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• W1982410119 abstract "Various anticancer drugs cause mitochondrial perturbations in association with apoptosis. Here we investigated the involvement of caspase- and Bcl-2-dependent pathways in doxorubicin-induced mitochondrial perturbations and apoptosis. For this purpose, we set up a novel three-color flow cytometric assay using rhodamine 123, annexin V-allophycocyanin, and propidium iodide to assess the involvement of the mitochondria in apoptosis caused by doxorubicin in the breast cancer cell line MTLn3. Doxorubicin-induced apoptosis was preceded by up-regulation of CD95 and CD95L and a collapse of mitochondrial membrane potential (Δψ) occurring prior to phosphatidylserine externalization. This drop in Δψ was independent of caspase activity, since benzyloxycarbonyl-Val-Ala-dl-Asp-fluoromethylketone did not inhibit it. Benzyloxycarbonyl-Val-Ala-dl-Asp-fluoromethylketone also blocked activation of caspase-8, thus excluding an involvement of the death receptor pathway in Δψ dissipation. Furthermore, although overexpression of Bcl-2 in MTLn3 cells inhibited apoptosis, dissipation of Δψ was still observed. No decrease in Δψ was observed in cells undergoing etoposide-induced apoptosis. Immunofluorescent analysis of Δψ and cytochrome c localization on a cell-to-cell basis indicates that the collapse of Δψ and cytochromec release are mutually independent in both normal and Bcl-2-overexpressing cells. Together, these data indicate that doxorubicin-induced dissipation of the mitochondrial membrane potential precedes phosphatidylserine externalization and is independent of a caspase- or Bcl-2-controlled checkpoint. Various anticancer drugs cause mitochondrial perturbations in association with apoptosis. Here we investigated the involvement of caspase- and Bcl-2-dependent pathways in doxorubicin-induced mitochondrial perturbations and apoptosis. For this purpose, we set up a novel three-color flow cytometric assay using rhodamine 123, annexin V-allophycocyanin, and propidium iodide to assess the involvement of the mitochondria in apoptosis caused by doxorubicin in the breast cancer cell line MTLn3. Doxorubicin-induced apoptosis was preceded by up-regulation of CD95 and CD95L and a collapse of mitochondrial membrane potential (Δψ) occurring prior to phosphatidylserine externalization. This drop in Δψ was independent of caspase activity, since benzyloxycarbonyl-Val-Ala-dl-Asp-fluoromethylketone did not inhibit it. Benzyloxycarbonyl-Val-Ala-dl-Asp-fluoromethylketone also blocked activation of caspase-8, thus excluding an involvement of the death receptor pathway in Δψ dissipation. Furthermore, although overexpression of Bcl-2 in MTLn3 cells inhibited apoptosis, dissipation of Δψ was still observed. No decrease in Δψ was observed in cells undergoing etoposide-induced apoptosis. Immunofluorescent analysis of Δψ and cytochrome c localization on a cell-to-cell basis indicates that the collapse of Δψ and cytochromec release are mutually independent in both normal and Bcl-2-overexpressing cells. Together, these data indicate that doxorubicin-induced dissipation of the mitochondrial membrane potential precedes phosphatidylserine externalization and is independent of a caspase- or Bcl-2-controlled checkpoint. mitochondrial membrane potential 7-amino-4-methylcoumarin allophycocyanin annexin V confocal laser-scanning microscopy MitotrackerTM Red CMXRos 3,3′-dihexyloxacarbocyanine neomycin-resistant cells propidium iodide phosphatidylserine rhodamine 123 benzyloxycarbonyl-Val-Ala-dl-Asp fluoromethylketone α-minimal essential medium fetal bovine serum phosphate-buffered saline 1,4-piperazinediethanesulfonic acid Upon anticancer drug treatment, a number of cellular stress response pathways are activated. Some of these pathways are linked to mitochondrial perturbations that are often associated with apoptosis. Thus, the release of proapoptotic factors from the intermembrane space into the cytosol, including cytochrome c, apoptosis-inducing factor, and Smac/DIABLO, occurs after cytostatic treatment (1Engels I.H. Stepczynska A. Stroh C. Lauber K. Berg C. Schwenzer R. Wajant H. Janicke R.U. Porter A.G. Belka C. Gregor M. Schulze-Osthoff K. Wesselborg S. Oncogene. 