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Rapid Turnover of Mcl-1 Couples Translation to Cell Survival and Apoptosis

2007; Elsevier BV; Volume: 282; Issue: 9 Linguagem: Inglês

10.1074/jbc.m610643200

ISSN

1083-351X

Autores

Kenneth W. Adams, Geoffrey M. Cooper,

Tópico(s)

Autophagy in Disease and Therapy

Resumo

Inhibition of translation plays a role in apoptosis induced by a variety of stimuli, but the mechanism by which it promotes apoptosis has not been established. We have investigated the hypothesis that selective degradation of anti-apoptotic regulatory protein(s) is responsible for apoptosis resulting from translation inhibition. Induction of apoptosis by cycloheximide was detected within 2–4 h and blocked by proteasome inhibitors, indicating that degradation of short-lived protein(s) was required. Caspase inhibition and overexpression of Bcl-xL blocked cycloheximide-induced apoptosis. In addition, cycloheximide induced rapid activation of Bak and Bax, which required proteasome activity. Mcl-1 was degraded by the proteasome with a half-life of ∼30 min following inhibition of protein synthesis, preceding Bak/Bax activation and the onset of apoptosis. Overexpression of Mcl-1 blocked apoptosis induced by cycloheximide, whereas RNA interference knockdown of Mcl-1 induced apoptosis. Knockdown of Bim and Bak, downstream targets of Mcl-1, inhibited cycloheximide-induced apoptosis, as did knockdown of Bax. Apoptosis resulting from inhibition of translation thus involves the rapid degradation of Mcl-1, leading to activation of Bim, Bak, and Bax. Because of its rapid turnover, Mcl-1 may serve as a convergence point for signals that affect global translation, coupling translation to cell survival and the apoptotic machinery. Inhibition of translation plays a role in apoptosis induced by a variety of stimuli, but the mechanism by which it promotes apoptosis has not been established. We have investigated the hypothesis that selective degradation of anti-apoptotic regulatory protein(s) is responsible for apoptosis resulting from translation inhibition. Induction of apoptosis by cycloheximide was detected within 2–4 h and blocked by proteasome inhibitors, indicating that degradation of short-lived protein(s) was required. Caspase inhibition and overexpression of Bcl-xL blocked cycloheximide-induced apoptosis. In addition, cycloheximide induced rapid activation of Bak and Bax, which required proteasome activity. Mcl-1 was degraded by the proteasome with a half-life of ∼30 min following inhibition of protein synthesis, preceding Bak/Bax activation and the onset of apoptosis. Overexpression of Mcl-1 blocked apoptosis induced by cycloheximide, whereas RNA interference knockdown of Mcl-1 induced apoptosis. Knockdown of Bim and Bak, downstream targets of Mcl-1, inhibited cycloheximide-induced apoptosis, as did knockdown of Bax. Apoptosis resulting from inhibition of translation thus involves the rapid degradation of Mcl-1, leading to activation of Bim, Bak, and Bax. Because of its rapid turnover, Mcl-1 may serve as a convergence point for signals that affect global translation, coupling translation to cell survival and the apoptotic machinery. Most signals that control survival of mammalian cells modulate the activity of Bcl-2 family members, which regulate the mitochondrial pathway of apoptosis (1Danial N.N. Korsmeyer S.J. Cell. 2004; 116: 205-219Abstract Full Text Full Text PDF PubMed Scopus (3991) Google Scholar, 2Willis S.N. Adams J.M. Curr. Opin. Cell Biol. 2005; 17: 617-625Crossref PubMed Scopus (638) Google Scholar). Anti-apoptotic members of the Bcl-2 family, including Bcl-2, Bcl-xL, and Mcl-1, maintain cell survival by inhibiting the pro-apoptotic Bcl-2 proteins Bak and Bax