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• W2022114707 abstract "NOXA is a BH3-only protein whose expression is induced by certain p53-depenent or independent apoptotic stimuli. Both NOXA and Bim are avid binders of Mcl-1, but a functional linkage between these BH3-only proteins has not yet been reported. In this study, we demonstrate that Mcl-1 binding of endogenously induced NOXA interferes with the ability of Mcl-1 to efficiently sequester endogenous Bim, as Bim is displaced from its complex with Mcl-1. Induced NOXA significantly enhances the UV sensitivity of cells, and the ensuing mitochondrial depolarization is entirely abrogated by Bim knockdown. These results demonstrate a Mcl-1-mediated cross-talk between endogenous NOXA and Bim that occurs upstream of the Bak/Bax-dependent execution of UV-induced mitochondrial depolarization. The current findings demonstrate that the mitochondrial response to an induced expression of NOXA is executed by endogenous Bim and suggest a plausible mechanism for the observed NOXA-Bim linkage. NOXA is a BH3-only protein whose expression is induced by certain p53-depenent or independent apoptotic stimuli. Both NOXA and Bim are avid binders of Mcl-1, but a functional linkage between these BH3-only proteins has not yet been reported. In this study, we demonstrate that Mcl-1 binding of endogenously induced NOXA interferes with the ability of Mcl-1 to efficiently sequester endogenous Bim, as Bim is displaced from its complex with Mcl-1. Induced NOXA significantly enhances the UV sensitivity of cells, and the ensuing mitochondrial depolarization is entirely abrogated by Bim knockdown. These results demonstrate a Mcl-1-mediated cross-talk between endogenous NOXA and Bim that occurs upstream of the Bak/Bax-dependent execution of UV-induced mitochondrial depolarization. The current findings demonstrate that the mitochondrial response to an induced expression of NOXA is executed by endogenous Bim and suggest a plausible mechanism for the observed NOXA-Bim linkage. Mitochondrial involvement in programmed cell death is mediated by mitochondrial alterations that lead to the release of caspase activators and caspase-independent death effectors, which are cumulatively responsible for eventual cell death. The permeability of the mitochondrial outer membrane is regulated by pro- and anti-apoptotic Bcl-2 family members (1.Danial N.N. Korsmeyer S.J. Cell. 2004; 116: 205-219Abstract Full Text Full Text PDF PubMed Scopus (3991) Google Scholar, 2.Lucken-Ardjomande S. Martinou J.C. Crit. Rev. Biol. 2005; 328: 616-631Google Scholar, 3.Donovan M. Cotter T.G. Biochim. Biophys. Acta. 2004; 1644: 133-147Crossref PubMed Scopus (198) Google Scholar, 4.Harris M.H. Thompson C.B. Cell Death Differ. 2000; 7: 1182-1191Crossref PubMed Scopus (432) Google Scholar). Bcl-2 proteins can be divided into three sub-classes on the basis of their function and the number of Bcl-2 homology (BH) 3The abbreviations used are: BH, Bcl-2 homology domain; Ab, antibody; mAb, monoclonal antibody; GSI, γ-secretase inhibitor; MOPS, 4-morpholinepropanesulfonic acid; nt, nucleotide(s); RNAi, RNA interference; siRNA, small interference RNA; JC-1, 1,1′3,3′-tetraethylbenzamidazolocarbocyanin iodide; Tet, tetracycline.3The abbreviations used are: BH, Bcl-2 homology domain; Ab, antibody; mAb, monoclonal antibody; GSI, γ-secretase inhibitor; MOPS, 4-morpholinepropanesulfonic acid; nt, nucleotide(s); RNAi, RNA interference; siRNA, small interference RNA; JC-1, 1,1′3,3′-tetraethylbenzamidazolocarbocyanin iodide; Tet, tetracycline. domains included in their structure. The anti-apoptotic members, including Bcl-2, Bcl-XL, Bcl-W, A1, and Mcl-1 contain three or four BH domains; the pro-apoptotic members, including Bax and Bak, possess three BH domains; and the “BH3-only” pro-apoptotic members, such as Bid, Bim, NOXA, and Puma, share homology only within the BH3 domain (5.Cory S. Huang D.C. Adams J.M. Oncogene. 