2000; 19: 4563-4573Crossref PubMed Scopus (232) Google Scholar, 2Zhuang J. Cohen G.M. Toxicol. Lett. 1998; 102: 121-129Crossref PubMed Scopus (35) Google Scholar, 3Susin 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 (3463) Google Scholar, 4Adrain C. Creagh E.M. Martin S.J. EMBO J. 2001; 20: 6627-6636Crossref PubMed Scopus (362) Google Scholar). The mechanisms regulating the release of cytochrome c include specific pore formation in the outer mitochondrial membrane and opening of the permeability transition pore (reviewed in Ref. 5Harris M.H. Thompson C.B. Cell Death Differ. 2000; 7: 1182-1191Crossref PubMed Scopus (443) Google Scholar). As a consequence of both the loss of the electrochemical gradient caused by pore opening and rupture of the outer mitochondrial membrane, the mitochondrial membrane potential (Δψ)1 generally collapses. There is general agreement that cytosolic cytochromec interacts with Apaf-1, ATP, and procaspase-9, resulting in the activation of the latter, followed by caspase-3 activation and initiation of a proteolytic cascade (6Li 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 (6259) Google Scholar). However, the exact sequence of events resulting in disruption of mitochondrial function and release of cytochrome c from the mitochondria in apoptosis caused by anticancer drugs is not yet clear. There are two prominent pathways that may cause mitochondrial dysfunction during apoptosis. First, death receptor activation through CD95/CD95L and caspase-8 activation during apoptosis is under several circumstances upstream from mitochondrial perturbations. Thus, active caspase-8 may cause cleavage of the proapoptotic Bcl-2 member Bid and, as a consequence, mitochondrial dysfunction (7Li H. Zhu H., Xu, C.J. Yuan J. Cell. 1998; 94: 491-501Abstract Full Text Full Text PDF PubMed Scopus (3798) Google Scholar, 8Luo X. Budihardjo I. Zou H. Slaughter C. Wang X. Cell. 1998; 94: 481-490Abstract Full Text Full Text PDF PubMed Scopus (3085) Google Scholar, 9Slee E.A. Keogh S.A. Martin S.J. Cell Death Differ. 2000; 7: 556-565Crossref PubMed Scopus (244) Google Scholar). Several anticancer agents, including doxorubicin and etoposide, can up-regulate CD95 and CD95L (10Friesen C. Herr I. Krammer P.H. Debatin K.M. Nat. Med. 1996; 2: 574-577Crossref PubMed Scopus (958) Google Scholar, 11Fulda S. Sieverts H. Friesen C. Herr I. Debatin K.M. Cancer Res. 1997; 57: 3823-3829PubMed Google Scholar, 12Kaufmann S.H. Earnshaw W.C. Exp. Cell Res. 2000; 256: 42-49Crossref PubMed Scopus (1071) Google Scholar). However, there is controversy on the relative importance of this pathway in anticancer drug-induced mitochondrial perturbations and apoptosis. Thus, in some cell types, inhibition of this pathway using either Fas-linked interleukin-β-converting enzyme inhibitory protein, CrmA, or dominant negative Fas-associated death domain abrogates apoptosis, whereas in other cell types little effect was observed (reviewed in Ref. 12Kaufmann S.H. Earnshaw W.C. Exp. Cell Res. 2000; 256: 42-49Crossref PubMed Scopus (1071) Google Scholar). Although up-regulation of death receptor pathway components was found in some solid tumor cell lines, the extent of subsequent involvement of the mitochondrial pathway remains unclear (11Fulda S. Sieverts H. Friesen C. Herr I. Debatin K.M. Cancer Res. 1997; 57: 3823-3829PubMed Google Scholar, 13Fulda S. Los M. Friesen C. Debatin K.M. Int. J. Cancer. 