through protein-protein interactions. Bak and Bax are typically activated by a second set of pro-apoptotic Bcl-2 proteins called BH3-only proteins that associate with anti-apoptotic Bcl-2 proteins through interactions that displace and activate Bak and Bax. Once activated, Bak and Bax permeabilize the mitochondrial outer membrane, resulting in the release of cytochrome c and other pro-apoptotic factors that induce caspase activation and cell death.Signaling pathways that regulate apoptosis can directly modify Bcl-2 family proteins, as well as alter the expression of Bcl-2 family members at both the transcriptional and translational levels. Many signaling pathways that regulate apoptosis target specific BH3-only proteins. For example, p53-mediated apoptosis involves transcriptional induction of the BH3-only proteins PUMA (3Nakano K. Vousden K.H. Mol. Cell. 2001; 7: 683-694Abstract Full Text Full Text PDF PubMed Scopus (1854) Google Scholar, 4Yu J. Zhang L. Hwang P.M. Kinzler K.W. Vogelstein B. Mol. Cell. 2001; 7: 673-682Abstract Full Text Full Text PDF PubMed Scopus (1080) Google Scholar) and Noxa (5Oda 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), whereas PI 3The abbreviations used are: PI, phosphatidylinositol; 4E-BP1, eIF4E-binding protein; PBS, phosphate-buffered saline; eIF, eukaryotic initiation factor; TUNEL, deoxynucleotidyltransferase-mediated dUTP nick end labeling; siRNA, small interfering RNA; fmk, fluoromethyl ketone; NS, nonspecific; PARP, poly(ADP-ribose) polymerase; mTOR, mammalian target of rapamycin.3The abbreviations used are: PI, phosphatidylinositol; 4E-BP1, eIF4E-binding protein; PBS, phosphate-buffered saline; eIF, eukaryotic initiation factor; TUNEL, deoxynucleotidyltransferase-mediated dUTP nick end labeling; siRNA, small interfering RNA; fmk, fluoromethyl ketone; NS, nonspecific; PARP, poly(ADP-ribose) polymerase; mTOR, mammalian target of rapamycin. 3-kinase/Akt signaling inhibits apoptosis through transcriptional repression of the BH3-only protein Bim (8Dijkers P.F. Medema R.H. Lammers J.-W.J. Koenderman L. Coffer P.J. Curr. Biol. 2000; 10: 1201-1204Abstract Full Text Full Text PDF PubMed Scopus (828) Google Scholar) and phosphorylation of the BH3-only protein Bad, resulting in its sequestration by 14-3-3 proteins (6Datta S.R. Dudek H. Tao X. Masters S. Fu H. Gotoh Y. Greenberg M.E. Cell. 1997; 91: 231-241Abstract Full Text Full Text PDF PubMed Scopus (4915) Google Scholar, 7del Peso L. González-García M. Page C. Herrera R. Nuñez G. Science. 1997; 278: 687-689Crossref PubMed Scopus (1978) Google Scholar).In addition to regulating Bcl-2 family proteins, many of the signaling pathways that control apoptosis affect global translational activity, generally by regulation of the initiation factors eIF2, eIF2B, and eIF4E (9Dever T.E. Cell. 2002; 108: 545-556Abstract Full Text Full Text PDF PubMed Scopus (605) Google Scholar, 10Holcik M. Sonenberg N. Nat. Rev. Mol. Cell Biol. 2005; 6: 318-327Crossref PubMed Scopus (1016) Google Scholar, 11Morley S.J. Coldwell M.J. Clemens M.J. Cell Death Differ. 2005; 12: 571-584Crossref PubMed Scopus (80) Google Scholar). A variety of stimuli that induce cell stress inhibit translation via phosphorylation of eIF2, which brings the initiating methionyl-tRNA to the ribosome. Inhibition of eIF2 is mediated by four eIF2α kinases that are activated in response to different stress stimuli: the double-stranded RNA-activated protein kinase PKR, which is activated during viral infection; GCN2, which is activated under conditions of amino acid starvation; PERK, which is activated by accumulation of unfolded proteins in the endoplasmic reticulum; and HRI, which couples globin synthesis to heme availability in reticulocytes. Although inhibition of translation can promote cell survival under conditions of endoplasmic reticulum stress or amino acid starvation, the phosphorylation of eIF2α by PKR plays a pro-apoptotic role in response to viral infection. Activation of PKR plays a central role in the antiviral response, which includes induction of apoptosis in response to interferon and double-stranded RNA (12Barber G.N. Cell Death Differ. 