Early Mitochondrial Activation and Cytochrome c Up-regulation during Apoptosis
2002; Elsevier BV; Volume: 277; Issue: 52 Linguagem: Inglês
10.1074/jbc.m207622200
ISSN1083-351X
AutoresDhyan Chandra, Jun Wei Liu, Dean G. Tang,
Tópico(s)ATP Synthase and ATPases Research
ResumoApoptosis induced by many stimuli requires the mitochondrial respiratory chain (MRC) function. While studying the molecular mechanisms underlying this MRC-dependent apoptotic pathway, we find that apoptosis in multiple cell types induced by a variety of stimuli is preceded by an early induction of MRC proteins such as cytochrome c (which is encoded by a nuclear gene) and cytochrome c oxidase subunit II (COX II) (which is encoded by the mitochondrial genome). Several non-MRC proteins localized in the mitochondria, e.g. Smac, Bim, Bak, and Bcl-2, are also rapidly up-regulated. The up-regulation of many of these proteins (e.g. cytochrome c, COX II, and Bim) results from transcriptional activation of the respective genes. The up-regulated cytosolic cytochrome c rapidly translocates to the mitochondria, resulting in an accumulation of holocytochrome c in the mitochondria accompanied by increasing holocytochrome c release into the cytosol. The increased cytochrome c transport from cytosol to the mitochondria does not depend on the mitochondrial protein synthesis or MRC per se. In contrast, cytochrome c release from the mitochondria involves dynamic changes in Bcl-2 family proteins (e.g. up-regulation of Bak, Bcl-2, and Bcl-xL), opening of permeability transition pore, and loss of mitochondrial membrane potential. Overexpression of cytochrome c enhances caspase activation and promotes cell death in response to apoptotic stimulation, but simple up-regulation of cytochrome c using an ecdysone-inducible system is, by itself, insufficient to induce apoptosis. Taken together, these results suggest that apoptosis induced by many stimuli involves an early mitochondrial activation, which may be responsible for the subsequent disruption of MRC functions, loss of Δψm, cytochrome c release, and ultimately cell death. Apoptosis induced by many stimuli requires the mitochondrial respiratory chain (MRC) function. While studying the molecular mechanisms underlying this MRC-dependent apoptotic pathway, we find that apoptosis in multiple cell types induced by a variety of stimuli is preceded by an early induction of MRC proteins such as cytochrome c (which is encoded by a nuclear gene) and cytochrome c oxidase subunit II (COX II) (which is encoded by the mitochondrial genome). Several non-MRC proteins localized in the mitochondria, e.g. Smac, Bim, Bak, and Bcl-2, are also rapidly up-regulated. The up-regulation of many of these proteins (e.g. cytochrome c, COX II, and Bim) results from transcriptional activation of the respective genes. The up-regulated cytosolic cytochrome c rapidly translocates to the mitochondria, resulting in an accumulation of holocytochrome c in the mitochondria accompanied by increasing holocytochrome c release into the cytosol. The increased cytochrome c transport from cytosol to the mitochondria does not depend on the mitochondrial protein synthesis or MRC per se. In contrast, cytochrome c release from the mitochondria involves dynamic changes in Bcl-2 family proteins (e.g. up-regulation of Bak, Bcl-2, and Bcl-xL), opening of permeability transition pore, and loss of mitochondrial membrane potential. Overexpression of cytochrome c enhances caspase activation and promotes cell death in response to apoptotic stimulation, but simple up-regulation of cytochrome c using an ecdysone-inducible system is, by itself, insufficient to induce apoptosis. Taken together, these results suggest that apoptosis induced by many stimuli involves an early mitochondrial activation, which