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• W2018119239 abstract "In the present work, Jurkat cells undergoing anti-Fas antibody (anti-Fas)-triggered apoptosis exhibited in increasing proportion a massive release of cytochrome cfrom mitochondria, as revealed by double-labeling confocal immunofluorescence microscopy. The cytochrome c release was followed by a progressive reduction in the respiratory activity of the last respiratory enzyme, cytochrome c oxidase (COX), and with a little delay, by a decrease in overall endogenous respiration rate, as measured in vivo in the whole cell population. Furthermore, in vivo titration experiments showed that an ∼30% excess of COX capacity over that required to support endogenous respiration, found in naive cells, was maintained in anti-Fas-treated cells having lost ∼40% of their COX respiratory activity. This observation strongly suggested that only a subpopulation of anti-Fas-treated cells, which maintained the excess of COX capacity, respired. Fractionation of cells on annexin V-coated paramagnetic beads did indeed separate a subpopulation of annexin V-binding apoptotic cells with fully released cytochrome c and completely lacking respiration, and a nonbound cell subpopulation exhibiting nearly intact respiration and in their great majority preserving the mitochondrial cytochrome c localization. The above findings showed a cellular mosaicism in cytochrome c release and respiration loss, and revealed the occurrence of a rate-limiting step preceding cytochrome c release in the apoptotic cascade. Furthermore, the striking observation that controlled digitonin treatment caused a massive and very rapid release of cytochrome c and complete loss of respiration in the still respiring anti-Fas-treated cells, but not in naive cells, indicated that the cells responding to digitonin had already been primed for apoptosis, and that this treatment bypassed or accelerated the rate-limiting step most probably at the level of the mitochondrial outer membrane. In the present work, Jurkat cells undergoing anti-Fas antibody (anti-Fas)-triggered apoptosis exhibited in increasing proportion a massive release of cytochrome cfrom mitochondria, as revealed by double-labeling confocal immunofluorescence microscopy. The cytochrome c release was followed by a progressive reduction in the respiratory activity of the last respiratory enzyme, cytochrome c oxidase (COX), and with a little delay, by a decrease in overall endogenous respiration rate, as measured in vivo in the whole cell population. Furthermore, in vivo titration experiments showed that an ∼30% excess of COX capacity over that required to support endogenous respiration, found in naive cells, was maintained in anti-Fas-treated cells having lost ∼40% of their COX respiratory activity. This observation strongly suggested that only a subpopulation of anti-Fas-treated cells, which maintained the excess of COX capacity, respired. Fractionation of cells on annexin V-coated paramagnetic beads did indeed separate a subpopulation of annexin V-binding apoptotic cells with fully released cytochrome c and completely lacking respiration, and a nonbound cell subpopulation exhibiting nearly intact respiration and in their great majority preserving the mitochondrial cytochrome c localization. The above findings showed a cellular mosaicism in cytochrome c release and respiration loss, and revealed the occurrence of a rate-limiting step preceding cytochrome c release in the apoptotic cascade. Furthermore, the striking observation that controlled digitonin treatment caused a massive and very rapid release of cytochrome c and complete loss of respiration in the still respiring anti-Fas-treated cells, but not in naive cells, indicated that the cells responding to digitonin had already been