2005; 12: 563-570Crossref PubMed Scopus (69) Google Scholar). The best characterized substrate of PKR is eIF2α, and its phosphorylation leads to inhibition of protein synthesis in virus-infected cells. This inhibition of global translation is critical to induction of apoptosis by PKR, because expression of mutant nonphosphorylatable S51A-eIF2α blocks apoptosis induced by PKR overexpression (13Gil J. Alcamí J. Esteban M. Mol. Cell Biol. 1999; 19: 4653-4663Crossref PubMed Google Scholar) as well as apoptosis induced by several stress stimuli that activate PKR, including double-stranded RNA, interferon, tumor necrosis factor α, serum deprivation, and lipopolysaccharide (14Hsu L.-C. Park J.M. Zhang K. Luo J.-L. Maeda S. Kaufman R.J. Eckmann L. Guiney D.G. Karin M. Nature. 2004; 428: 341-345Crossref PubMed Scopus (317) Google Scholar, 15Scheuner D. Patel R. Wang F. Lee K. Kumar K. Wu J. Nilsson A. Karin M. Kaufman R.J. J. Biol. Chem. 2006; 281: 21458-21468Abstract Full Text Full Text PDF PubMed Scopus (114) Google Scholar, 16Srivastava S.P. Kumar K.U. Kaufman R.J. J. Biol. Chem. 1998; 273: 2416-2423Abstract Full Text Full Text PDF PubMed Scopus (322) Google Scholar).Although activation of PKR induces apoptosis through eIF2 inhibition, growth factor signaling through the PI 3-kinase/Akt pathway promotes cell survival in part by maintaining eIF2 activity through regulation of its guanine nucleotide exchange factor, eIF2B (17Pap M. Cooper G.M. Mol. Cell Biol. 2002; 22: 578-586Crossref PubMed Scopus (147) Google Scholar). One of the targets of PI 3-kinase/Akt signaling involved in regulation of cell survival is the pro-apoptotic protein kinase GSK-3β, which is inhibited by Akt phosphorylation (18Cross D.A.E. Alessi D.R. Cohen P. Andjelkovich M. Hemmings B.A. Nature. 1995; 378: 785-789Crossref PubMed Scopus (4326) Google Scholar, 19Frame S. Cohen P. Biochem. J. 2001; 359: 1-16Crossref PubMed Scopus (1266) Google Scholar, 20Jope R.S. Johnson G.V.W. Trends Biochem. Sci. 2004; 29: 95-102Abstract Full Text Full Text PDF PubMed Scopus (1318) Google Scholar, 21Pap M. Cooper G.M. J. Biol. Chem. 1998; 272: 19929-19932Abstract Full Text Full Text PDF Scopus (954) Google Scholar). The substrates of GSK-3β include eIF2B, which is inhibited as a result of GSK-3β phosphorylation (22Pavitt G.D. Biochem. Soc. Trans. 2005; 33: 1487-1492Crossref PubMed Scopus (115) Google Scholar, 23Wang X. Paulin F.E.M. Campbell L.E. Gomez E. O'Brien K. Morrice N. Proud C.G. EMBO J. 2001; 20: 4349-4359Crossref PubMed Scopus (98) Google Scholar, 24Welsh G.I. Miller C.M. Loughlin A.J. Price N.T. Proud C.G. FEBS Lett. 1998; 421: 125-130Crossref PubMed Scopus (248) Google Scholar). Growth factor deprivation and inhibition of PI 3-kinase leads to activation of GSK-3β, which then phosphorylates and inhibits eIF2B, resulting in inhibition of translation initiation. Expression of nonphosphorylatable eIF2B mutants suppresses apoptosis induced by GSK-3β overexpression, PI 3-kinase inhibition, or growth factor deprivation, indicating that inhibition of eIF2B contributes to apoptosis resulting from inhibition of PI 3-kinase/Akt signaling (17Pap M. Cooper G.M. Mol. Cell Biol. 2002; 22: 578-586Crossref PubMed Scopus (147) Google Scholar).PI 3-kinase/Akt signaling also activates mTOR, which promotes the activity of multiple proteins involved