1998; 76: 105-114Crossref PubMed Scopus (165) Google Scholar). Up-regulation and/or translocation of Bax and/or other proapoptotic Bcl-2 family members to the mitochondria is a second major pathway for mitochondrial perturbation preceding the onset of apoptosis. At the mitochondria, Bax invokes cytochrome crelease and loss of Δψ (14Gross A. Jockel J. Wei M.C. Korsmeyer S.J. EMBO J. 1998; 17: 3878-3885Crossref PubMed Scopus (968) Google Scholar), possibly via direct pore formation (15Schlesinger P.H. Gross A. Yin X.M. Yamamoto K. Saito M. Waksman G. Korsmeyer S.J. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 11357-11362Crossref PubMed Scopus (443) Google Scholar, 16Antonsson B. Conti F. Ciavatta A. Montessuit S. Lewis S. Martinou I. Bernasconi L. Bernard A. Mermod J.J. Mazzei G. Maundrell K. Gambale F. Sadoul R. Martinou J.C. Science. 1997; 277: 370-372Crossref PubMed Scopus (932) Google Scholar) or by association with the voltage-dependent anion channel (17Tsujimoto Y. Shimizu S. Cell Death Differ. 2000; 7: 1174-1181Crossref PubMed Scopus (267) Google Scholar, 18Shimizu S. Narita M. Tsujimoto Y. Nature. 1999; 399: 483-487Crossref PubMed Scopus (1928) Google Scholar). Importantly, the antiapoptotic Bcl-2 family members Bcl-2 and Bcl-xL generally inhibit these mitochondrial perturbations (19Vander Heiden M.G. Thompson C.B. Nat. Cell Biol. 1999; 1: E209-E216Crossref PubMed Scopus (602) Google Scholar, 20Green D.R. Reed J.C. Science. 1998; 281: 1309-1312Crossref PubMed Google Scholar). Various tumor cells have increased expression of proteins that inhibit either the death receptor pathways (e.g. Fas-linked interleukin-β-converting enzyme inhibitory protein) or apoptosis caused by Bax (e.g. Bcl-2 or Bcl-xL) (21Bullani R.R. Huard B. Viard-Leveugle I. Byers H.R. Irmler M. Saurat J.H. Tschopp J. French L.E. J. Invest Dermatol. 2001; 117: 360-364Abstract Full Text Full Text PDF PubMed Google Scholar, 22Kamihira S. Yamada Y. Hirakata Y. Tomonaga M. Sugahara K. Hayashi T. Dateki N. Harasawa H. Nakayama K. Br. J. Haematol. 2001; 114: 63-69Crossref PubMed Scopus (94) Google Scholar, 23Ryu B.K. Lee M.G. Chi S.G. Kim Y.W. Park J.H. J. Pathol. 2001; 194: 15-19Crossref PubMed Scopus (128) Google Scholar, 24Tsujimoto Y. Croce C.M. Proc. Natl. Acad. Sci. U. S. A. 1986; 83: 5214-5218Crossref PubMed Scopus (1068) Google Scholar, 25Vaux D.L. Cory S. Adams J.M. Nature. 1988; 335: 440-442Crossref PubMed Scopus (2737) Google Scholar). These tumor cells are generally more resistant to anticancer drug-induced apoptosis. It is unclear whether such resistance includes mitochondrial protection. Therefore, we have investigated the relative roles of caspase activation and Bcl-2-dependent pathways in mitochondrial perturbations and apoptosis caused by the anticancer drug doxorubicin. Doxorubicin is often used in the treatment of solid tumors, including breast, liver, and bone tumors (26Hortobagyi G.N. Drugs. 1997; 54 Suppl. 4: 1-7Crossref PubMed Scopus (620) Google Scholar). It causes DNA damage and formation of reactive oxygen species, eventually resulting in apoptosis (27Gewirtz D.A. Biochem. Pharmacol. 