primed for apoptosis, and that this treatment bypassed or accelerated the rate-limiting step most probably at the level of the mitochondrial outer membrane. cytochrome c oxidase anti-Fas antibody 4,6-diamidino-2-phenylindole dinitrophenol N,N,N′,N′-tetramethyl-1,4-phenylenediamine maximum COX capacity z-Val-Ala-Asp(OMe)-CH2F fluorescein isothiocyanate phosphate-buffered saline phosphate-buffered saline with Tween 20 horse serum in phosphate-buffered saline Recent studies on apoptosis, a highly controlled form of cell death triggered by a variety of stimuli, have demonstrated that mitochondria function as a common regulator of apoptotic self-destruction (1Kroemer G. Reed J.C. Nat. Med. 2000; 6: 513-519Crossref PubMed Scopus (2785) Google Scholar). In particular, mitochondria serve as a reservoir of apoptogenic molecules, such as cytochrome c (2Liu X. Kim C.N. Yang J. Jemmerson R. Wang X. Cell. 1996; 86: 147-157Abstract Full Text Full Text PDF PubMed Scopus (4484) Google Scholar, 3Zhivotovsky B. Orrenius S. Brustugun O.T. Doskeland S.O. Nature. 1998; 391: 449-450Crossref PubMed Scopus (274) Google Scholar), apoptosis-inducing factor (4Susin S.A. Lorenzo H.K. Zamzami N. Marzo I. Snow B.E. Brothers G.M. Mangion J. Jacotot E. Costantini P. Loeffler M. Larochette N. Goodlett D.R. Aebersold R. Siderovski D.P. Penninger J.M. Kroemer G. Nature. 1999; 397: 441-446Crossref PubMed Scopus (3464) Google Scholar), and Smac/Diablo (5Du C. Fang M. Li Y. Li L. Wang X. Cell. 2000; 102: 33-42Abstract Full Text Full Text PDF PubMed Scopus (2941) Google Scholar, 6Verhagen A.M. Ekert P.G. Pakusch M. Silke J. Connolly L.M. Reid G.E. Moritz R.L. Simpson R.J. Vaux D.L. Cell. 2000; 102: 43-53Abstract Full Text Full Text PDF PubMed Scopus (1985) Google Scholar), and their release from these organelles is controlled by many of pro- and anti-apoptotic Bcl-2 family proteins (6Verhagen A.M. Ekert P.G. Pakusch M. Silke J. Connolly L.M. Reid G.E. Moritz R.L. Simpson R.J. Vaux D.L. Cell. 2000; 102: 43-53Abstract Full Text Full Text PDF PubMed Scopus (1985) Google Scholar, 7Gross A. McDonnell J.M. Korsmeyer S.J. Genes Dev. 1999; 13: 1899-1911Crossref PubMed Scopus (3268) Google Scholar, 8Vander Heiden M.G. Thompson C.B. Nat. Cell Biol. 1999; 1: E209-E216Crossref PubMed Scopus (602) Google Scholar). Cytochrome c, when released from the mitochondrial intermembrane space into the cytosol, participates, together with Apaf-1 and pro-caspase-9, in activation of the apoptotic protease cascade (9Li P. Nijhawan D. Budihardjo I. Srinivasula S.M. Ahmad M. Alnemri E.S. Wang X. Cell. 1997; 91: 479-489Abstract Full Text Full Text PDF PubMed Scopus (6261) Google Scholar). The mechanism of cytochrome c release is largely unknown, though several models have been proposed (1Kroemer G. Reed J.C. Nat. Med. 2000; 6: 513-519Crossref PubMed Scopus (2785) Google Scholar, 10Green D.R. Reed J.C. Science. 1998; 281: 1309-1312Crossref PubMed Google Scholar, 11Martinou J.-C. Nature. 1999; 399: 411-412Crossref PubMed Scopus (87) Google Scholar). As cells progress through apoptosis, mitochondria undergo many changes. Specifically, mitochondrial alkalization and swelling, loss of electrochemical potential across the mitochondrial inner membrane, outer membrane rupture, and permeability transition have been reported (1Kroemer G. Reed J.C. Nat. Med. 2000; 6: 513-519Crossref PubMed Scopus (2785) Google Scholar, 10Green D.R. Reed J.C. Science. 1998; 281: 1309-1312Crossref PubMed Google Scholar, 12Matsuyama S. Llopis J. Deveraux Q.L. Tsien R.Y. Reed J.C. Nat. Cell Biol. 2000; 2: 318-325Crossref PubMed Scopus (636) Google Scholar). Although cytochrome c release precedes the loss of the mitochondrial inner membrane potential in many systems (13Kluck R.M. Bossy-Wetzel E. Green D.R. Newmeyer D.D. Science. 