in translation (25Sarbassov D.D. Ali S.M. Sabatini D.M. Curr. Opin. Cell Biol. 2005; 17: 596-603Crossref PubMed Scopus (1298) Google Scholar). mTOR regulates the activity of eIF4E (which binds to the 5′ cap of mRNAs) by phosphorylating eIF4E-binding protein 1 (4E-BP1). In the absence of mTOR signaling, 4E-BP1 binds to eIF4E and inhibits translation initiation. Phosphorylation of 4E-BP1 by mTOR prevents its interaction with eIF4E, coupling PI 3-kinase/Akt and mTOR signaling to translation of capped mRNAs. Overexpression of eIF4E has been shown to inhibit apoptosis induced by several stimuli (26Li S. Takasu T. Perlman D.M. Peterson M.S. Burrichter D. Avdulov S. Bitterman P.B. Polunovsky V.A. J. Biol. Chem. 2003; 278: 3015-3022Abstract Full Text Full Text PDF PubMed Scopus (99) Google Scholar, 27Polunovsky V.A. Rosenwald I.B. Tan A.T. White J. Chiang L. Sonenberg N. Bitterman P.B. Mol. Cell Biol. 1996; 16: 6573-6581Crossref PubMed Scopus (149) Google Scholar, 28Tan A. Bitterman P. Sonenberg N. Peterson M. Polunovsky V. Oncogene. 2000; 19: 1437-1447Crossref PubMed Scopus (89) Google Scholar), whereas overexpression of 4E-BP1 can promote apoptosis (29Li S. Sonenberg N. Gingras A.-C. Peterson M. Avdulov S. Polunovsky V.A. Bitterman P.B. Mol. Cell Biol. 2002; 22: 2853-2861Crossref PubMed Scopus (94) Google Scholar, 30Polunovsky V.A. Gingras A.-C. Sonenberg N. Peterson M. Tan A. Rubins J.B. Manivel J.C. Bitterman P.B. J. Biol. Chem. 2000; 275: 24776-24780Abstract Full Text Full Text PDF PubMed Scopus (93) Google Scholar). Inhibition of mTOR with rapamycin also induces apoptosis of some tumor cells (31Huang S. Houghton P.J. Curr. Opin. Pharmacol. 2003; 3: 371-377Crossref PubMed Scopus (398) Google Scholar), although recent studies suggest that this may result from inhibition of Akt activation rather than from direct effects of mTOR on translation (32Sarbassov D.D. Ali S.M. Sengupta S. Sheen J.-H. Hsu P.P. Bagley A.F. Markhard A.L. Sabatini D.M. Mol. Cell. 2006; 22: 159-168Abstract Full Text Full Text PDF PubMed Scopus (2152) Google Scholar). mTOR also stimulates translation by phosphorylation of p70 S6 kinase (25Sarbassov D.D. Ali S.M. Sabatini D.M. Curr. Opin. Cell Biol. 2005; 17: 596-603Crossref PubMed Scopus (1298) Google Scholar) and regulation of eEF2 kinase (33Browne G.J. Proud C.G. Mol. Cell Biol. 2004; 24: 2986-2997Crossref PubMed Scopus (217) Google Scholar), thereby maintaining activity of the elongation factor eEF2. Although roles for the S6 kinases and eEF2 in apoptosis have not been established, the regulation of these additional factors by mTOR demonstrates the complexity of translation regulation by this pathway.Despite its role in apoptosis induced by several stimuli, the mechanism by which translation inhibition contributes to apoptosis has not been established. One effect of inhibition of translation initiation factors is a selective increase in the translation of some mRNAs containing internal ribosome entry sites or upstream open reading frames (9Dever T.E. Cell. 2002; 108: 545-556Abstract Full Text Full Text PDF PubMed Scopus (605) Google Scholar, 10Holcik M. Sonenberg N. Nat. Rev. Mol. Cell Biol. 2005; 6: 318-327Crossref PubMed Scopus (1016) Google Scholar, 34Spriggs K.A. Bushell M. Mitchell S.A. Willis A.E. Cell Death Differ. 