1999; 57: 727-741Crossref PubMed Scopus (1832) Google Scholar). Although doxorubicin causes mitochondrial injury in cardiac muscle cells (28Gille L. Nohl H. Free Radic. Biol. Med. 1997; 23: 775-782Crossref PubMed Scopus (185) Google Scholar) and some other cell types, these effects were studied primarily in lymphoid cells (29Gamen S. Anel A. Perez-Galan P. Lasierra P. Johnson D. Pineiro A. Naval J. Exp. Cell Res. 2000; 258: 223-235Crossref PubMed Scopus (120) Google Scholar, 30Decaudin D. Geley S. Hirsch T. Castedo M. Marchetti P. Macho A. Kofler R. Kroemer G. Cancer Res. 1997; 57: 62-67PubMed Google Scholar). The molecular mechanism of mitochondrial injury and its role in the induction of apoptosis in adenocarcinoma cells remains, however, largely unclear. As discussed above, the dissipation of Δψ is one of the markers for mitochondrial involvement in apoptosis. So far, dissipation of Δψ caused by doxorubicin has been determined either in the total cell population (30Decaudin D. Geley S. Hirsch T. Castedo M. Marchetti P. Macho A. Kofler R. Kroemer G. Cancer Res. 1997; 57: 62-67PubMed Google Scholar, 31Fulda S. Susin S.A. Kroemer G. Debatin K.M. Cancer Res. 1998; 58: 4453-4460PubMed Google Scholar, 32Bossy-Wetzel E. Newmeyer D.D. Green D.R. EMBO J. 1998; 17: 37-49Crossref PubMed Scopus (1107) Google Scholar) or in “viable” cells based on scatter properties (33Denecker G. Dooms H. Van Loo G. Vercammen D. Grooten J. Fiers W. Declercq W. Vandenabeele P. FEBS Lett. 2000; 465: 47-52Crossref PubMed Scopus (88) Google Scholar). These methods do not allow proper distinction of the exact cell population in which the changes in Δψ occurred: genuinely viable, apoptotic or (secondary) necrotic cells. As a consequence, the identification of the exact sequence of events in doxorubicin-induced apoptosis was precluded. In the present study, we set up three-color flow cytometry with rhodamine 123, annexin V-allophycocyanin (APC), and propidium iodide to assess the involvement of the mitochondria in doxorubicin-induced apoptosis. For this purpose, we used the rat mammary adenocarcinoma cell line MTLn3, which is often used as a model to study molecular mechanisms of metastasis formation (34Welch D.R. Neri A. Nicolson G.L. Invasion Metastasis. 1983; 3: 65-80PubMed Google Scholar, 35Kiley S.C. Clark K.J. Goodnough M. Welch D.R. Jaken S. Cancer Res. 1999; 59: 3230-3238PubMed Google Scholar) and responses to drug therapy both in vitro andin vivo (36Toyota N. Strebel F.R. Stephens L.C. Matsuda H. Oshiro T. Jenkins G.N. Bull J.M. Int. J. Cancer. 1998; 76: 499-505Crossref PubMed Scopus (17) Google Scholar, 37Kakeji Y. Maehara Y. Ikebe M. Teicher B.A. Int. J. Radiat. Oncol. Biol. Phys. 1997; 37: 1115-1123Abstract Full Text PDF PubMed Scopus (48) Google Scholar, 38Huigsloot M. Tijdens I.B. Mulder G.J. van de Water B. Biochem. Pharmacol. 2001; 62: 1087-1097Crossref PubMed Scopus (35) Google Scholar). We have previously characterized in detail the induction of apoptosis by anticancer drugs in these cells (38Huigsloot M. Tijdens I.B. Mulder G.J. van de Water B. Biochem. Pharmacol. 2001; 62: 1087-1097Crossref PubMed Scopus (35) Google Scholar). In the present study, we show that the doxorubicin-mediated collapse of Δψ is a primary event preceding PS externalization. Moreover, despite the fact that doxorubicin causes up-regulation of CD95 and CD95L, prevention of caspase-8 activation does not prevent loss of Δψ. Furthermore, although Bcl-2 inhibits apoptosis, dissipation of Δψ is still observed. Analysis of Δψ and cytochromec localization on a cell-to-cell basis indicates that the collapse of Δψ and cytochrome c release are mutually independent in both normal and Bcl-2-overexpressing cells. Together, these data indicate that doxorubicin-induced dissipation of the mitochondrial membrane potential precedes PS externalization and is independent of a caspase- or Bcl-2-controlled checkpoint. α-Modified minimal essential medium with ribonucleosides and deoxyribonucleosides (α-MEM), fetal bovine serum (FBS), penicillin/streptomycin, LipofectAMINE Plus, and Geneticin (G418 sulfate) were from Invitrogen. Collagen (type I, rat tail) was from Upstate Biotechnology, Inc. (Lake Placid, NY). Doxorubicin, propidium iodide (PI), 7-amino-4-methylcoumarin (AMC), DiOC6, rhodamine 