may be responsible for the subsequent disruption of MRC functions, loss of Δψm, cytochrome c release, and ultimately cell death. mitochondrial respiratory chain actinomycin D 7-amino-4-trifluoromethylcoumarin a hydroxamic acid compound cycloheximide cytochrome c oxidase subunit II cyclosporin A 4′,6-diamidino-2-phenylindole green fluorescence protein mitochondrial activation-dependent apoptotic pathway poly(ADP-ribose) polymerase permeability transition pore reactive oxygen species reverse transcriptase voltage-dependent anion channel etoposide respiration-deficient cells tumor necrosis factor-α 7-amino-4-trifluoromethylcoumarin 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid benzyloxycarbonyl fluoromethyl ketone fetal bovine serum phosphate-buffered saline enhanced green fluorescent protein inner mitochondrial membrane outer mitochondrial membrane mitochondria apoptosis-induced channel N-acetylcysteine Mitochondria generate ATP through the mitochondrial respiratory chain (MRC),1 which is composed of four multisubunit respiration complexes (I–IV) and two mobile electron carriers (i.e. cytochrome c and ubiquinone). Electrons from reducing substrates such as NADH and succinate are transferred from complex I (NADH dehydrogenase) or complex II (succinate dehydrogenase), respectively, to ubiquinone, to complex III (cytochrome c reductase), to cytochromec, to complex IV (cytochrome c oxidase or COX), and finally to O2. The electron transport through complexes I, III, and IV is accompanied by the pumping of protons from the matrix to the intermembrane space, where the protons establish a mitochondrial membrane potential (Δψm) by forming a proton and a pH gradient. The reverse flow of the protons from the intermembrane space into the matrix drives another multiprotein complex, F0F1-ATPase (or complex V), to produce ATP. The protein subunits in complexes I, III, IV, and V are encoded by both nuclear and mitochondrial genomes, thus necessitating smooth communications between and coordinated gene expressions from two genomes (1Poyton R.O. McEwen J.E. Annu. Rev. 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Genes Dev. 2002; 16: 33-45Crossref PubMed Scopus (431) Google Scholar).Apoptosis induced by many stimuli seems to depend on MRC. For example, MRC function has been reported to be required for apoptosis induced by TNF-α (31Schultze-Osthoff K. Beyaert R. Vandevoorde V. Haegeman G. Fiers W. EMBO J. 1993; 12: 3095-3104Crossref PubMed Scopus (548) Google Scholar, 32Higuchi M. Aggarwal B.B. Yeh E.T.H. J. Clin. Invest. 1997; 99: 1751-1758Crossref PubMed Scopus (133) Google Scholar, 33Deshpande S.S. AngKeow P. Huang J. Ozaki M. Iran K. FASEB J. 2000; 14: 1705-1714Crossref PubMed Scopus (203) Google Scholar), lipoxygenase inhibitor nordihydroguaiaretic acid (34Tang D.G. Honn K.V. J. Cell. Physiol. 1997; 172: 155-170Crossref PubMed Scopus (79) Google Scholar), butyrate, and some other short chain fatty acids (35Heerdt B.G. Houston M.A. Augenlicht L.H. Cell Growth Differ. 1997; 8: 523-532PubMed Google Scholar), ceramide (36Quillet-Mary A. Jaffrezou J.-P. Mansat V. Bordier C. Naval J. Laurent G. 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Schmitz M.L. Oncogene. 1999; 18: 747-757Crossref PubMed Scopus (294) Google Scholar), and Ca2+ overloading (43Chauvin C., De Oliveira F. Ronot X. Mousseau M. Leverve X. Fontaine E. J. Biol. Chem. 2001; 276: 41394-41398Abstract Full Text Full Text PDF PubMed Scopus (117) Google Scholar). Thus, in these apoptotic model systems, MRC-deficient ρ0 cells are more resistant to apoptosis, and MRC inhibitors (such as rotenone and antimycin A) block or inhibit apoptosis. Also in support of the dependence of apoptosis on MRC, complex I deficiency in leukemia cells results in apoptosis resistance (44Kataoka A. Kubota M. Watanabe K. Sawada M. Koishi S. Lin W.W. Usami I. Akiyama V. Kitoh T. Furusho K. Cancer Res. 1997; 57: 5243-5245PubMed Google Scholar). Deficiency in complex IV in colon carcinoma cells renders them resistant to apoptosis induction (35Heerdt B.G. Houston M.A. Augenlicht L.H. Cell Growth Differ. 