2003; 22: 8590-8607Crossref PubMed Scopus (1292) Google Scholar). Structural studies have suggested that the hydrophobic face of the α-helix formed by the BH3 domain of BH3-only proteins inserts into the hydrophobic groove formed by the BH1, BH2, and BH3 domains of the anti-apoptotic Bcl-2 members (6.Petros A.M. Olejniczak E.T. Fesik S.W. Biochim. Biophys. Acta. 2004; 1644: 83-94Crossref PubMed Scopus (595) Google Scholar). In response, Bax and Bak form homo-oligomers that are capable of permeabilizing the mitochondrial outer membrane for an egress of mitochondrial apoptotic proteins. Bid and Bim appear to function upstream of Bax and Bak; ectopic expression of these proteins in cells lacking both Bax and Bak could not induce the mitochondrial release of cytochrome c (7.Wei M.C. Zong W.X. Cheng E.H. Lindsten T. Panoutsakopoulou V. Ross A.J. Roth K.A. MacGregor G.R. Thompson C.B. Korsmeyer S.J. Science. 2001; 292: 727-730Crossref PubMed Scopus (3317) Google Scholar, 8.Zong W.X. Lindsten T. Ross A.J. MacGregor G.R. Thompson C.B. Genes Dev. 2001; 15: 1481-1486Crossref PubMed Scopus (709) Google Scholar). The function of Bcl-2 proteins is also closely tied (in a yet unresolved manner) to the loss in mitochondrial membrane potential that represents an early phase in the apoptotic process (9.Green D.R. Kroemer G. Science. 2004; 305: 626-629Crossref PubMed Scopus (2775) Google Scholar, 10.Halestrap A.P. McStay G.P. Clarke S.J. Biochimie (Paris). 2002; 84: 153-166Crossref PubMed Scopus (619) Google Scholar). The electrochemical gradient across the inner mitochondrial membrane is critical for normal mitochondrial function and therefore its loss is ultimately associated with cell death.The BH3-only proteins NOXA and Puma have been identified as transcription targets of p53 that are involved in DNA damage-induced apoptosis (11.Villunger A. Michalak E.M. Coultas L. Mullauer F. Bock G. Ausserlechner M.J. Adams J.M. Strasser A. Science. 2003; 302: 1036-1038Crossref PubMed Scopus (1082) Google Scholar). NOXA was originally identified as an adult T-cell leukemia-derived phorbol 12-myristate 13-acetate-responsive gene (12.Hijikata M. Kato N. Sato T. Kagami Y. Shimotohno K. J. Virol. 1990; 64: 4632-4639Crossref PubMed Google Scholar) and more recently as a p53-induced gene in x-ray-irradiated murine embryo fibroblasts (13.Oda E. Ohki R. Murasawa H. Nemoto J. Shibue T. Yamashita T. Tokino T. Taniguchi T. Tanaka N. Science. 2000; 288: 1053-1058Crossref PubMed Scopus (1690) Google Scholar). Expression of NOXA is induced by phorbol ester, p53, UV radiation, and other DNA-damaging agents, such as etoposide and doxorubicin (13.Oda E. Ohki R. Murasawa H. Nemoto J. Shibue T. Yamashita T. Tokino T. Taniguchi T. Tanaka N. Science. 2000; 288: 1053-1058Crossref PubMed Scopus (1690) Google Scholar, 14.Schuler M. Maurer U. Goldstein J.C. Breitenbucher F. Hoffarth S. Waterhouse N.J. Green D.R. Cell Death Differ. 2003; 10: 451-460Crossref PubMed Scopus (95) Google Scholar, 15.Shibue T. Takeda K. Oda E. Tanaka H. Murasawa H. Takaoka A. Morishita Y. Akira S. Taniguchi T. Tanaka N. Genes Dev. 2003; 17: 2233-2238Crossref PubMed Scopus (272) Google Scholar, 16.Sesto A. Navarro M. Burslem F. Jorcano J.L. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 2965-2970Crossref PubMed Scopus (161) Google Scholar). Recently, it was reported that proteasome inhibitors induce NOXA in a p53-independent manner (17.Fernandez Y. Verhaegen M. Miller T.P. Rush J.L. Steiner P. Opipari Jr., A.W. Lowe S.W. Soengas M.S. Cancer Res. 2005; 65: 6294-6304Crossref PubMed Scopus (191) Google Scholar, 18.Qin J.Z. Stennett L. Bacon P. Bodner B. Hendrix M.J. Seftor R.E. Seftor E.A. Margaryan N.V. Pollock P.M. Curtis A. Trent J.M. Bennett F. Miele L. Nickoloff B.J. Mol. Cancer Ther. 2004; 3: 895-902PubMed Google Scholar, 19.Qin J.Z. Ziffra J. Stennett L. Bodner B. Bonish B.K. Chaturvedi V. Bennett F. Pollock P.M. Trent J.M. Hendrix M.J. Rizzo P. Miele L. Nickoloff B.J. Cancer Res. 2005; 65: 6282-6293Crossref PubMed Scopus (261) Google Scholar). The human NOXA gene is located on chromosome 18q21 and encodes a 54-amino acid protein with a single BH3 motif, which is in contrast to the murine NOXA protein that possesses two BH3 domains (13.Oda E. Ohki R. Murasawa H. Nemoto J. Shibue T. Yamashita T. Tokino T. Taniguchi T. Tanaka N. Science. 2000; 288: 1053-1058Crossref PubMed Scopus (1690) Google Scholar). Both the human and the mouse proteins contain a mitochondrial targeting domain. Following its translocation to the mitochondria, NOXA binds preferentially to Mcl-1, and not to Bcl-2 or Bcl-XL (20.Willis S.N. Chen L. Dewson G. Wei A. Naik E. Fletcher J.I. Adams J.M. Huang D.C. Genes Dev. 2005; 19: 1294-1305Crossref PubMed Scopus (1042) Google Scholar).Our previous studies have documented the sequestration of Bim by Mcl-1 under non-apoptotic conditions (21.Han J. Goldstein L.A. Gastman B.R. Froelich C.J. Yin X.M. Rabinowich H. J. Biol. Chem. 2004; 279: 22020-22029Abstract Full Text Full Text PDF PubMed Scopus (91) Google Scholar, 22.Han J. Goldstein L.A. Gastman B.R. Rabinovitz A. Rabinowich H. J. Biol. Chem. 2005; 280: 16383-16392Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar, 23.Han J. Goldstein L.A. Gastman B.R. Rabinowich H. J. Biol. Chem. 2006; 281: 10153-10163Abstract Full Text Full Text PDF PubMed Scopus (161) Google Scholar). As Mcl-1 is also a preferred binding target of NOXA, we investigated the relationships between NOXA and Bim in Mcl-1 binding and in mediating mitochondrial apoptotic susceptibility. The current study demonstrates a functional linkage between NOXA and Bim, which is suggested by the apoptotic involvement of Bim downstream of NOXA induction.EXPERIMENTAL PROCEDURESReagents−Anti-human Mcl-1 Abs were from Oncogene (Boston, MA; mouse clone RC13 generated against recombinant Mcl-1), and from Santa Cruz Biotechnology (Santa Cruz, CA; a polyclonal Ab generated against a 20-amino acid residue synthetic peptide of human Mcl-1). Anti-β-actin mAb (clone AC-15) was purchased from Sigma; anti-Cox IV mAb was from Molecular Probes (Eugene, OR); rabbit anti-Bim Ab was from ProSci (Poway, CA); Bim-specific rat mAb was from Apoptech (San Diego, CA; Clone 14A8); anti-NOXA Ab was from Imgenex (San Diego, CA). Abs to AIF, Bcl-2, Bcl-XL, p53 (clone DO-1), and β-tubulin were from Santa Cruz Biotechnology; anti-Xpress mouse mAb for lacZ was from Invitrogen; [35S]methionine, Protein A-Sepharose beads, and Protein G-Sepharose beads were from Amersham Biosciences. The γ-secretase inhibitor (GSI) consisting of a tripeptide aldehyde, N-benzyloxycarbonyl-Leu-Leu-norleucine-CHO was from Calbiochem.Cell Lines, Cell Lysates, and Cell Extracts−Jurkat T leukemic cells were grown in RPMI 1640 medium containing 10% fetal calf serum, 20 mm HEPES, 2 mml-glutamine, and 100 units/ml each of penicillin and streptomycin. HeLa, breast carcinoma CAMA-1, colon cancer Hct116, and T-REx-293 cells were grown in Dulbecco’s modified Eagle’s medium containing 15% fetal calf serum, 20 mml-glutamine, and 100 units/ml each of penicillin and streptomycin. Transfected T-REx-293 clonal cell lines were maintained in the presence of blasticidin (5 μg/ml) and Zeocin (20 μg/ml). Cell lysates were prepared with 1% Nonidet P-40, 20 mm Tris-HCl, pH 7.4, 137 mm NaCl, 10% glycerol, 1 mm phenylmethylsulfonyl fluoride, 10 μg/ml leupeptin, and 10 μg/ml aprotinin. To prepare cell extracts, cultured 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. 