123, and RNase A were from Sigma. Benzyloxycarbonyl-Val-Ala-dl-Asp-fluoromethylketone (zVAD-fmk) and acetyl-Asp-Glu-Val-Asp-7-amino-4-methylcoumarin were from Bachem (Bubendorf, Switzerland). Annexin V was from Roche Molecular Biochemicals. Hoechst 33258, the AlexaTM 488 protein labeling kit, and MitotrackerTM Red CMXRos were from Molecular Probes (Leiden, The Netherlands). APC was from Prozyme (San Leandro, CA). All other chemicals were of analytical grade. MTLn3 rat mammary adenocarcinoma cells were originally developed by Dr. D. R. Welch (Jake Gittlen Cancer Research Institute, The Pennsylvania State University College of Medicine, Hershey, PA) and used between passages 46 and 56. They were cultured in α-MEM supplemented with 5% (v/v) FBS (complete medium). For experiments, cells were plated at a density of 4 × 103 cells/cm2 in Corning plates (Acton, MA) and grown for 3 days in complete medium supplemented with 50 units of penicillin/liter and 50 mg of streptomycin/liter (penicillin/streptomycin). Cells were exposed to doxorubicin for 1 h in Hanks' balanced salt solution (137 mm NaCl, 5 mm KCl, 0.8 mmMgSO4·7H2O, 0.4 mmNa2HPO4·2H2O, 0.4 mmKH2PO4, 1.3 mm CaCl2, 4 mm NaHCO3, 25 mm HEPES, 5 mmd-glucose, pH 7.4). After removal of doxorubicin, cells were recovered in α-MEM containing 2.5% (v/v) FBS and penicillin/streptomycin for the indicated periods. In some experiments, cells were recovered in α-MEM containing 2.5% (v/v) FBS, penicillin/streptomycin and 100 μm zVAD-fmk. Subconfluent MTLn3 cells were transfected with pcDNA3 (Neo) or pcDNA3 containing human Bcl-2 (gift from Dr. James L. Stevens) using LipofectAMINE Plus reagent, and after reaching confluence they were selected for neomycin resistance (G418; 100 μg/ml). For both vectors, three clones were selected and used for up to six passages, during which they stably expressed Bcl-2 in over 95% of cells based on immunofluorescence. In some experiments, we used porcine renal proximal tubular cell line LLC-PK1 expressing either Bcl-2 (pkBCL-2 clone 6) or the empty vector (pkNEO clone 1) that have been described previously (39Zhan Y. van de Water B. Wang Y. Stevens J.L. Oncogene. 1999; 18: 6505-6512Crossref PubMed Scopus (73) Google Scholar). LLC-PK1 cells were cultured in Dulbecco's modified Eagle's medium containing 10% (v/v) FBS and penicillin/streptomycin. For experiments, cells were plated overnight in Dulbecco's modified Eagle's medium plus penicillin/streptomycin without FBS on collagen-coated 6-cm culture dishes to form a subconfluent monolayer as described previously (40van de Water B. Houtepen F. Huigsloot M. Tijdens I.B. J. Biol. Chem. 2001; 276: 36183-36193Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar). Thereafter, cells were treated with varying concentrations of doxorubicin in Dulbecco's modified Eagle's medium/penicillin/streptomycin for 24 h. For annexin V/propidium iodide (AV/PI) staining, cells were washed twice in PBS (137 mmNaCl, 2.7 mm KCl, 4.3 mmNa2HPO4·2H2O, 1.4 mmKH2PO4, pH 7.4) containing 1 mmEDTA (PBS-EDTA) and subsequently trypsinized with 0.13 g/liter trypsin in PBS-EDTA. Medium, washes, and cells were combined and centrifuged (5 min, 200 × g, 4 °C), and the pellet was washed once with PBS-EDTA. Cells were allowed to recover from trypsinization in complete medium (30 min, 37 °C). Externalized phosphatidylserine (PS) was labeled (15 min, 0 °C) with AlexaTM488-conjugated annexin V in AV buffer (10 mm HEPES, 145 mm NaCl, 5 mm KCl, 1.0 mmMgCl2·6H2O, 1.8 mmCaCl2·2H2O, pH 7.4). Propidium iodide (2 μm) in AV buffer was added 1 min prior to analysis on a FACScalibur flow cytometer (BD PharMingen). For cell cycle analysis, trypsinized and floating cells were pooled, washed with PBS-EDTA, and fixated in 70% (v/v) ethanol (30 min, −20 °C). After two washes with PBS-EDTA, cells were incubated with PBS-EDTA containing 50 μg/ml RNase A and 7.5 μm PI (45 min, room temperature) and subsequently analyzed by flow cytometry. Caspase-3-like activity was determined as described previously (38Huigsloot M. Tijdens I.B. Mulder G.J. van de Water B. Biochem. Pharmacol. 