). Loss in cardiolipin, indicating loss in mitochondria matrix integrity, was detected in sensitive cells but not in resistant cells treated with either anti-Fas Ab or VP-16 (Fig. 6B). Also, no generation of reactive oxygen species was detected in resistant Jurkat cells, as assessed by flow cytometric analysis of HE staining (Fig. 6C).

It has been reported that the mAb APO2.7 detects an apoptosis-associated increase in expression of a p38 mitochondrial antigen (36). A population of cells expressing this antigen was detected in sensitive, but not in resistant, Jurkat cells following Fas or VP-16 stimulation (Fig. 6D). These results suggest that the block in the VP-16-induced cascade is at the level of the mitochondria, since mitochondria-specific apoptotic events did not occur in resistant Jurkat cells treated with VP-16.

Involvement of the mitochondria in apoptosis has been associated with release of cytochrome c to the cytoplasm, where it initiates a caspase cascade (5, 6). Indeed, in wild-type Jurkat cells treated with either anti-Fas Ab or VP-16, translocation of cytochrome c to the cytosol was observed (Fig. 7). However, no cytochrome c release was detected in resistant Jurkat cells treated by each of these stimuli. These observations suggest that in resistant Jurkat cells the apoptotic mechanism responsible for cytochrome c release is blocked.



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Fig. 7.   Release of cytochrome c from mitochondria to the cytoplasm in response to anti-Fas Ab, staurosporin, or VP-16 is blocked in resistant Jurkat cells. Wild-type and resistant Jurkat cells treated with anti-Fas Ab (200 ng/ml), staurosporin (0.5 µM), or VP-16 (20 µM) for 8 h were assessed for redistribution of cytochrome c. Following treatment, Jurkat cells were lysed and separated to an HM fraction, which contains mitochondria, and a mitochondria-free S-100 fraction. The proteins were resolved by 15% SDS-PAGE and immunoblotted by a cytochrome c-specific Ab. Equal protein loading was ensured by protein quantification, and by reprobing the stripped membranes with anti-beta -actin. Cytochrome c oxidase IV served as a marker for mitochondrial fraction. The results shown are representative of at least five independent experiments.

Analysis of Intermembrane Protein Release from Purified Mitochondria-- It has recently been reported that the recombinant proteins, Bax and tBid, induce cytochrome c release when directly applied to purified mitochondria. To further characterize the block in cytochrome c release in resistant cells treated with VP-16, we investigated the response of purified mitochondria from sensitive and resistant Jurkat cells to recombinant Bax (GST-BaxDelta TM) or tBid (His-tBid). These recombinant proteins have been previously demonstrated to efficiently induce cytochrome c release from mitochondria (47-52). Purified mitochondria from sensitive or resistant Jurkat cells were incubated for 30 min at 30 °C with recombinant Bax or tBid. Following removal of mitochondria, the resulting supernatant was tested by immunoblot analysis for the presence of cytochrome c. As expected, cytochrome c was released from mitochondria obtained from sensitive Jurkat cells in response to either exogenous recombinant Bax or tBid (Fig. 8, A and B). No release of cytochrome c was detected in supernatants of resistant mitochondria treated with these recombinant proteins. These results suggest that the mitochondrial mechanism responsible for release of cytochrome c in response to Bid, Bax, or cell stimulation with VP-16 is impaired in mitochondria of resistant Jurkat cells.



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Fig. 8.   Bid and Bax induce release of cytochrome c from purified mitochondria of sensitive but not resistant Jurkat cells. Purified mitochondria from wild-type or resistant Jurkat cells were incubated with the indicated doses of recombinant tBid (A and C) or recombinant Bax (B) for 1 h at 30 °C. Mitochondria were then pelleted by centrifugation, and the resulting supernatant (Mit-Sup) was subjected to SDS-PAGE immunoblot analysis with anti-cytochrome c Ab (A and B) and anti-AIF Ab (C). Input of an equivalent amount of mitochondria used for each treatment was verified by immunoblot analysis of cytochrome c or AIF in control pellets of wild-type and resistant Jurkat cells (Mit). The results shown are representative of at least five independent experiments.