2005; 12: 585-591Crossref PubMed Scopus (134) Google Scholar). Many proteins that are selectively induced by this mechanism during cell stress either inhibit apoptosis, such as caspase inhibitors XIAP (35Holcik M. Lefebvre C. Yeh C. Chow T. Korneluk R.G. Nat. Cell Biol. 1999; 1: 190-192Crossref PubMed Scopus (269) Google Scholar) and HIAP2 (36Warnakulasuriyarachchi D. Cerquozzi S. Cheung H.H. Holcík M. J. Biol. Chem. 2004; 279: 17148-17157Abstract Full Text Full Text PDF PubMed Scopus (119) Google Scholar), or initiate an adaptive program to the cellular stress, such as the transcription factor ATF4 in response to oxidative stress (37Harding H.P. Zhang Y. Zeng H. Novoa I. Lu P.D. Calfon M. Sadri N. Yun C. Popko B. Paules R. Stojdl D.F. Bell J.C. Hettmann T. Leiden J.M. Ron D. Mol. Cell. 2003; 11: 619-633Abstract Full Text Full Text PDF PubMed Scopus (2312) Google Scholar). In addition, some pro-apoptotic proteins have been reported to be induced during translation inhibition, such as Fas and Bax during translation inhibition mediated by PKR (38Balachandran S. Kim C.N. Yeh W.-C. Mak T.W. Bhalla K. Barber G.N. EMBO J. 1998; 17: 6888-6902Crossref PubMed Scopus (306) Google Scholar), suggesting that selective translation of pro-apoptotic proteins may be one mechanism by which global translation inhibition promotes apoptosis.However, apoptosis is also induced by inhibition of protein synthesis with cycloheximide in a variety of cells (17Pap M. Cooper G.M. Mol. Cell Biol. 2002; 22: 578-586Crossref PubMed Scopus (147) Google Scholar, 39Alessenko A.V. Boikov P.Y. Filippova G.N. Khrenov A.V. Loginov A.S. Makarieva E.D. FEBS Lett. 1997; 416: 113-116Crossref PubMed Scopus (43) Google Scholar, 40Blom W.M. de Bont H.J.G.M. Meijerman I. Mulder G.J. Nagelkerke J.F. Biochem. Pharmacol. 1999; 58: 1891-1898Crossref PubMed Scopus (31) Google Scholar, 41Martin S.J. Lennon S.V. Bonham A.M. Cotter T.G. J. Immunol. 1990; 145: 1859-1867PubMed Google Scholar, 42Tang D. Lahti J.M. Grenet J. Kidd V.J. J. Biol. Chem. 1999; 274: 7245-7252Abstract Full Text Full Text PDF PubMed Scopus (120) Google Scholar). Because protein synthesis is completely inhibited by cycloheximide, increased translation of pro-apoptotic proteins cannot be responsible for cell death under these conditions. One possibility is that translation inhibition could induce apoptosis by activating a cell stress pathway. Another possibility is that general turnover of cell constituents might lead to deterioration of the cell in an apoptosis-like manner. Alternatively, apoptosis could result from the selective loss of rapidly degraded anti-apoptotic regulatory protein(s).In the present study, we have focused on induction of apoptosis by cycloheximide to analyze the events responsible for apoptosis resulting from inhibition of protein synthesis. We show that translation inhibition activates the mitochondrial pathway of apoptosis because of the loss of a regulatory protein(s) via proteasome-mediated degradation and identify the anti-apoptotic Bcl-2 family member Mcl-1 as a key regulatory protein that couples cell survival to translational regulation.EXPERIMENTAL PROCEDURESCell Culture—Rat-1 fibroblasts were grown in Dulbecco's modified Eagle's medium supplemented with 10% calf serum. PC12 rat pheochromocytoma cells were grown in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum and 5% horse serum. HeLa cells were grown in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum. T98G human glioblastoma cells were grown in Eagle's minimal essential medium supplemented with 10% fetal bovine serum. U937 human promyelocytic leukemia cells were grown in suspension in RPMI 1640 medium supplemented with 10% fetal bovine serum.Apoptosis Assay by DNA Fragmentation—Rat-1, PC12, and T98G cells (5 × 106 cells/100-mm dish) were plated and U937 cells (3 × 106 cells/10 ml) suspended 24 h before treatment with cycloheximide (10 μg/ml) and other inhibitors as indicated in the figure legends. The cells were harvested, washed once with phosphate-buffered saline (PBS), and cytosolic nucleic acids isolated as previously described (43Hockenbery D. Nuñez G. Milliman C. Schreiber R.D. Korsmeyer S.J. Nature. 