1997; 275: 1132-1136Crossref PubMed Scopus (4289) Google Scholar, 14Yang 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 (4422) Google Scholar, 15Bossy-Wetzel E. Newmeyer D.D. Green D.R. EMBO J. 1998; 17: 37-49Crossref PubMed Scopus (1107) Google Scholar), very little is known about the relationship of cytochrome crelease with respiratory changes in the apoptotic cell (8Vander Heiden M.G. Thompson C.B. Nat. Cell Biol. 1999; 1: E209-E216Crossref PubMed Scopus (602) Google Scholar, 10Green D.R. Reed J.C. Science. 1998; 281: 1309-1312Crossref PubMed Google Scholar, 16Reed J.C. Cell. 1997; 91: 559-562Abstract Full Text Full Text PDF PubMed Scopus (702) Google Scholar). Since cytochrome c functions as a mobile electron carrier of the respiratory chain, it seems plausible to predict that the complete release of cytochrome c from mitochondria would cause loss of respiration. Very recently, however, it has been reported that HeLa cells induced to undergo apoptosis by UV irradiation released rapidly their cytochrome c into the cytosol, but still maintained an azide-sensitive membrane potential, indicative of a functional cytochrome c oxidase (COX),1 if caspase activation was blocked (17Goldstein J.C. Waterhouse N.J. Juin P. Evan G.I. Green D.R. Nat. Cell Biol. 2000; 2: 156-162Crossref PubMed Scopus (886) Google Scholar). In the present work, measurements of respiration and cytochrome c localization in intact Jurkat cells induced to undergo anti-Fas antibody (anti-Fas)-mediated apoptosis and cell sorting experiments have unambiguously shown a massive release of cytochrome c associated with a complete loss of COX respiratory activity and of endogenous respiration in a subpopulation of cells. This subpopulation increased with time after the apoptotic stimulus, with nearly all remaining cells exhibiting normal cytochrome c localization and respiration. These observations have pointed to the existence of a rate-limiting step preceding cytochrome c release in the apoptotic cascade. Most significantly, we obtained strong evidence that this rate-limiting step occurred in cells already primed for apoptosis. Exogenous cytochrome crestored the COX respiratory activity in digitonin-treated cells nearly completely, but, surprisingly, the glutamate/malate- or succinate-dependent respiration only partially. Jurkat cells, a lymphoblastoma-derived T-cell line (TIB 152, ATCC), were grown in RPMI 1640 medium with 10 mm Hepes, 2 mm l-glutamine, and 10% fetal bovine serum. Individual cultures were maintained at a cell concentration between 105/ml and 106/ml for no longer than 2 months. For apoptosis induction, cells were transferred to fresh medium, and, after 16 h, 50 ng/ml anti-Fas IgM (clone CH-11, Kamiya Biomedical Co.) was added to a culture containing ∼106 cells/ml. Cells were fixed on coverslips by formaldehyde and methanol treatment (as described below), washed in TD buffer (137 mm NaCl, 5 mm KCl, 0.7 mm Na2HPO4, 25 mmTris-HCl, pH 7.4 at 25 °C), and stained with 1 μg/ml dsDNA-binding fluorochrome 4,6-diamidino-2-phenylindole (DAPI, Sigma) in TD buffer for 5 min. With DAPI staining, normal cells show homogenous staining of their nuclei, whereas apoptotic cells show irregular staining as a result of chromatin condensation and nuclear fragmentation (18Finucane D.M. Bossy-Wetzel E. Waterhouse N.J. Cotter T.G. Green D.R. J. Biol. Chem. 1999; 274: 2225-2233Abstract Full Text Full Text PDF PubMed Scopus (653) Google Scholar). Both normal and apoptotic nuclei were counted using fluorescence microscopy. Apoptosis was also determined by fluorescence microscopy of cells stained with FITC-labeled annexin V (Kamiya Biomedical Co.), according to the manufacturer's protocol. Previous work in this laboratory had shown that the osteosarcoma-derived 143B TK− cell line respires in TD buffer at the same rate as in Dulbecco's modified Eagle's medium lacking glucose (19Villani G. Attardi G. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 1166-1171Crossref PubMed Scopus (175) Google Scholar). Therefore, the respiration rate was continuously measured in an oxygraph (Yellow Springs Instruments, model 5300) in a suspension at 107cells/ml in TD buffer, before and after each of the following sequential additions: 17 μm dinitrophenol (DNP), 20 nm antimycin A (Sigma), 10 mm ascorbate + 400 μm N,N,N′,N′-tetramethyl-1,4-phenylenediamine (TMPD, Fluka), essentially as described (19Villani G. Attardi G. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 1166-1171Crossref PubMed Scopus (175) Google Scholar). The concentration of DNP specified above was one that, in preliminary tests (data not shown), produced the highest stimulation of the endogenous respiration rate in naive Jurkat cells. Since ascorbate/TMPD autooxidation causes significant oxygen consumption, the oxygen consumption rate of ascorbate and TMPD in the absence of cells was subtracted from the oxygen consumption rate measured in the presence of cells, DNP, antimycin A, ascorbate, and TMPD. Oxygen consumption rate was expressed in nanomoles of oxygen consumed per min and mg of cellular protein, as determined by the Bradford procedure (Bio-Rad). Cells were resuspended at 1.5–2.0 × 107/ml in TD buffer containing either 17 μm DNP, for KCN titration of “integrated” COX activity, or 17 μm DNP, 20 nm antimycin A, 10 mm ascorbate, and 200 μm TMPD, for KCN titration of “isolated” COX activity, and transferred into two chambers connected in parallel, as described (19Villani G. Attardi G. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 1166-1171Crossref PubMed Scopus (175) Google Scholar). If anti-Fas-treated cells were analyzed, the cell concentration was increased to yield respiration rates similar to those of naive cells. The KCN titration measurements and the determination of maximum COX capacity (COXR(max)), relative to the uncoupled endogenous respiration rate, from the threshold plots (i.e. plots of relative endogenous respiration rate versus the percentage of inhibition of isolated COX activity at the same KCN concentration in DNP-uncoupled intact cells) were performed as described (19Villani G. Attardi G. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 1166-1171Crossref PubMed Scopus (175) Google Scholar). Cell culture samples (in some experiments after digitonin treatment, see below) were centrifuged onto glass coverslips, and then sequentially incubated in 2% formaldehyde in PBS (140 mm NaCl, 3.8 mmNaH2PO4, 16.2 mmNa2HPO4), PBS, anhydrous methanol, PBS, 2% horse serum in PBS (HSPBS) containing 0.5% Triton X-100 (20Samali A. Cai J. Zhivotovsky B. Jones D.P. Orrenius S. EMBO J. 1999; 18: 2040-2048Crossref PubMed Scopus (463) Google Scholar). The coverslips were then incubated with mouse anti-cytochrome cmonoclonal antibody 6H2.B4 (PharMingen), diluted 1:15 in HSPBS, and rabbit anti-Hsp60 antiserum (StressGen Biotechnologies Corp.), diluted 1:50, for 1 h at 37 °C in a humidified chamber. After three washes in HSPBS, the coverslips were incubated with 1:50-diluted FITC-conjugated goat anti-mouse IgG (Jackson Immunoresearch Laboratories) and 1:100-diluted lyssamine-rhodamine-conjugated goat anti-rabbit IgG (Jackson Immunoresearch Laboratories) for 1 h at room temperature. After three washes in PBS, the coverslips were mounted onto microscope slides in FluoroGuard antifade reagent (Bio-Rad), and analyzed on a Zeiss 310 laser-scanning microscope equipped with 488-nm argon and 543-nm helium neon lasers. Cells with diffuse cytosolic cytochrome c staining (green) and punctate mitochondrial Hsp60 staining (red) were counted as cells carrying cytochrome c released from mitochondria into cytosol. Cells with