2001; 62: 1087-1097Crossref PubMed Scopus (35) Google Scholar). Briefly, cells were trypsinized as described for AV/PI, washed once in PBS-EDTA, and resuspended in lysis buffer (10 mm HEPES, 40 mm β-glycerophosphate, 50 mm NaCl, 2 mm MgCl2, 5 mm EGTA, pH 7.0). Cells were lysed by four cycles of freezing and thawing followed by centrifugation (30 min, 13,000 × g, 4 °C). To 10 μg of cell lysate protein, 80 μl assay buffer was added (100 mm HEPES, 10% (w/v) sucrose, 0.1% (v/v) Nonidet P40, 10 mm dithiothreitol, 25 μmacetyl-Asp-Glu-Val-Asp-7-amino-4-methylcoumarin, pH 7.25), and the release of AMC was monitored (45 min, 37 °C) in a fluorescence plate reader (HTS 7000 bioassay reader; PerkinElmer Life Sciences). Free AMC was used as a standard, and caspase activity was expressed as pmol of AMC/min/mg of protein. Mitochondrial membrane potential was essentially performed as described before (41van de Water B. Zoeteweij J.P. de Bont H.J. Mulder G.J. Nagelkerke J.F. J. Biol. Chem. 1994; 269: 14546-14552Abstract Full Text PDF PubMed Google Scholar, 42Zoeteweij J.P. van de Water B. de Bont H.J. Mulder G.J. Nagelkerke J.F. J. Biol. Chem. 1993; 268: 3384-3388Abstract Full Text PDF PubMed Google Scholar) with some modifications. Briefly, cells were harvested as described for AV/PI staining. Following recovery in complete medium, cells were incubated with 1 μm rhodamine 123 (Rho123) and APC-conjugated annexin V (43Hardy R.R. Weir D.M. Herzenberg L.A. Blackwell C. Purification and Coupling of Fluorescent Proteins for Use in Flow Cytometry. Blackwell Scientific Publications, Boston1986: 31.1-31.12Google Scholar) in AV buffer (30 min, 37 °C). Cells were centrifuged (30 s, 400 × g, room temperature), and the pellet was resuspended in AV buffer containing 2 μm propidium iodide 1 min prior to analysis by confocal laser-scanning microscopy (CLSM; Bio-Rad) or flow cytometry. As an alternative method to determine the mitochondrial membrane potential, we used DiOC6 (0.1 μm) instead of Rho123 (30Decaudin D. Geley S. Hirsch T. Castedo M. Marchetti P. Macho A. Kofler R. Kroemer G. Cancer Res. 1997; 57: 62-67PubMed Google Scholar, 44Finucane D.M. Waterhouse N.J. Amarante-Mendes G.P. Cotter T.G. Green D.R. Exp. Cell Res. 1999; 251: 166-174Crossref PubMed Scopus (123) Google Scholar, 45Petit P.X. O'Connor J.E. Grunwald D. Brown S.C. Eur. J. Biochem. 1990; 194: 389-397Crossref PubMed Scopus (213) Google Scholar). Selective localization of DiOC6 at the mitochondria was confirmed by CLSM. MTLn3 Neo and Bcl-2 cells were treated with varying concentrations of doxorubicin as described above. After 24 h, cells were trypsinized, and viable cells (trypan blue exclusion) were counted. Next, 12,500 cells were plated in 1 ml of top agar (0.33% (w/v) agarose in complete medium in the presence of amphotericin B (250 ng/ml)) on top of 2.5 ml of bottom agar (0.66% (w/v) agarose in complete medium in the presence of amphotericin B (250 ng/ml)) in duplicate in six-well plates, as described by Kiley et al. (35Kiley S.C. Clark K.J. Goodnough M. Welch D.R. Jaken S. Cancer Res. 1999; 59: 3230-3238PubMed Google Scholar). After 1 week, a top layer of 2.5 ml of bottom agar was added. After 14 days, 150 μl of a 5 μg/ml 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide solution in medium was added to the wells, and after overnight incubation (37 °C), digital images of the wells were taken with a Nikon CCD camera. Colonies were counted using