1990; 348: 334-336Crossref PubMed Scopus (3523) Google Scholar). The resulting nucleic acids were electrophoresed through a 1.5% agarose gel containing ethidium bromide. The gels were treated with 20 μg/ml RNase A for 3 h at 37 °C prior to visualization by UV transillumination.Apoptosis Assay by Sub-G1 Analysis—Following treatment, the cells were harvested (adherent cells were trypsinized to provide a single cell suspension) and centrifuged. The cell pellets were washed twice in 1 ml of PBS and fixed in 1 ml of prechilled 70% ethanol (in PBS) overnight at 4 °C. The following day, the cells were washed once with 1 ml of PBS and suspended in 0.5 ml of propidium iodide solution (50 μg/ml propidium iodide, 40 μg/ml RNase A in PBS). The cell suspensions were incubated 30 min at 37 °C in the dark, followed by DNA content analysis by flow cytometry.Immunoblot Analysis—Proteins were separated by SDS-polyacrylamide gel electrophoresis and transferred to polyvinylidene difluoride membranes. Antibodies included anti-Bak NT (06-536; Upstate Biotechnology), anti-Bax (2772; Cell Signaling), anti-Mcl-1 (554103; PharMingen), anti-Bcl-2 (610538; BD Transduction Laboratories), anti-Bcl-x (610746; BD Transduction Laboratories), anti-Bcl-xL (2762; Cell Signaling), anti-Bim (4582; Cell Signaling), anti-PARP (9542; Cell Signaling), and anti-β-actin (A5441; Sigma). Western blots were developed using a chemiluminescence reagent (PerkinElmer Life Sciences).Bak and Bax Activation Assays—U937 cells (20 × 106/50 ml) were suspended in growth medium 24 h before treatment. Following treatment with cycloheximide, the cells were washed once in PBS, suspended in 1 ml of hypotonic RSB buffer (10 mm NaCl, 1.5 mm MgCl2, 10 mm Tris-HCl, pH 7.5), and incubated on ice for 10 min. The cells were ruptured with 20–30 strokes with a tight fitting pestle in a Dounce homogenizer, 0.8 ml of 2.5× MS buffer (525 mm mannitol, 175 mm sucrose, 12.5 mm Tris-HCl, pH 7.5, 2.5 mm EDTA, pH 7.5) was added, and mitochondria were isolated by differential centrifugation. The samples were centrifuged at 1,300 × g for 10 min at 4 °C, the supernatants were transferred to clean tubes, and centrifugation was repeated twice. The resulting supernatants were then centrifuged at 17,000 × g for 20 min at 4 °C to pellet mitochondria. The mitochondrial pellet was washed once with 0.5 ml of 1× MS buffer and recentrifuged. Cross-linking reactions were then performed on mitochondrial pellets suspended in 675 μl of PBS by treatment with 10 mm bismaleimidohexane (Pierce) for 30 min at room temperature (44Wei M.C. Lindsten T. Mootha V.K. Weiler S. Gross A. Ashiya M. Thompson C.B. Korsmeyer S.J. Genes Dev. 2000; 14: 2060-2071Crossref PubMed Google Scholar). The mitochondria were pelleted and suspended in sample buffer supplemented with 10 mm dithiothreitol to quench the cross-linking reaction. The samples were then subjected to immunoblot analysis to detect oligomeric Bak and Bax.Transient Transfections and TUNEL Assays—HeLa cells (5 × 105/60-mm plate) were plated 24 h before transfection. The transfections were performed using 6 μl of TransIT-LT1 reagent (Mirus)/60-mm plate according to the manufacturer's instructions. The cells were transfected with 1 μg of pcDNA3 or the indicated Mcl-1, Bcl-xL, or Bcl-2 expression construct plus 1 μg of pCMV-DsRed (BD Biosciences), which expresses a red fluorescent protein. The cells were treated with cycloheximide 24–48 h after transfection, and TUNEL assays were performed using the Fluorescein FragEL DNA fragmentation detection kit (Calbiochem; catalog number QIA39). The cells were trypsinized and processed according to the manufacturer's recommended protocol for "Fluorescein-FragEL of cell suspensions for flow cytometry." Transfected cells