1997; 8: 523-532PubMed Google Scholar). Similarly, F0F1-ATPase is required for Bax-induced apoptosis (45Matsuyama S., Xu, Q. Velours J. Reed J.C. Mol. Cell. 1998; 1: 327-336Abstract Full Text Full Text PDF PubMed Scopus (250) Google Scholar).The molecular mechanisms underlying this MRC-dependent apoptosis remain unclear. Here we report that apoptosis induced by BMD188, a chemical that causes cell death in an MRC-dependent manner (37Joshi B., Li, L. Taffe B.G. Zhu Z. Wahl S. Tian H. Ben-Josef E. Taylor J.D. Porter A.T. Tang D.G. Cancer Res. 1999; 59: 4343-4355PubMed Google Scholar), involves an early up-regulation of MRC proteins (in particular, cytochrome c) prior to caspase activation and cell death. Importantly, this phenomenon seems to represent a general early apoptotic response as it is also observed in multiple cell types induced to die by a variety of stimuli.MATERIALS AND METHODSCells and ReagentsHuman osteosarcoma cells 143B(TK−) (143B) and fibroblasts GM701.2-8C (GM701) and their respective mtDNA-less, respiration-deficient ρ0 derivatives 143B206 and GM701.2–8C (2Eguchi Y. Shimizu S. Tsujimoto Y. Cancer Res. 1997; 57: 1835-1840PubMed Google Scholar) cells (46King M.P. Attardi G. Science. 1989; 246: 500-503Crossref PubMed Scopus (1438) Google Scholar) were kindly provided by Dr. M. King (Thomas Jefferson University). 143B and GM701 cells were cultured in Dulbecco's minimum essential medium (Invitrogen) supplemented with 10% heat-inactivated fetal bovine serum (FBS) and antibiotics. 143B206 and GM701.2–8C (2Eguchi Y. Shimizu S. Tsujimoto Y. Cancer Res. 1997; 57: 1835-1840PubMed Google Scholar) cells were cultured in Dulbecco's minimum essential medium with high glucose supplemented with 100 μg/ml pyruvate, 200 ng/ml ethidium bromide, 50 μg/ml uridine, 10% FBS, and antibiotics. Human prostate cancer cells, PC3 and LNCaP, and breast carcinoma cells, MDA-MB231, were purchased from ATCC (Manassas, VA) and cultured in RPMI 1640 supplemented with 5 and 10% FBS, respectively. The ρ0 PC3 clone 6 cells (37Joshi B., Li, L. Taffe B.G. Zhu Z. Wahl S. Tian H. Ben-Josef E. Taylor J.D. Porter A.T. Tang D.G. Cancer Res. 1999; 59: 4343-4355PubMed Google Scholar) were cultured as for 143B206 cells.The primary antibodies used were listed in Table I. Secondary antibodies, i.e. goat, donkey, or sheep anti-mouse or rabbit IgG conjugated to horseradish peroxidase, fluorescein isothiocyanate, or rhodamine, together with ECL (enhanced chemiluminescence) reagents were acquired from Amersham Biosciences. Fluorogenic caspase substrates DEVD-AFC and LEHD-AFC, pan-caspase inhibitor z-VAD-fmk, and caspase-3/6/7 inhibitor, z-DEVD-fmk, were bought from Biomol (Plymouth Meeting, PA). Ponasterone and Zeocin were purchased from Invitrogen. Annexin V conjugated to AlexaFluor 568 and mitochondrial dyes were purchased from Molecular Probes (Eugene, OR). Liposome FuGENE 6 was bought from Roche Molecular Biochemicals. All other chemicals were purchased from Sigma unless specified otherwise.Table IPrimary antibodies used in this studyAb1-aThe abbreviations used are: Ab, antibodies; COX I/II/IV, cytochrome c oxidase subunit I/II/IV; cyt-c, cytochrome c; GFP, green fluorescence protein; PARP, poly-(ADP-ribose); polymerase; mAb, monoclonal antibody; pAb, polyclonal antibody; Rb, rabbit; HSP60, heat shock protein 60.TypeSource (catalog no.)RemarksActinMouse mAbICN (69100)BadMouse mAbSanta Cruz Biotechnology (sc-8044)BakRb pAbSanta Cruz Biotechnology (sc-832)BakRb