To evaluate whether the resistance mechanism was specific for cytochrome c or applied also to other intermembrane proteins, we examined release of AIF from sensitive and resistant mitochondria in response to recombinant tBid. As shown in Fig. 8C, AIF was released from sensitive, but not resistant, mitochondria. These results suggest that the mitochondrial mechanism responsible for release of intermembrane proteins is blocked in resistant Jurkat cells.

To assess the ability of endogenous Bax or tBid to translocate to the mitochondria, we examined the expression of these proteins within the mitochondria of resistant cells. To this end, purified mitochondria from sensitive and resistant Jurkat cells were treated with 0.1 M Na2CO3 (pH 11.5) for 20 min on ice, to remove alkali-sensitive proteins that are attached but not inserted into the mitochondrial outer membrane (39). The presence of Bax within the alkali-resistant fraction of the treated mitochondria was tested for by Western blot analysis. As shown in Fig. 9A, similar levels of endogenous Bax were expressed in mitochondria of sensitive or resistant Jurkat cells. The expression of mitochondrial VDAC and the absence of cytosolic beta -actin in the alkali-treated mitochondria (Ins) further suggest that the fractions tested represent pure mitochondria, and therefore, the translocation of endogenous Bax to the mitochondria is not impaired in resistant cells. These results also demonstrate that in Jurkat cells, as shown previously for HeLa cells (39) and for the hematopoietic cells FL5.12 (53), a basal level of Bax is present within the mitochondria prior to induction of apoptosis. To assess the presence of endogenous tBid in the mitochondria, extracts of sensitive or resistant Jurkat cells were treated with recombinant caspase-8 (100 nM) for 1 h at 37 °C to generate endogenous tBid. The extracts were then separated into cytosol (S-100) and mitochondria (HM). As shown in Fig. 9B, endogenous tBid (15 kDa) generated by exogenous recombinant caspase-8 was detected in mitochondrial fractions from both sensitive and resistant Jurkat cells. The use of beta -actin as a marker for cytosolic proteins and VDAC as a marker for mitochondrial outer membrane proteins provides evidence for the presence of endogenous tBid in the mitochondria. To further determine whether exogenous tBid added to purified mitochondria could translocate into the mitochondria, purified mitochondria from sensitive or resistant Jurkat cells were incubated with recombinant tBid for 30 min at 37 °C. The mitochondria were then treated with 0.1 M Na2CO3, and alkali-sensitive (attached) or alkali-resistant (membrane-inserted) fractions were assessed for the presence of tBid by Western blot analysis. As demonstrated in Fig. 9C, tBid was found inserted into the mitochondria membrane in both sensitive and resistant cells. In these studies, cytochrome c oxidase IV, a mitochondrial inner membrane enzyme, and VDAC, a mitochondrial outer membrane protein, served as markers for the mitochondrial matrix as well as a gel loading control. These results suggest that the block in cytochrome c or AIF release in resistant mitochondria is not mediated by impaired translocation of either Bax or tBid to the mitochondria.



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Fig. 9.   Expression of Bax or tBid in mitochondria of resistant Jurkat cells. A, purified mitochondria from sensitive or resistant cells were treated with 0.1 M Na2CO3 (pH 11.5) for 20 min on ice. The alkali-resistant mitochondrial fraction (insert (Ins)) was lysed, resolved by SDS-PAGE, and immunoblotted for the presence of Bax. Results obtained with mitochondria purified in two different experiments are shown. The blot membrane was stripped and reprobed with anti-VDAC mAb, stripped again, and probed with anti-beta -actin. No beta -actin was detected in purified mitochondria following alkali treatment (not shown). B, extracts of sensitive or resistant Jurkat cells were treated with recombinant caspase-8 (100 nM) for 1 h at 37 °C. The extracts were separated into cytosol (S-100) and mitochondrial (HM) fractions as described under "Experimental Procedures." These two cellular fractions were resolved by SDS-PAGE and probed by immunoblotting for the presence of tBid. The blot membrane was stripped and reprobed with anti-beta -actin mAb and subsequently with anti-VDAC mAb, which served as cellular fractionation controls. C, purified mitochondria from sensitive or resistant Jurkat cells were incubated with recombinant His-tBid (100 nM) for 1 h at 30 °C. Treated and untreated mitochondria were washed in MIB and further treated with 0.1 M Na2CO3 (pH 11.5) for 20 min on ice. Alkali-sensitive (attached (Att)) and alkali-resistant (insert (Ins)) fractions were lysed, resolved by SDS-PAGE, and immunoblotted for the presence of tBid. The membrane was stripped and reprobed with anti-cytochrome c oxidase IV Ab and subsequently with anti-VDAC mAb as markers for mitochondrial matrix and as loading controls.