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.Cellular Fractionation and Mitochondria Purification−To obtain an enriched mitochondrial fraction, Hct116 or T-REx-293 cells were suspended in mitochondrial buffer composed of 0.3 m sucrose, 10 mm MOPS, 1 mm EDTA, and 4 mm KH2PO4 (pH 7.4) and lysed by Dounce homogenization as previously described (21.Han J. Goldstein L.A. Gastman B.R. Froelich C.J. Yin X.M. Rabinowich H. J. Biol. Chem. 2004; 279: 22020-22029Abstract Full Text Full Text PDF PubMed Scopus (91) Google Scholar). Briefly, nuclei and debris were removed by a 10-min centrifugation at 650 × g, and a pellet containing mitochondria was obtained by two successive spins at 10,000 × g for 12 min. To obtain the S-100 fraction, the postnuclear supernatant was further centrifuged at 100,000 × g for 1 h at 4°C. To obtain the enriched mitochondrial fraction, the mitochondria containing pellet was resuspended in mitochondrial buffer and layered on a Percoll gradient consisting of four layers of 10%, 18%, 30%, and 70% Percoll in mitochondrial buffer. After centrifugation for 30 min at 15,000 × g, the mitochondrial fraction was collected at the 30/70 interface. Mitochondria were diluted in mitochondrial buffer containing 1 mg/ml bovine serum albumin (at least a 10-fold dilution required to remove Percoll). The mitochondrial pellet was obtained by a 40-min spin at 20,000 × g and used immediately. Purity was assessed by electron microscopy and by enzyme marker analysis (23.Han J. Goldstein L.A. Gastman B.R. Rabinowich H. J. Biol. Chem. 2006; 281: 10153-10163Abstract Full Text Full Text PDF PubMed Scopus (161) Google Scholar). For enzyme analysis, the following enzymes were assayed: aryl sulfatase (lysosomes/granules); N-acetyl-β-d-glucosaminidase, α-l-fucosidase, and β-glucoronidase (lysosome); lactate dehydrogenase (cytosol); cytochrome oxidase or monoamine oxidase (mitochondria); thiamine pyrophosphatase (Golgi); NADH oxidase (endoplasmic reticulum); and dipeptidylpeptidase IV (plasma membrane). The purity was assessed at 95%, with ∼5% or less contamination from the microsomal fraction.Molecular Cloning of Tet-inducible NOXA Expression Plasmid−Total RNA was isolated from Jurkat T-cells using RNA STAT-60 Reagent (Tel-Test B, Inc.). Reverse transcription was carried out with 5 μg of total RNA using an oligo(dT)12–18 primer and SuperScript II RNase H– reverse transcriptase (Invitrogen). PCR was performed using the Expand Long Template PCR System kit (Roche Applied Science). A NOXA amplicon containing its open reading frame was generated with the following primer pair (forward and reverse): 5′-CGCGGATCCGCGGAGATGCCTGGGAAG-3′, extends 6 nucleotides (nt) into 5′-untranslated region and 5′-ACGCGTCGACCATGAATGCACCTTCACATTC-3′, complementary to nt 437–457 in 3′-untranslated region (extends 119 nt into the 3′-untranslated region). The putative NOXA amplicon was size-selected using a 1.4% agarose gel, and DNA was purified with the QIAquick gel extraction kit (Qiagen). The purified amplicon was digested with the restriction enzymes BamHI and SalI and ligated into the Tet-inducible vector, pcDNA4/TO (Invitrogen) that had been previously digested with BamH1 and XhoI. Following transformation (Escherichia coli TOP 10F′, Invitrogen), plasmids from randomly picked colonies underwent automated DNA sequence analysis (University of Pittsburgh DNA Sequencing Core Facility) to confirm sequence integrity.Stable Transfection of T-REx-293 Cells with Tet-inducible Plasmids pcDNA4/TO, pcDNA4/TO/lacZ, and pcDNA4/TO/NOXA−The cell line T-REx-293 (Invitrogen) comprises human embryonic kidney cells that stably express the Tet repressor. To generate stable clones that harbor the pcDNA4/TO vector control, pcDNA4/TO/lacZ expression control (Invitrogen) and pcDNA4/TO/NOXA, all plasmids were linearized using the restriction enzymes FspI (pcDNA4/TO and pcDNA4/TO/NOXA) and ScaI (pcDNA4/TO/lacZ). Transfection was carried out using 30 μl of GenePorter Transfection Reagent (Gene Therapy Systems Inc., San Diego, CA) following the manufacturer’s directions and 5 μg of linearized plasmid in 6-well plates with 0.8 × 106 cells per well (∼60% confluency) that were maintained in the presence of 5 μg/ml blasticidin. At 48 h post-transfection Zeocin was added to a final concentration of 100–200 μg/ml. After ∼2 weeks individual colonies were selected, expanded, and harvested. Induction of NOXA and lacZ expression by individual clones was confirmed by immunoblotting after the addition of 1 μg/ml tetracycline for 16 h. Confirmation of stable clones containing integrated pcDNA4/TO vector was obtained by reverse transcription-PCR of their total RNA. We utilized a primer pair that delimits the Zeocin resistance gene open reading frame in pcDNA4/TO (forward and reverse): 5′-ACCATGGCCAAGTTGACCAGT-3′, corresponds to nt 2247–2267 and 5′-GAAATCTCGTAGCACGTGTCA-3′, complementary to nt 2622–2642. Multiple such clones were utilized in the studies to confirm the described findings.RNAi Using NOXA, Mcl-1, Bim, Bak, and Bax siRNAs and a Non-targeting siRNA−NOXA, Mcl-1, and Bim siRNAs were obtained as duplexes in purified and desalted form (Option C) from Dharmacon. The three siRNAs had the following sense strand sequences: NOXA, 5′-GUCGAGUGUGCUACUCAACUdTdT-3′; Mcl-1, 5′-GAAACGCGGUAAUCGGACUdTdT-3′; and Bim, 5′-GACCGAGAAGGUAGACAAUUGdTdT-3′. Bak and Bax siRNAs were also obtained from Dharmacon but as siGENOME SMARTpool reagents M-003305-01 and M-003308-00, respectively. The non-targeting siRNA control used in our RNAi experiments is the siCONTROL Non-Targeting siRNA #1 (D-001210-01) from Dharmacon. Hct116 or T-REx-293 cells (2.5 × 105) were plated in a 6-well plate, and following 16 h (at ∼30% confluency) were transfected with 200 nm siRNA in Opti-MEM medium (Invitrogen) without fetal calf serum using Oligofectamine reagent (Invitrogen) according to the manufacturer’s transfection protocol. After 4 h, fetal calf serum was added to a final concentration of 10%. At 40 h, the medium over the cells was adjusted to 1 ml before the addition of an apoptotic agent.UV Irradiation−T-REx-293 cells were irradiated with UV light from a germicidal lamp, which emitted radiation primarily in the UVC region, with a peak wavelength of 254 nm. The total dose of irradiation was 60 J/m2 as measured by a Black-Ray UV meter, model J225 (UVP Inc., Upland, CA).In Vitro Transcription-Translation−Mcl-1, NOXA, Bim, and Bak cDNAs were expressed in the TnT T7 transcription-translation reticulocyte lysate system (Promega, Madison, WI). Each coupled transcription-translation reaction contained 1 μg of plasmid DNA in a final volume of 50 μl in a methionine-free reticulocyte lysate reaction mixture supplemented with 35S-labeled methionine according to the manufacturer’s instructions. After incubation at 30 °C for 90 min, the reaction products were immediately used or stored at –78 °C.Western Blot Analysis−Proteins in cell lysates, cell extracts, mitochondria, or S-100 were resolved by SDS-PAGE and transferred to polyvinylidene difluoride membranes, as previously described (24.Johnson D.E. Gastman B.R. Wieckowski E. Wang G.Q. Amoscato A. Delach S.M. Rabinowich H. Cancer Res. 2000; 60: 1818-1823PubMed Google Scholar). Following probing with a specific primary Ab and horseradish peroxidase-conjugated secondary Ab, the protein bands were detected by enhanced chemiluminescence (Pierce).Flow Cytometry−Cytofluorometric analyses of apoptosis were performed by co-staining with propidium iodide and fluorescein isothiocyanate-Annexin V conjugates (BD Biosciences). Mitochondrial membrane potential depolarization was measured by utilizing a fluorescent cationic dye, 1,1′3,3′-tetraethylbenzamidazolocarbocyanin iodide (JC-1, Molecular Probes). The staining was performed according to the manufacturer’s procedures, and loss of mitochondrial membrane potential was quantified by flow cytometric analysis of the decrease in the 590 nm (red)/527 nm (green) fluorescence intensity emission ratio utilizing a Beckman Coulter Epics XL-MCL and analyzed with the EXPO32 software.RESULTSInvolvement of Mcl-1, NOXA, and Bim in GSI-mediated Changes in Mitochondrial Membrane Potential−To investigate the role of NOXA in mitochondrial apoptotic events, we treated several tumor cell lines with GSI, which was recently reported to mediate apoptosis in association with induced expression of NOXA (18.Qin J.Z. Stennett L. Bacon P. Bodner B. Hendrix M.J. Seftor R.E. Seftor E.A. Margaryan N.V. Pollock P.M. Curtis A. Trent J.M. Bennett F. Miele L. Nickoloff B.J. Mol. Cancer Ther. 2004; 3: 895-902PubMed Google Scholar). This drug is a tripeptide aldehyde that was initially identified as an inhibitor of Notch signaling (25.Curry C.L. Reed L.L. Golde T.E. Miele L. Nickoloff B.J. Foreman K.E. Oncogene. 2005; 24: 6333-6344Crossref PubMed Scopus (192) Google Scholar, 26.Kang J.H. Lee da H. Lee J.S. Kim H.J. Shin J.W. Lee Y.H. Lee Y.S. Park C.S. Chung I.Y. Eur. J. Immunol. 2005; 35: 2982-2990Crossref PubMed Scopus (22) Google Scholar) and was recently shown to induce apoptosis in Kaposi’s sarcoma and melanoma cell lines (18.Qin J.Z. Stennett L. Bacon P. Bodner B. Hendrix M.J. Seftor R.E. Seftor E.A. Margaryan N.V. Pollock P.M. Curtis A. Trent J.M. Bennett F. Miele L. Nickoloff B.J. Mol. Cancer Ther. 2004; 3: 895-902PubMed Google Scholar, 25.Curry C.L. Reed L.L. Golde T.E. Miele L. Nickoloff B.J. Foreman K.E. Oncogene. 2005; 24: 6333-6344Crossref PubMed Scopus (192) Google Scholar). In our studies, this aldehyde inhibitor induced a significant level of apoptosis in most of the treated tumor cell lines (Fig. 1, A and B). GSI-induced apoptosis was associated with NOXA induction in four of five treated cell lines, whereas only a low level of apoptosis was detected in CAMA-1 cells, where NOXA was not induced. Induction of p53 accompanied that of NOXA in WT Hct116, but NOXA up-regulation can also be independent of p53, as was determined in a p53 knock-out-Hct116 clonal cell line (p53–/–) and in Jurkat cells (p53-null). Moreover, despite p53 expression, there was no induction of NOXA by GSI in CAMA-1 cells. GSI enhanced the Mcl-1 expression levels in both Hct116 clonal cell lines (WT and p53–/–), in CAMA-1 and in HeLa cells, whereas in Jurkat cells it mediated Mcl-1 degradation. Bim was up-regulated following treatment with GSI in Hct116 clonal cell lines, unchanged in CAMA-1 cells, and down-regulated in Jurkat and HeLa cells. These observations illustrate the occurrence of multiple changes in the expression levels of mitochondrial apoptosis