pAbUpstate Biotechnology (06–536)Recognizes activated BakBaxRb pAbSanta Cruz Biotechnology (554104)BaxRb pAbUpstate Biotechnology (06–499)Recognizes activated BaxBcl-2Mouse mAbPharmingen (14831A)Bcl-xRb pAbPharmingen (610211)BidRb pAbDr. X WangRecognizes both t Bid and uncleaved BidBimRb pAbCalbiochem (202000)COX IMouse mAbMolecular Probes (A-6403)COX IIMouse mAbMolecular Probes (A-6404)COX IVMouse mAbMolecular Probes (A-6431)Cyt-cMouse mAb (clone 6H2.B4)Pharmingen (556432)Recognizes holocytochromec on immunofluorescenceCyt-cMouse mAb (clone 7H8.2C12)Pharmingen (65981A)Recognizes apo- and holo-cyt-c on Western blotCyt-cMouse mAbR & D Systems (6380-MC-100)Recognizes holocytochrome c only on Western blotCaspase-3Mouse mAbTransduction Laboratories (C31720)Recognizes the proform onlyCaspase-9Rb pAbChemicon (AB16970)GFPMouse mAbClontech (8362–1)PARPRb pAbRoche Molecular Biochemicals (1 835 238)SmacRb pAbDr. X. WangHSP60Mouse mAbChemicon (mAb3514)1-a The abbreviations used are: Ab, antibodies; COX I/II/IV, cytochrome c oxidase subunit I/II/IV; cyt-c, cytochrome c; GFP, green fluorescence protein; PARP, poly-(ADP-ribose); polymerase; mAb, monoclonal antibody; pAb, polyclonal antibody; Rb, rabbit; HSP60, heat shock protein 60. Open table in a new tab Subcellular Fractionation and Western BlottingMitochondria were prepared using differential centrifugation (37Joshi B., Li, L. Taffe B.G. Zhu Z. Wahl S. Tian H. Ben-Josef E. Taylor J.D. Porter A.T. Tang D.G. Cancer Res. 1999; 59: 4343-4355PubMed Google Scholar, 47Tang D.G., Li, L. Zhu Z. Joshi B. Biochem. Biophys. Res. Commun. 1998; 242: 380-384Crossref PubMed Scopus (116) Google Scholar, 48Liu J.-W. Chandra D. Tang S.-H. Chopra D. Tang D.G. Cancer Res. 2002; 62: 2976-2981PubMed Google Scholar) with slight modifications. Briefly, cells were treated with various chemicals or vehicle (ethanol or Me2SO) control. In some experiments, cells were pretreated with protein synthesis inhibitor cycloheximide (CHX), RNA synthesis inhibitor actinomycin D (A/D), or mitochondrial protein synthesis inhibitor tetracycline before apoptosis induction. At the end of the treatment, cells were harvested by scraping, washed twice in ice-cold PBS, and resuspended in 600 μl of homogenizing buffer (20 mmHEPES-KOH, pH 7.5, 10 mm KCl, 1.5 mmMgCl2, 1 mm sodium EDTA, 1 mmsodium EGTA, and 1 mm dithiothreitol) containing 250 mm sucrose and a mixture of protease inhibitors (1 mm phenylmethylsulfonyl fluoride, 1% aprotinin, 1 mm leupeptin, 1 μg/ml pepstatin A, and 1 μg/ml chymostatin). After 30 min of incubation on ice, cells were homogenized in the same buffer using a glass Pyrex homogenizer (type A pestle, 140 strokes). Unbroken cells, large plasma membrane pieces, and nuclei were removed by centrifuging the homogenates at 500 × g for 5 min at 4 °C. The resulting supernatant was centrifuged at 10,000 × g for 20 min to obtain mitochondria. The remaining supernatant was again subjected to centrifugation at 100,000 × g for 1 h to obtain the cytosolic fraction (i.e. S100 fraction). The mitochondrial pellet was washed three times in homogenizing buffer, and then solubilized in TNC buffer (10 mm Tris acetate, pH 8.0, 0.5% Nonidet P-40, 5 mm CaCl2) containing protease inhibitors. Protein concentration was determined by Micro-BCA kit (Pierce).For Western blotting, 25 μg of proteins (mitochondrial or cytosolic fractions) was loaded in each lane of a 15% SDS-polyacrylamide gel. After gel electrophoresis and protein transfer, the membrane was probed or reprobed, after stripping, with various primary and corresponding secondary antibodies (37Joshi B., Li, L. Taffe B.G. Zhu Z. Wahl S. Tian H. Ben-Josef E. Taylor J.D. Porter A.T. Tang D.G. Cancer Res. 1999; 59: 4343-4355PubMed Google Scholar, 47Tang D.G., Li, L. Zhu Z. Joshi B. Biochem. Biophys. Res. Commun. 