a particle-counting option in Image Pro (Media Cybernetics, Silver Spring, MD). Attached cells were scraped in ice-cold TSE+ (10 mm Tris-HCl, 250 mm sucrose, 1 mm EGTA, pH 7.4, containing 1 mmdithiothreitol, 10 μg/ml leupeptin, 10 μg/ml aprotinin, 1 mm sodium vanadate, 50 mm sodium fluoride, and 1 mm phenylmethylsulfonyl fluoride). Floating cells in the medium and in one wash of PBS were pelleted (5 min, 200 ×g, 4 °C) and pooled with scraped cells in TSE+. The protein concentration in the supernatant was determined using the Bio-Rad protein assay using IgG as a standard. Fifteen μg of total cellular protein was separated by SDS-PAGE and transferred to polyvinylidene difluoride membrane (Millipore Corp., Etten Leur, The Netherlands). Blots were blocked with 5% (w/v) nonfat dry milk in TBS-T (0.5 m NaCl, 20 mm Tris-HCl, 0.05% (v/v) Tween 20, pH 7.4) and probed for Bcl-2 (C-2; Santa Cruz Biotechnology, Inc., Santa Cruz, CA), p53 (polyclonal antibody 240; Santa Cruz Biotechnology), CD95 (FL335; Santa Cruz Biotechnology), CD95L (clone 33; Transduction Laboratories), caspase-8 (kindly provided by Prof. J. Borst (46Boesen-de Cock J.G. Tepper A.D. de Vries E. van Blitterswijk W.J. Borst J. J. Biol. Chem. 1999; 274: 14255-14261Abstract Full Text Full Text PDF PubMed Scopus (113) Google Scholar)), active caspase-3 (CM-1; kindly provided by Dr. A. Srinivasan (47Srinivasan A. Roth K.A. Sayers R.O. Shindler K.S. Wong A.M. Fritz L.C. Tomaselli K.J. Cell Death Differ. 1998; 5: 1004-1016Crossref PubMed Scopus (352) Google Scholar)), or protein kinase Cδ (δ14K; kindly provided by Dr. S. Jaken (48Kiley S.C. Clark K.J. Duddy S.K. Welch D.R. Jaken S. Oncogene. 1999; 18: 6748-6757Crossref PubMed Scopus (97) Google Scholar)), followed by incubation with secondary antibody containing horseradish peroxidase and visualization with ECL reagent (Amersham Biosciences AB, Uppsala, Sweden). Cytosolic fractions were prepared as described by Boesen-de Cock (46Boesen-de Cock J.G. Tepper A.D. de Vries E. van Blitterswijk W.J. Borst J. J. Biol. Chem. 1999; 274: 14255-14261Abstract Full Text Full Text PDF PubMed Scopus (113) Google Scholar). Briefly, floating cells in the medium and one wash of mitobuffer (50 mm PIPES-KOH (pH 7.4), 220 mmmannitol, 68 mm sucrose, 50 mm KCl, 5 mm EGTA, 2 mm MgCl2, 1 mm dithiothreitol, and 1 mmphenylmethylsulfonyl fluoride) were pelleted (5 min, 200 × g, 4 °C) and pooled with attached cells scraped in 100 μl of ice-cold mitobuffer. Cells were pelleted (1 min, 400 × g, room temperature), resuspended in 100 μl of mitobuffer, and allowed to swell on ice for 30 min. Cells were homogenized by passing the suspension through a 25-gauge needle (10 strokes). Homogenates were centrifuged (15 min, 13,000 × g, 4 °C), and supernatants were collected. Thirty μg of cytosolic protein was separated on a 15% SDS-PAGE gel and transferred to polyvinylidene difluoride membrane. Cytochrome c was detected with anti-cytochrome c monoclonal antibody (7H8.2C12; BD PharMingen) using the Western Star kit (Tropix, Bedford, MA). Cells were cultured on 12-mm collagen-coated glass coverslips and fixated in fresh 4% (w/v) paraformaldehyde in PBS. Coverslips were blocked in TBP (0.5% (w/v) bovine serum albumin and 0.05% (v/v) Tween 20 in PBS, pH 7.4) (1 h, room temperature) and subsequently incubated with primary antibody in TBP (1 h, room temperature). Coverslips were washed twice in PBS containing 0.05% Tween 20 and incubated with AlexaTM 488-, Cy3- or Cy5-conjugated secondary antibodies in TBP (45 min, room temperature). After washing, coverslips were incubated with 2 μg/ml Hoechst 33258 in PBS (15 min, room temperature), washed in PBS, and mounted in Aqua PolyMount (Polysciences, Warrington, PA). In some experiments, cells were incubated with 200 nm