regulatory proteins in response to a single drug and suggest that these differential alterations are cell-type specific.Because GSI induced a significant apoptotic level in Hct116 cells in association with up-regulated expression of the mitochondrial Bcl-2 members, Mcl-1, NOXA, and Bim, we investigated the roles of these proteins in the regulation of mitochondrial membrane potential in these cells. To this end, RNAi was carried out on WT Hct116 cells with siRNA specific for Mcl-1, NOXA, and Bim or with a non-targeting siRNA control. During the last 16 h of the siRNA transfection, the cells were treated with GSI and assessed by immunoblotting for the expression levels of the targeted proteins and by JC-1 flow cytometry for changes in the mitochondrial membrane potential (Fig. 1, C and D). JC-1 is a lipophilic fluorochrome that, upon mitochondrial binding, emits a green fluorescent signal, which is further processed to an additional red fluorescent signal only in mitochondria with preserved membrane potential. Thus, mitochondria with intact membrane potential emit high green and high red fluorescence, whereas loss of mitochondrial membrane potential results in reduced emission of red fluorescence while maintaining the high green fluorescence. Mcl-1 siRNA increased the loss in red fluorescence in response to GSI, whereas knockdown of either NOXA or Bim significantly inhibited the GSI-mediated loss in mitochondrial membrane potential. These observations suggest that the changes in mitochondrial membrane potential in response to GSI are jointly regulated by Mcl-1, NOXA, and Bim. However, because of the multiple concurrent changes in the expression of these mitochondrial apoptosis regulators, the drug-induced cell is not an optimal system for analyzing the functional relationship between NOXA and Bim.Elucidation of NOXA Apoptotic Implications in Tet-induced NOXA-transfected Cells−To elucidate a potential role for NOXA in mitochondrial apoptotic events, we utilized a tetracycline-regulated expression (T-REx) system consisting of the T-REx-293 (human embryonic kidney epithelial cells stably transfected with the Tet repressor) clonal cell line stably transfected with NOXA or the empty inducible vector whose expression is switched on by the presence of tetracycline. Similar to GSI-induced NOXA, Tet-induced NOXA accumulated exclusively in the mitochondria (Fig. 2, A and B). In contrast to GSI-induced NOXA, Tet-induced NOXA was not associated with increased expression of Bim, p53, or Mcl-1 (Figs. 2B and 3C). Furthermore, induction of NOXA alone did not result in cell death or a release of mitochondrial apoptogenic proteins.FIGURE 2Apoptotic implications of mitochondrial translocation of GSI- or Tet-induced NOXA. A, mitochondrial translocation of GSI-induced NOXA is associated with cytochrome c release. GSI-treated Jurkat cells (5 μm, 16 h) were disrupted by Dounce homogenization followed by a purification process for mitochondria and S-100 fractions. The extract, mitochondrial (Mit) and S-100 fractions of either control or GSI-treated cells were assessed for the expression of the indicated proteins. Expression of β-actin (cytosolic protein) and Cox IV (mitochondrial protein) serve as markers for equal loading and purity of the cellular fractions. B, mitochondrial translocation of Tet-induced NOXA is not associated with release of apoptogenic proteins or changes in expression of Bim or p53. T-REx-293 cells stably transfected with either Tet-inducible NOXA or its vector control were treated with tetracycline (1μg/ml for 16 h). The cells were then disrupted by Dounce homogenization and subcellular fractionated as described