punctate cytochrome c staining that overlapped with Hsp60 staining were counted as cells with mitochondrial cytochrome c staining. Apoptotic cells were separated from nonapoptotic cells by magnetic enrichment using the Apoptotic Cell Isolation Kit (Miltenyi Biotec), according to the manufacturer's protocol. Cells (4 × 107) were incubated with 80 μl of annexin V-Microbeads in 400 μl of binding buffer for 15 min at 12 °C and, after a 20-fold dilution with binding buffer, centrifuged. The cells, resuspended in the same buffer, were then passed through the magnetic separation column for positive selection (VS+), which was placed in the magnetic field of the Vario MACS magnetic separator. The flow-through fraction was centrifuged and resuspended in TD buffer. After removal of the column from the magnetic field, the magnetically retained cells were eluted, centrifuged, and resuspended in TD buffer. In a separate experiment, the flow-through fraction of the cell population, manipulated as described above, but without addition of annexin V-microbeads, was also collected to obtain the mock-fractionated cell fraction. In a typical experiment, cells were resuspended in a measurement buffer at ∼1.2 × 107/ml, transferred into the 1.9-ml oxygraphic chamber, and the cell number was then determined by counting. After taking four aliquots from the chamber for protein determination, the oxygen consumption in the presence of DNP (endogenous uncoupled respiration) was measured. Then, digitonin was added from 5% stock solution in Me2SO to permeabilize the cells. The concentration of digitonin that, in preliminary tests (data not shown), produced the highest stimulation of the glutamate/malate-dependent respiration rate in naive Jurkat cells (5 μg/106 cells) was used. If not indicated otherwise, two min after the addition of digitonin a substrate was added to support the respiration. The glutamate/malate-dependent respiration was measured, in the presence of 17 μm DNP, 5 mm glutamate, and 5 mm malate, in respiration medium I (21Villani G. Greco M. Papa S. Attardi G. J. Biol. Chem. 1998; 273: 31829-31836Abstract Full Text Full Text PDF PubMed Scopus (190) Google Scholar) (75 mmsucrose, 20 mm d-glucose, 5 mmKPi, 40 mm KCl, 0.5 mm EDTA, 3 mm MgCl2, 30 mm Tris, pH 7.4), as oxygen consumption that was sensitive to 0.2 μm rotenone. The respiration medium I was chosen because the glutamate/malate-dependent respiration rate in digitonin-permeabilized naive cells in this measurement buffer was 1) almost constant during the course of experiment, and 2) similar to the endogenous respiration rate. The succinate-dependent respiration was measured, in the presence of 17 μm DNP or 0.5 mm ADP, 0.2 μm rotenone, and 5 mm succinate, in respiration medium I as oxygen consumption that was sensitive to 20 nm antimycin A. The TMPD-dependent respiration was measured in respiration medium II (250 mm sucrose, 20 mm Hepes, 10 mm MgCl2, 2 mm KPi, pH 7.1), since in this measurement buffer only a low rate of ascorbate/TMPD autooxidation occurred. In this medium, the respiration rate was determined in the presence of 17 μm DNP, 20 nm antimycin A, 10 mm ascorbate, and 400 μm TMPD, and then corrected by subtracting the nonspecific oxygen consumption rate due to autooxidation of ascorbate/TMPD in the same buffer. Cytochrome c (Sigma) was added from a 10 mm stock solution. In this form, cytochrome c was fully oxidized, since addition of potassium ferricyanide did not decrease the A550. Reduced cytochrome c was prepared by addition of few crystals of sodium hydrosulfite into a 10 mm stock solution of cytochrome c, and stirring for 1 h. Full reduction of cytochrome c was verified by showing that addition of extra sodium hydrosulfite did not increase the A550. Samples (pellet of intact cells or pellet and supernatant