were identified by expression of the DsRed protein with a flow cytometer using the FL2 channel, and the percentage of TUNEL-positive cells were quantified using the FL1 channel.RNA Interference—RNA interference transfections were conducted using HiPerfect reagent (Qiagen) and indicated pre-designed siRNAs (final concentration, 10 nm) from Ambion, Inc. For Mcl-1 RNA interference experiments, the transfection mixtures were prepared in 400 μl of total volume by adding 24 μl of HiPerfect and either nonspecific siRNA (Ambion negative control siRNA 1, catalog number 4611) or indicated Mcl-1 siRNA (Ambion siRNA I. D. numbers 6126, 6314, and 42844) to serum-free medium, mixed, and incubated at room temperature for 10 min. The transfection mixtures were then transferred to 60-mm plates and overlaid with 4.6 ml of a 105/ml cell suspension in serum-containing medium. The cells were then placed at 37 °C, 5% CO2 for the indicated times before harvest for immunoblot analysis, DNA fragmentation assays, or sub-G1 analysis. For Bim, Bax, and Bak RNA interference experiments, transfections were conducted as described above, using 200-μl total volumes and 12 or 24 μl of HiPerfect reagent for the transfection mixtures, which were overlaid with 2.3 ml of a 105/ml cell suspension on 35-mm plates.All of the siRNAs were purchased from Ambion. Each siRNA consisted of 19-nucleotide double-stranded RNA with two 3′-dT overhangs on each strand. siRNA sense sequences and Ambion ID numbers were as follows: Mcl-1 ID 6126, 5′-GGACACAAAGCCAAUGGGCTT-3′; Mcl-1 ID 6314, 5′-GGACUUUUAGAUUUAGUGATT-3′; Mcl-1 ID 42844, 5′-GGAGGCCUCGGCCCGGCGATT-3′; Bim ID 262307, 5′CCUUCUGAUGUAAGUUCUGTT-3′; Bak ID 120199, 5′-CCCAGAGAUGGUCACCUUATT; Bak ID 120201, 5′-GCUUUAGCAAGUGUGCACUTT-3′; and Bax ID 213259, 5′-GAGGUCUUUUUCCGAGUGGTT-3′.RESULTSApoptosis Induced by Translation Inhibition Requires Proteasome Activity—If apoptosis in response to translation inhibition occurs because of either the degradation of a specific regulatory protein(s) or nonspecific degradation of cellular constituents, then inhibition of proteolysis should block apoptosis. Because the proteasome is responsible for degradation of most proteins within the cell, the effect of proteasome inhibition on apoptosis resulting from inhibition of protein synthesis was examined in four cell lines: Rat-1 fibroblasts, PC12 rat pheochromocytoma cells, U937 human promyelocytic leukemia cells, and T98G human glioblastoma cells. Rat-1, PC12, and T98G cells were studied as models of growth factor-dependent rodent and human cells (45Tullai J.W. Schaffer M.E. Mullenbrock S. Kasif S. Cooper G.M. J. Biol. Chem. 2004; 279: 20167-20177Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar, 46Yao R. Cooper G.M. Oncogene. 1996; 13: 343-351PubMed Google Scholar), whereas U937 cells were used because they rapidly undergo apoptosis in response to a variety of stimuli and are therefore advantageous for biochemical analysis (47Erhardt P. Cooper G.M. J. Biol. Chem. 1996; 271: 17601-17604Abstract Full Text Full Text PDF PubMed Scopus (136) Google Scholar). Protein synthesis was inhibited by treatment with cycloheximide at a concentration (10 μg/ml) that inhibited [35S]methionine incorporation >95% (data not shown). The cells were pretreated with proteasome inhibitor MG132 for 30 min prior to treatment with cycloheximide for 0–6 h, after which fragmented cytosolic DNA was isolated and detected by agarose gel electrophoresis. Cycloheximide treatment resulted in rapid and time-dependent apoptosis, detected as early as 2 h after cycloheximide addition, which was blocked by proteasome inhibition with MG132 in all cell lines tested (Fig. 1A).DNA content analysis was performed on U937 cells by flow cytometry to