in A. The same membrane was stripped and reprobed successively for the indicated proteins.View Large Image Figure ViewerDownload Hi-res image Download (PPT)FIGURE 3Co-immunoprecipitation of NOXA with Mcl-1. A, GSI-induced NOXA co-immunoprecipitates with endogenous Mcl-1. Hct116 cells were treated with GSI (5 μm, 16 h). The lysates of control and GSI-treated cells were subjected to immunoprecipitation with Mcl-1-specific mouse mAb. The original lysates, depleted supernatants, and immunoprecipitated pellets were assessed by immunoblotting for the expression of Mcl-1 (using rabbit anti-Mcl-1 Ab, top). The membrane was stripped and reprobed with NOXA-specific mouse mAb (bottom). B, in vitro translated NOXA co-immunoprecipitates with in vitro translated Mcl-1. The 35S-labeled in vitro translated Mcl-1 was mixed with unlabeled in vitro translated NOXA and subjected to immunoprecipitation with rabbit anti-Mcl-1 Ab. The original mixture, depleted supernatant, and immunoprecipitated pellet were assessed by immunoblotting for the presence of Mcl-1 (top) and NOXA (middle), and by autoradiography for the radioactive signal generated by 35S-labeled Mcl-1 (bottom). C, Tet-induced NOXA co-immunoprecipitates with endogenous Mcl-1. T-REx-293 cells stably transfected with Tet-inducible control or with Tet-inducible NOXA were treated with tetracycline (1 μg/ml, 16 h). The cells were then lysed, and subjected to immunoprecipitation by rabbit anti-Mcl-1 Ab. The original lysates, depleted supernatants, and immunoprecipitated pellets were assessed by immunoblotting for expression of Mcl-1 and NOXA. β-Actin serves as an equal loading control. All immunoprecipitation procedures were performed after pre-clearance with protein A/G-Sepharose beads and nonspecific rabbit or mouse Ig. The ratio of cell lysate (Input) gel loading to that of the immunoprecipitation (IP) pellet was 1:4.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Recently, NOXA was reported to bind exclusively to Mcl-1, and not to Bcl-2 or Bcl-XL (20.Willis S.N. Chen L. Dewson G. Wei A. Naik E. Fletcher J.I. Adams J.M. Huang D.C. Genes Dev. 2005; 19: 1294-1305Crossref PubMed Scopus (1042) Google Scholar, 27.Chen L. Willis S.N. Wei A. Smith B.J. Fletcher J.I. Hinds M.G. Colman P.M. Day C.L. Adams J.M. Huang D.C. Mol. Cell. 2005; 17: 393-403Abstract Full Text Full Text PDF PubMed Scopus (1506) Google Scholar). To investigate the ability of drug-induced NOXA and Tet-induced NOXA to bind Mcl-1, we immunoprecipitated Mcl-1 in three experimental systems, including GSI-treated Hct116 cells (Fig. 3A), a mixture of in vitro translated Mcl-1 and NOXA proteins (Fig. 3B), and T-REx-293 cells expressing Tet-induced NOXA or Tet-vector control (Fig. 3C). In each of these experimental systems, Mcl-1 was efficiently immunoprecipitated, because it was completely depleted from the remaining supernatants (Fig. 3, A (lane 7 top), B (lane 4 top), and C (lanes 4–6 top)). GSI-induced NOXA was entirely co-immunoprecipitated with Mcl-1 (Fig. 3A, lanes 6–8 bottom), whereas Tet-induced NOXA was partly, although significantly, co-immunoprecipitated with Mcl-1 (Fig. 3C, lanes 3, 6, and 9). It is possible that the expression level of Tet-induced NOXA is excessive relative to that of endogenous Mcl-1, and thus a significant proportion of the induced NOXA remains Mcl-1-free.Previous studies performed by others (28.Opferman J.T. Iwasaki H. Ong C.C. Suh H. Mizuno S. Akashi K. Korsmeyer S.J. Science. 2005; 307: 1101-1104Crossref PubMed Scopus (468) Google Scholar, 29.Opferman J.T. Letai A. Beard C. Sorcinelli M.D. Ong C.C. Korsmeyer" @default.
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