    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

In the present study, we describe the selection of Jurkat leukemic T cells resistant to an intrinsic apoptotic pathway via elimination of cells susceptible to the extrinsic Fas-mediated apoptotic cascade. The block in the Fas cascade appears to be at the level of caspase-8, since no processing or activation of caspase-8 was observed following cross-linking of either Fas or TRAIL receptors, which were expressed at a similar level on sensitive and resistant cells. However, caspase-8 in resistant cells was processed in the presence of active recombinant caspase-3, suggesting that it is potentially activable. Activation of caspase-8 was not suppressed by up-regulation in expression of either FLIP-long or FLIP-short or by a differential expression of FADD, since similar levels of these proteins were detected in all sensitive and resistant clones. The resistance to Fas-mediated apoptosis and the block in caspase-8 activation was not affected by pretreatment of the cells with cycloheximide, a metabolism inhibitor that significantly increased susceptibility of sensitive Jurkat cells to Fas-mediated apoptosis. Although these findings suggest that caspase-8 inhibition is not mediated by a metabolized protein inhibitor, the mechanism responsible for the block in caspase-8 activation in these resistant cells is not yet known. It is possible that additional regulatory components may be involved in the activation of caspase-8, which are yet to be identified. Since caspase-8, the apical caspase in the death receptor cascade, was not activated, it was expected that downstream apoptotic events would not be executed. It was, however, surprising that elimination of Fas-susceptible T cells by anti-Fas Ab would result in selection of cells resistant to intrinsic apoptotic pathways.

The block in the intrinsic cascade was localized to the mitochondria, since no apoptosis-associated alterations were detected in resistant mitochondria treated with VP-16. We tested a broad range of mitochondrial apoptotic events, including changes in the inner membrane, the outer membrane, and the mitochondrial matrix. No changes in inner membrane permeability transition, loss of mitochondrial inner membrane cardiolipin, or reactive oxygen species generation were detected in resistant cell mitochondria. Further, we identified a block in the mechanism responsible for cytochrome c release in response to an apoptotic signaling by VP-16 or in cell-free mitochondria treated with recombinant Bax or tBid. The abrogation in release of cytochrome c was not due to impaired translocation of either Bax or tBid to the mitochondria. These proapoptotic Bcl-2 family members were found inserted, rather than attached, to the resistant mitochondria. The block in cytochrome c release applies also to another intermembrane protein, AIF, reported to be released in response to apoptotic signaling (54, 55). Our results suggest that the block in resistant mitochondria to specific death stimulation localizes to the mechanism responsible for generating specific pores in the outer mitochondrial membrane, which allow translocation of intermembrane proteins to the cytosol.

According to the currently accepted notion, which is based on genetic studies in knockout mouse models (7-10, 12), the initiation phases of the extrinsic and the intrinsic pathways of apoptosis are independent. Cross-talk between the pathways would occur mainly as an amplification loop once the cascade has been initiated. Fas-induced apoptosis is mediated either by direct activation of downstream effector caspases or via Bid-induced mitochondrial pathway (40). Thus, the observed cross-resistance of Jurkat clones to Fas- and VP-16-mediated apoptosis may be explained as two independent mechanisms involved in blocking the initiation of two independent pathways. Since the entire study was performed in clonal cell lines derived by limiting dilution, each of the clones would encompass two independent mechanisms of resistance. It is possible that in initial selection against cells susceptible to Fas signaling, accumulation of type II cells dependent on mitochondria for their Fas-mediated apoptosis took place (56), whereas continuous selection resulted in cells deficient in mitochondrial mechanism for releasing intermembrane proteins.