quantify the percentage of cells undergoing apoptosis. As shown in Fig. 1 (B and C), cycloheximide treatment resulted in 25 and 53% cells within the sub-G1 population at 2 and 4 h, respectively. However, pretreatment with MG132 largely blocked apoptosis, because only 5 and 10% cells were in the sub-G1 population at 2 and 4 h, respectively.To ensure that the effects of MG132 were due to its inhibition of the proteasome, U937 cells were pretreated with two other proteasome inhibitors, MG115 and proteasome inhibitor I prior to cycloheximide, both of which also blocked apoptosis through 6 h (Fig. 1D). Collectively, these studies show that apoptosis induced by cycloheximide requires proteasome activity, indicating that the loss of one or more proteins via proteasome-mediated degradation is responsible for apoptosis induced by translation inhibition.Translation Inhibition Activates the Mitochondrial Pathway of Apoptosis—Protein degradation upon translation inhibition could cause cell death because of a nonspecific global loss of protein, resulting in deterioration of the cell in an apoptosis-like manner. Alternatively, cell death could occur through caspase-dependent apoptosis because of the loss of a specific protein, or set of proteins, whose activity normally maintains cell survival by blocking caspase activation directly or indirectly. Treatment of U937 cells with cycloheximide for 6 h resulted in cleavage of the caspase substrate PARP, which was blocked by the general caspase inhibitor zVAD-fmk (Fig. 2A), indicating that translation inhibition induces caspase activation. To test whether cell death is dependent on caspase activity, the effect of zVAD-fmk on cycloheximide-induced DNA fragmentation was examined. Pretreatment of Rat-1, U937, and T98G cells with zVAD-fmk blocked apoptosis through 4 h of cycloheximide treatment (Fig. 2B), indicating that translation inhibition activates an apoptotic program leading to caspase-dependent cell death.FIGURE 2Apoptosis induced by translation inhibition is dependent on caspase activity and blocked by Bcl-xL overexpression. A, U937 cells were left untreated or treated with 10 μm zVAD-fmk for 30 min prior to treatment with 10 μg/ml cycloheximide for 6 h. The cells were harvested for immunoblot analysis to detect PARP. B, cells were treated with zVAD-fmk (Rat-1 and T98G cells, 100 μm; U937 cells, 10 μm) or left untreated for 30 min prior to treatment with 10 μg/ml cycloheximide (CHX) for 4 h. Cytosolic nucleic acids were isolated, and DNA fragmentation was assessed by gel electrophoresis. C, HeLa cells were transfected with 1 μg of pCMV-DsRed expression construct and 1 μg of pcDNA3 empty vector or pSG5-Bcl-xL expression construct. 48 h after transfection, the cells were treated with 10 μg/ml cycloheximide for 18 h and trypsinized, and apoptosis was assessed by TUNEL analysis. Transfected cells were identified by flow cytometry, and the data are presented as the percentages of transfected cells that were TUNEL-positive. The data represent the averages values of two independent cultures ± S.D.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Caspase activation during apoptosis typically occurs because of mitochondrial outer membrane permeabilization and release of pro-apoptotic factors including cytochrome c into the cytosol (1Danial N.N. Korsmeyer S.J. Cell. 2004; 116: 205-219Abstract Full Text Full Text PDF PubMed Scopus (3991) Google Scholar). Pap and Cooper (17Pap M. Cooper G.M. Mol. Cell Biol. 2002; 22: 578-586Crossref PubMed Scopus (147) Google Scholar) previously reported that cycloheximide induces cytochrome c release, suggesting that translation inhibition activates apoptosis through the

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