MitoTracker Red CMXRos in complete medium (15 min, 37 °C) prior to fixation. Primary antibodies used were Bcl-2 (C-2; Santa Cruz Biotechnology) and cytochrome c (6H2B4; BD PharMingen). Imaging occurred by confocal laser-scanning microscopy (Bio-Rad). Student's t test was used to determine whether there was a significant difference between two means (p < 0.05). When multiple means were compared, significance was determined by one-way analysis of variance (p < 0.05). For analysis of variance analysis, letter designations are used to indicate statistically significant differences. Means with a common letter designation within onefigure are not different; those with a different letter designation are significantly different from all other means with different letter designations. For example, a mean designated asa is significantly different from a mean designatedb, but neither is different from a mean designateda,b. The mitochondria are key organelles in the control of apoptosis. Therefore, we investigated the involvement of mitochondrial dysfunction in doxorubicin-induced apoptosis. The Δψ is a sensitive measure for mitochondrial functioning (reviewed by Kroemer and Reed (49Kroemer G. Reed J.C. Nat. Med. 2000; 6: 513-519Crossref PubMed Scopus (2785) Google Scholar)). Previously, we reported on the apoptotic effects of doxorubicin on MTLn3 cells (38Huigsloot M. Tijdens I.B. Mulder G.J. van de Water B. Biochem. Pharmacol. 2001; 62: 1087-1097Crossref PubMed Scopus (35) Google Scholar). Doxorubicin induced apoptosis in a time-dependent manner, as determined by annexin V-staining, analysis of DNA content, and caspase activity. The onset of apoptosis occurred between 8 and 16 h after exposure, with maximal caspase-3 activity at 24 h. In the present study, we analyzed Δψ after doxorubicin treatment by flow cytometry using Rho123. Initially, Rho123 fluorescence was only quantified in the PI (i.e. viable) population, gated as indicated in Fig. 1 A. Doxorubicin caused a decrease in Δψ in PI− cells (Fig.1 B). PI− cells, however, include two cell populations: genuinely viable cells as well as early apoptotic cells. These cells can be distinguished by the absence or presence of externalized PS (viable and apoptotic cells, respectively) as identified by annexin V staining. Therefore, the possibility existed that the drop in Δψ was mainly present in cells that were already apoptotic (i.e. have externalized PS). Alternatively, apoptotic cells may have a disturbed intracellular distribution of Rho123 that does not reflect the mitochondrial membrane potential. To investigate these possibilities in more detail, MTLn3 cells were stained with Rho123 and propidium iodide as well as APC-labeled AV. This enabled us to determine the relationship between loss of Δψ and onset of apoptosis in PI− cells. Using CLSM (Fig.2), we observed that in control cells, the Rho123 staining is intense and strictly located at the mitochondria; little variability is observed between cells (A–C). In contrast, in doxorubicin-treated cells, there is considerable variability in Rho123 staining (G–I). Thus, whereas in all AV−/PI− cells Rho123 is located at mitochondria, some of these cells have a markedly decreased Δψ. In contrast, in AV+/PI− cells the Rho123 staining is more diff" @default.
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• W1982410119 date "2002-09-01" @default.
• W1982410119 modified "2023-10-16" @default.
• W1982410119 title "Differential Regulation of Doxorubicin-induced Mitochondrial Dysfunction and Apoptosis by Bcl-2 in Mammary Adenocarcinoma (MTLn3) Cells" @default.
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• W1982410119 doi "https://doi.org/10.1074/jbc.m200378200" @default.
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