of digitonin-treated cells) derived from the same number of cells, as estimated from the amount of total cellular protein (350 μg), were analyzed by 15% SDS-PAGE. Proteins were then transferred to Immun-Blot polyvinylidene difluoride membrane (Bio-Rad) at 150 mA for 12 h in a buffer (0.037% SDS, 20 mmTris, 150 mm glycine, 20% methanol, pH 8.2 (25 °C)). After blocking of nonspecific binding in PBST (0.1% Tween 20, 3% nonfat milk in PBS) containing 3% bovine serum albumin for 3 h at room temperature, the membranes were incubated with mouse anti-cytochrome c monoclonal antibody 7H8.2C12 (PharMingen), diluted 1:500 in PBS containing 0.05% Tween 20 and 3% nonfat milk, for 17 h at 4 °C. The membranes, washed three times in PBST, were incubated with sheep anti-mouse IgG peroxidase-linked (Amersham Pharmacia Biotech), diluted 1:1000 in PBST, for 2 h at room temperature. The membranes were washed five times in PBST, and specific protein complexes were identified using the SuperSignal West Pico chemiluminescence reagent (Pierce) by autoradiography. Cells were washed in respiration medium I, counted, resuspended in the same medium at 107/ml, treated with digitonin (5 μg/106cells) for 7 min at 37 °C, and centrifuged at 400 ×g for 5 min. The resulting supernatant (digitonin supernatant) was then transferred to the oxygraphic chamber. Since the determination of cytochrome c release from mitochondria based on immunoblotting of mitochondrial and cytosolic fractions prepared from cellular homogenates have led to different conclusions even in the same system (22Krippner A. Matsuno-Yagi A. Gottlieb R.A. Babior B.M. J. Biol. Chem. 1996; 271: 21629-21636Abstract Full Text Full Text PDF PubMed Scopus (188) Google Scholar, 23Vander Heiden M.G. Chandel N.S. Williamson E.K. Schumacker P.T. Thompson C.B. Cell. 1997; 91: 627-637Abstract Full Text Full Text PDF PubMed Scopus (1242) Google Scholar, 24Scaffidi C. Fulda S. Srinivasan A. Friesen C. Li F. Tomaselli K.J. Debatin K.M. Krammer P.H. Peter M.E. EMBO J. 1998; 17: 1675-1687Crossref PubMed Scopus (2633) Google Scholar, 25Adachi S. Gottlieb R.A. Babior B.M. J. Biol. Chem. 1998; 273: 19892-19894Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar, 26Sun X.-M. MacFarlane M. Zhuang J. Wolf B.B. Green D.R. Cohen G.M. J. Biol. Chem. 1999; 274: 5053-5060Abstract Full Text Full Text PDF PubMed Scopus (780) Google Scholar, 27Linsinger G. Wilhelm S. Wagner H. Hacker G. Mol. Cell. Biol. 1999; 19: 3299-3311Crossref PubMed Scopus (55) Google Scholar), in the present work, double-labeling confocal immunofluorescence microscopy was used to analyze in situ this phenomenon in apoptotic Jurkat cells. The majority of the cells treated for 4 h with anti-Fas antibody exhibited diffused cytosolic cytochrome c staining, while staining of Hsp60, located in the mitochondrial matrix, was punctate (Fig. 1). In contrast, in untreated (naive) cells or in cells treated with anti-Fas antibody and the caspase inhibitor z-Val-Ala-Asp(OMe)-CH2F (z-VADfmk, Kamiya), cytochrome c staining was punctate and colocalized with the Hsp60 staining. These results confirmed earlier observations (23Vander Heiden M.G. Chandel N.S. Williamson E.K. Schumacker P.T. Thompson C.B. Cell. 1997; 91: 627-637Abstract Full Text Full Text PDF PubMed Scopus (1242) Google Scholar, 24Scaffidi C. Fulda S. Srinivasan A. Friesen C. Li F. Tomaselli K.J. Debatin K.M. Krammer P.H. Peter M.E. EMBO J. 1998; 17: 1675-1687Crossref PubMed Scopus (2633) Google Scholar, 26Sun X.