1998; 242: 380-384Crossref PubMed Scopus (116) Google Scholar). Western blotting was performed using ECL as described previously (37Joshi B., Li, L. Taffe B.G. Zhu Z. Wahl S. Tian H. Ben-Josef E. Taylor J.D. Porter A.T. Tang D.G. Cancer Res. 1999; 59: 4343-4355PubMed Google Scholar, 47Tang D.G., Li, L. Zhu Z. Joshi B. Biochem. Biophys. Res. Commun. 1998; 242: 380-384Crossref PubMed Scopus (116) Google Scholar).Measurement of ApoptosisApoptosis was measured using several biochemical and biological approaches.PARP CleavagePARP cleavage assays were performed as described previously (37Joshi B., Li, L. Taffe B.G. Zhu Z. Wahl S. Tian H. Ben-Josef E. Taylor J.D. Porter A.T. Tang D.G. Cancer Res. 1999; 59: 4343-4355PubMed Google Scholar, 47Tang D.G., Li, L. Zhu Z. Joshi B. Biochem. Biophys. Res. Commun. 1998; 242: 380-384Crossref PubMed Scopus (116) Google Scholar).Caspase-3 ActivationCaspase-3 cleavage (activation) was analyzed by Western blotting. Cells were lysed in TNC lysis buffer, and 100 μg of whole cell lysates was separated on 15% SDS-PAGE. After protein transfer, the blot was probed with a monoclonal antibody for caspase-3. The activation of caspase-3 was monitored by a decrease or disappearance of the ∼32-kDa procaspase-3 (37Joshi B., Li, L. Taffe B.G. Zhu Z. Wahl S. Tian H. Ben-Josef E. Taylor J.D. Porter A.T. Tang D.G. Cancer Res. 1999; 59: 4343-4355PubMed Google Scholar).DEVDase and LEHDase ActivityCells were washed twice in PBS, and the whole cell lysates were made in the lysis buffer (50 mm HEPES, 1% Triton X-100, 0.1% CHAPS, 1 mmdithiothreitol, and 0.1 mm EDTA). Forty μg of protein was added to a reaction mixture containing 30 μm fluorogenic peptide substrates, DEVD-AFC or LEHD-AFC, 50 mm of HEPES, pH 7.4, 10% glycerol, 0.1% CHAPS, 100 mm NaCl, 1 mm EDTA, and 10 mm dithiothreitol, in a total volume of 1 ml and incubated at 37 °C for 1 h. Production of 7-amino-4-trifluoromethylcoumarin (AFC) was monitored in a spectrofluorimeter (Hitachi F-2000 fluorescence spectrophotometer) using excitation wavelength 400 nm and emission wavelength 505 nm. The fluorescent units were converted into nanomoles of AFC released per h per mg of protein using a standard curve. The results were generally presented as fold activation over the control.DNA FragmentationFragmented DNA was extracted using SDS/RNase/proteinase K methods (37Joshi B., Li, L. Taffe B.G. Zhu Z. Wahl S. Tian H. Ben-Josef E. Taylor J.D. Porter A.T. Tang D.G. Cancer Res. 1999; 59: 4343-4355PubMed Google Scholar, 47Tang D.G., Li, L. Zhu Z. Joshi B. Biochem. Biophys. Res. Commun. 1998; 242: 380-384Crossref PubMed Scopus (116) Google Scholar), and 20 μg of DNA was run on 1.2% agarose gel.Quantification of Apoptotic Nuclei Using DAPI StainingCells were plated on glass coverslips (4 × 104 cells/18-mm2 coverslip) and the next day treated with vehicle control (i.e. ethanol or Me2SO) or various inducers. Thereafter, cells were incubated live with DAPI (0.5 μg/ml) for 10 min at 37 °C followed by washing. The percentage of cells exhibiting apoptotic nuclei, as judged by chromatin condensation or nuclear fragmentation, was assessed by fluorescence microscopy (49Tang D.G., Li, L. Chopra D.P. Porter A.T. Cancer Res. 1998; 58: 3466-3479PubMed Google Scholar). An average of 600–700 cells was counted for each condition.Immunofluorescence Analysis of Cytochrome c Distribution, Mitochondrial Membrane Potential, and ApoptosisCells grown on glass coverslips were treated for various time intervals. Fifteen min prior to the end of the treatment, cells were incubated live with MitoTracker Orange CMTMRos to label mitochondria (37Joshi B., Li, L. Taffe B.G. Zhu Z. Wahl S. Tian H. Ben-Josef E. Taylor J.D. Porter A.T. Tang D.G. Cancer Res. 1999; 59: 4343-4355PubMed Google Scholar). Then cells