The identification of BAR, an apoptosis regulator, which can mediate cross-talk between components of the "independent" pathways, may suggest that in certain cells or apoptotic conditions, common regulators of the two pathways may be functional. BAR was identified by using a yeast-based screen for inhibitors of Bax-induced cell death (57). The BAR protein contains a SAM domain, which is required for its interaction with Bcl-2 and Bcl-XL and for suppression of Bax-induced cell death in both mammalian cells and yeast. In addition, BAR contains a DED-like domain responsible for its interaction with DED-containing procaspases and suppression of Fas-induced apoptosis. Furthermore, BAR can bridge procaspase-8 and Bcl-2 into a protein complex. Since BAR may serve as an inhibitor of Bax and at the same time bind to procaspase-8, we examined by reverse transcription-polymerase chain reaction its level of expression in sensitive and resistant Jurkat cells. We detected similar levels of BAR mRNA in sensitive and resistant Jurkat cells (data not shown), suggesting that BAR is not involved in the mechanism of resistance in the Jurkat cells.

A Fas-interacting serine/threonine kinase capable of inducing phosphorylation of FADD has recently been identified as one of the homeodomain-interacting protein kinases (homeodomain-interacting protein kinase 3) involved in regulation of apoptosis via p53 (58, 59). It is conceivable that such a common regulator of both apoptotic pathways may be linked to the observed mechanism of cross-resistance (58).

A common regulator of Fas- and gamma -irradiation-induced apoptotic cascade was also suggested in a previous study (60). Clones of Jurkat cells were reported to have a deficiency in caspase-8 activation in response to Fas cross-linking and in cytochrome c release following irradiation. In the present study, we identified the mitochondrial mechanism responsible for the release of intermembrane proteins as the target of the block observed in the intrinsic apoptotic cascade. The biochemical nature of this block and the mechanism responsible for the cross-resistance between the mitochondrial and the death receptor cascades remain to be elucidated.


    ACKNOWLEDGEMENT

We thank Dr. Daniel E. Johnson (University of Pittsburgh) for Jurkat cells transfected with CrmA or Bcl-2.


    FOOTNOTES

* This work was supported by National Institutes of Health (NIH) Grant RO1 CA 84134-01 (to H. R.), NCI, NIH Grant PO1DE 12321-01 (to H. R.), a grant from The Pittsburgh Foundation (to H. R.), American Cancer Society Grant RPG-98-288-01-CIM (to H. R.), Department of Defense Grant BC981056 (to H. R.), and a grant from the Pennsylvania Department of Health (to H. R.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§ These two authors contributed equally to this work.

** To whom correspondence should be addressed: University of Pittsburgh Cancer Inst., W952 Biomedical Science Tower, 200 Lothrop St., Pittsburgh, PA 15213. Tel.: 412-624-0289; Fax: 412-624-7737; E-mail: rabinow@pitt.edu.

Published, JBC Papers in Press, November 3, 2000, DOI 10.1074/jbc.M006222200


    ABBREVIATIONS

The abbreviations used are: TRAIL, tumor necrosis factor-related apoptosis-inducing ligand; AIF, apoptosis-inducing factor; Apaf-1, apoptosis protease-activating factor 1; BAR, bifunctional apoptosis regulator; CMXRos, chloromethyl X-rosamine; DISC, death-inducing signaling complex; FADD, Fas-associated death domain; FLIP, FLICE-inhibitory protein; HE, hydroethidine/hydroethidium; HM, heavy membrane; NAO, nonyl acridine orange; PARP, poly(ADP-ribose) polymerase; tBid, truncated Bid; VDAC, voltage-dependent anion channel(s); XIAP, X-linked inhibitor of apoptosis; Ab, antibody; mAb, monoclonal antibody; MIB, mitochondria buffer; MOPS, 4-morpholinepropanesulfonic acid.


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