-M. MacFarlane M. Zhuang J. Wolf B.B. Green D.R. Cohen G.M. J. Biol. Chem. 1999; 274: 5053-5060Abstract Full Text Full Text PDF PubMed Scopus (780) Google Scholar), made by immunoblotting experiments, that, in Jurkat cells treated with anti-Fas antibody, cytochrome c is released in a caspase-dependent manner from mitochondria into the cytosol. Furthermore, the physical integrity of the inner mitochondrial membrane was preserved under these conditions, as indicated by the behavior of Hsp60. In addition, an analysis of multiple optical sections of individual cells anti-Fas-treated for 4 h showed that the majority of the cells displayed either only punctate mitochondrial cytochrome c staining, or diffused cytosolic cytochrome c staining without detectable mitochondrial staining (data not shown). These observations indicated the existence of a mechanism for a rapid cytochrome c release from all mitochondria of individual cells, as recently reported for HeLa cells induced to undergo apoptosis by UV irradiation or staurosporine treatment (17Goldstein J.C. Waterhouse N.J. Juin P. Evan G.I. Green D.R. Nat. Cell Biol. 2000; 2: 156-162Crossref PubMed Scopus (886) Google Scholar). Furthermore, there was clearly a marked cellular heterogeneity in cytochrome c release from mitochondria (Fig. 1). To investigate the possible effects of cytochrome c release on respiration, the rate of oxygen consumption in naive Jurkat cells and in cells induced to undergo apoptosis by anti-Fas antibody was measured both in the absence and in the presence of the uncoupler DNP. Fig. 2 Apresents the results of a representative experiment showing that Jurkat cells, treated with anti-Fas antibody for increasing time periods, exhibited a progressively lower rate of both uncoupled and coupled endogenous respiration. The uncoupled respiration rate decreased to ∼62% of the rate of naive cells after 4 h of treatment, ∼53% after 6 h, and ∼43% after 8 h. The uncoupled endogenous respiration of both naive and anti-Fas-treated cells was 98% antimycin A-sensitive. The progressive decrease in endogenous respiration rate correlated well with an increase in number of apoptotic cells, which exhibited a characteristic shrunk and fragmented appearance of their nuclei, as determined by staining with the dsDNA-binding fluorochrome DAPI (Fig. 2 A). To dissect further the anti-Fas-induced changes in respiration, the effect of anti-Fas antibody on COX respiratory activity of intact cells was measured. The rate of oxygen consumption, in the presence of the membrane-permeant electron donor TMPD, of ascorbate as primary reducing agent, and of antimycin A to block the electron flux upstream of COX, is known to depend on both cytochrome c and COX, providing a measure of COX-dependent oxygen consumption that is isolated from the upstream segment of the respiratory chain (19Villani G. Attardi G. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 1166-1171Crossref PubMed Scopus (175) Google Scholar). Fig.2 A shows that the TMPD-dependent respiration rate decreased progressively with increasing time of cell treatment with anti-Fas antibody. The kinetics of decrease in TMPD-dependent respiration rate during the first 8 h of treatment was very similar to the kinetics of decrease in endogenous uncoupled respiration rate. However, it was apparently faster in the first few hours. In fact, a comparison of the kinetics of decrease in the endogenous and TMPD-dependent respiration rates in the first 3 h after anti-Fas-induction revealed that the reduction in endogenous respiration rate was slightly, but significantly, delayed with respect to the decrease in TMPD-dependent respiration rate. In particular, as shown by a representative experiment in Fig.2 B, after 1 h of anti-Fas-induction, the endogenous respiration rate was almost unchanged, whereas the TMPD-dependent respiration rate was decreased by ∼12% relative to the control. It was also found that z-VADfmk fully prevented the anti-Fas-induced decrease in endogenous coupled, endogenous uncoupled, and TMPD-dependent respiration rates (Fig. 2 C), indicating a requirement for caspase activation