were fixed in 4% paraformaldehyde for 10 min followed by permeabilization in 1% Triton X-100 for 10 min. After washing in PBS, cells were first blocked in 20% goat whole serum for 30 min at 37 °C and then incubated with monoclonal anti-cytochromec antibody (clone 6H2.B4, 1:500) for 1 h at 37 °C. Finally, cells were incubated with fluorescein isothiocyanate-conjugated goat anti-mouse IgG (1:1000) for 1 h at 37 °C. After thorough washing, coverslips were mounted on slides using Vectashield mounting medium (Vector Laboratories, Inc., Burlingame, CA) and observed under an Olympus BX40 epifluorescence microscope. Images were captured with MagnaFire software and processed in Adobe Photoshop. In a separate set of samples, following apoptotic treatments, cells were washed once with 1× PBS and then incubated in the Annexin-Binding Buffer containing annexin V-AlexaFluor conjugates for 30 min followed by washing. Images were captured on an Olympus IX50 inverted fluorescence microscope.RT-PCR Analysis of Cytochrome c and COX II mRNA ExpressionTotal RNA was isolated using Tri-Reagent (Invitrogen). RT was performed using 2 μg of total RNA (at 42 °C for 2 h) in a total 20-μl reaction volume containing random hexamers and Superscript II reverse transcriptase (Invitrogen). PCR primers, designed based on the published cytochrome c and COX II cDNA sequences, are 5′-TTTGGATCCAATGGGTGATGTTGAG-3′ (cytochromec, sense), 5′-TTTGAATTCCTCATTAGTAGCTTTTTTGAG-3′ (cytochromec, antisense), 5′-CCATCCCTACGCATCCTTTAC-3′; (COX II, sense), and 5′-GTTTGCTCCACAGATTTCAGAG-3′ (COX II, antisense). For PCR, 1 μl of cDNA was used in a 25-μl reaction containing 0.5 μm primers, dNTPs and Taq, using the cycling profile 94 °C × 45 s, 56 °C × 1 min, and 72 °C × 1 min for 14 cycles for COX II and 23 cycles for cytochrome c with a final extension at 72 °C for 10 min. PCR products were analyzed on 1% agarose gel. RT-PCR of glyceraldehyde 3-phosphate dehydrogenase (50Tang D.G. Tokumoto Y.M. Raff M.C. J. Cell Biol. 2000; 148: 971-984Crossref PubMed Scopus (111) Google Scholar) was used as a control.Transient Transfection with pEGFP-Cytochrome cThe pEGFP-cytochrome c expression plasmid, in which the full-length rat cytochrome c cDNA was cloned into the NheI and XhoI sites of the pEGFP-N1 (Clontech), was kindly provided by Dr. A.-L. Nieminen (Case Western Reserve University; see Ref. 51Heiskanen K.M. Bhat M.B. Wang H.W., Ma, J. Nieminen A.-L. J. Biol. Chem. 1999; 274: 5654-5658Abstract Full Text Full Text PDF PubMed Scopus (305) Google Scholar). GM701 cells, plated 1 day earlier on 10-cm culture dishes to achieve 50–60% confluence, were transfected, using FuGENE 6, with 15 μg of either empty vector alone or pEGFP-cytochrome c. Twenty four h after transfection, cells were treated with BMD188 for various times and then harvested and used for subcellular fractionations as described above. To assess apoptosis, GM701 cells grown on coverslips were transfected with 1 μg of plasmids. Twenty four h later, cells were treated with BMD188 followed by DAPI staining as described above. The percentage of GFP-positive and -negative cells with apoptotic nuclear morphology was determined by fluorescence microscopy (49Tang D.G., Li, L. Chopra D.P. Porter A.T. Cancer Res. 1998; 58: 3466-3479PubMed Google Scholar). At least 600–700 cells were counted for each condition.Up-regulation of Cytochrome c Using the Ecdysone-inducible SystemPlasmids pVgRXR and pIND were obtained from Invitrogen. pIND/cyt-c-GFP was prepared by cloning theNheI/NotI fragment of the rat cytochromec-GFP fusion cDNA from pEGFP-cytochrome c(51Heiskanen K.M. Bhat M.B. Wang H.W., Ma, J. Nieminen A.-L. J. Biol. Chem. 1999; 274: 5654-5658Abstract Full Text Full Text PDF PubMed Scopus (305) Go
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