in anti-Fas-triggered loss of respiration in intact cells. In Fig.3, the changes in respiration in apoptotic Jurkat cells were correlated with the kinetics of cytochrome c release. Quantification of cytochrome c release from images such as those shown in Fig. 1 revealed that about 25% of the cells released massively cytochrome c into the cytosol after less than 1 h of treatment with anti-Fas antibody (Fig. 3). After 2 h of treatment, 58% of the cells had released cytochrome c, while the endogenous uncoupled and TMPD-dependent respiration rates had decreased only by 20 and 22%, respectively, relative to those measured in naive cells. After 4 h of treatment, 71% of cells had released cytochrome c, while the endogenous uncoupled and TMPD-dependent respiration rates had decreased only by 40% and 39%, respectively. Cytochrome c remained mitochondria-localized in ∼20% of the cells even after 8 h of treatment with anti-Fas antibody, when the uncoupled and TMPD-dependent respiration rates were decreased by ∼60% and ∼57%, respectively (Fig. 3). The faster kinetics of increase in the percentage of cells with released cytochrome c and the faster kinetics of respiration loss during the first 4 h indicated the presence of a subpopulation of cells responding faster to the apoptotic stimulus. These results also suggested a sequence of events in which loss of cytochrome c from a cell would precede a decrease in COX-dependent oxygen consumption and endogenous respiration. The observation mentioned above of a slight, although apparently significant, delay in the kinetics of decrease in the endogenous respiration rate relative to the kinetics of decrease in TMPD-dependent respiration rate could reflect an excess of COX capacity over that required to support the normal endogenous respiration. In fact, recent studies have demonstrated that, in a variety of human cell types analyzed, including fibroblasts and myoblasts, there is in vivo a relatively low excess of COX capacity (19Villani G. Attardi G. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 1166-1171Crossref PubMed Scopus (175) Google Scholar, 21Villani G. Greco M. Papa S. Attardi G. J. Biol. Chem. 1998; 273: 31829-31836Abstract Full Text Full Text PDF PubMed Scopus (190) Google Scholar). In the present work, the TMPD-dependent respiration rate had been measured in naive uncoupled Jurkat cells, and found to be ∼33% higher than the endogenous uncoupled respiration rate (Fig. 2, A and B, and data not shown). Thus, it seemed plausible to assume that the release of cytochrome c from mitochondria would cause initially a decrease in COX-dependent oxygen consumption, without affecting the endogenous respiration rate. To obtain a deeper insight into the role of the respiratory flux control by COX in the apoptosis-related events, the relative COX capacity in intact naive Jurkat cells was determined. For this purpose, the COX activity in DNP-uncoupled intact cells was titrated with the specific COX inhibitor KCN both as isolated step, in the presence of antimycin A, ascorbate, and TMPD, and as respiratory chain-integrated step (endogenous respiration) (19Villani G. Attardi G. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 1166-1171Crossref PubMed Scopus (175) Google Scholar). In the low range of KCN con" @default.
• W2018119239 created "2016-06-24" @default.
• W2018119239 creator A5044553741 @default.
• W2018119239 creator A5051767634 @default.
• W2018119239 creator A5061536209 @default.
• W2018119239 date "2001-01-01" @default.
• W2018119239 modified "2023-09-27" @default.
• W2018119239 title "Rate-limiting Step Preceding Cytochrome c Release in Cells Primed for Fas-mediated Apoptosis Revealed by Analysis of Cellular Mosaicism of Respiratory Changes" @default.
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