MitochondrialDysfunctionImpairsTumorSuppressorp53 ... · PDF fileMitochondrialDysfunctionImpairsTumorSuppressorp53 Expression/Function* S Receivedforpublication,July23,2010,andinrevisedform,March29,2011

Mitochondrial Dysfunction Impairs Tumor Suppressor p53Expression/Function*□S

Received for publication, July 23, 2010, and in revised form, March 29, 2011 Published, JBC Papers in Press, April 18, 2011, DOI 10.1074/jbc.M110.163063

Shannon Compton‡, Chul Kim‡, Nicholas B. Griner§, Prasanth Potluri¶, Immo E. Scheffler¶, Sabyasachi Sen‡§�,D. Joseph Jerry‡§, Sallie Schneider‡§, and Nagendra Yadava‡�**1

From the ‡Pioneer Valley Life Sciences Institute, Springfield, Massachusetts 01107, the �Division of Endocrinology, Diabetes, andMetabolism, Baystate Medical Center, Tufts University School of Medicine, Springfield, Massachusetts 01199, the Departments of**Biology and §Veterinary and Animal Sciences, University of Massachusetts, Amherst, Massachusetts 01003, and the ¶Section ofMolecular Biology, University of California San Diego, La Jolla, California 92093

Recently, mitochondria have been suggested to act in tumorsuppression. However, the underlying mechanisms by whichmitochondria suppress tumorigenesis are far from being clear.In this study, we have investigated the link between mitochon-drial dysfunction and the tumor suppressor protein p53 using aset of respiration-deficient (Res�)mammalian cellmutantswithimpaired assembly of the oxidative phosphorylationmachinery.Our data suggest that normal mitochondrial function isrequired for �-irradiation (�IR)-induced cell death, which ismainly a p53-dependent process. The Res� cells are protectedagainst �IR-induced cell death due to impaired p53 expression/function. We find that the loss of complex I biogenesis in theabsence of the MWFE subunit reduces the steady-state level ofthe p53 protein, although there is no effect on the p53 proteinlevel in the absence of the ESSS subunit that is also essential forcomplex I assembly. The p53 protein level was also reduced toundetectable levels in Res� cells with severely impaired mito-chondrial protein synthesis. This suggests that p53 proteinexpression is differentially regulated depending upon the typeof electron transport chain/respiratory chain deficiency. More-over, irrespective of the differences in the p53 protein expres-sion profile, �IR-induced p53 activity is compromised in allRes� cells. Using two different conditional systems for complexI assembly, we also show that the effect of mitochondrial dys-function on p53 expression/function is a reversible phenome-non.We believe that these findings will havemajor implicationsin the understanding of cancer development and therapy.

Mitochondrial dysfunction is associated with aging, degen-erative diseases, and cancer (1–3). One of the key functions ofmitochondria is to make ATP by the process of oxidative phos-

phorylation (OxPhos),2 which is carried out by four electrontransport chain (ETC)/respiratory chain (RC) complexes(I–IV) and the ATP synthase (complex V). The OxPhosmachinery consists of over 100 nuclear and mitochondrialDNA-encoded proteins (4). Thirteen proteins encoded bymitochondrial (mt) DNA are core proteins of complexes I andIII–V that are synthesized inside mitochondria. Complex II isan exception as all of its subunits are encoded by nuclear genes.Mutations in bothmtDNAandnuclear genes encodingOxPhoscomplexes are associated with almost all types of cancers (1, 3).Somatic mutations in complex I subunit-encoding genes arefrequently associated with oncocytomas (5, 6). A recent studywith head and neck cancers suggests that there are no muta-tional hot spots in the mtDNA (7). However, other studies sug-gest that mtDNA polymorphisms may predispose certainpopulations to cancer (8–12). The association of germ linemutations in complex II subunits with hereditary paragan-gliomas and pheochromocytomas constitute the strongestevidence for implicating mitochondrial metabolism intumorigenesis (13, 14). Furthermore, the association ofreduced expression of several subunits of OxPhos complexeswith various cancers also suggests a possible role of mito-chondrial metabolism in tumor progression (15, 16).Mitochondria have been recently proposed to act as tumor

suppressors (17, 18). However, the exact molecular mecha-nisms by which mitochondria suppress tumorigenesis are notclear. In tumors associated with mutations in complex II sub-units, accumulation of succinate is suggested to be the under-lying cause of tumorigenesis because it promotes hypoxicadaptations via regulating prolyl hydroxylases (19, 20). Theassociation of mutations in fumarate hydratase, another TCAcycle enzyme capable of modulating succinate metabolism, inleiomyomas, renal cell carcinomas, and Leydig cell tumors alsosupports the role of TCA cycle intermediates in tumorigenesis(21–23). It has been observed that succinate and fumarate canimpair the activities of prolyl hydroxylases, which regulate thehypoxia-inducible factor 1 alpha (HIF1�) (22). Furthermore,the inactivation of tumor suppressors (e.g. PTEN, phosphatase

* This work was supported, in whole or in part, by National Institutes of HealthGrants R21NS057224 (to N. Y.) and R01-CA105452, R01-ES015739 (toD. J. J.). This work was also supported in part by funds from a Rays of Hopegrant from the Baystate Health Foundation and start-up funds from Centerof Excellence in Apoptosis Research.

The nucleotide sequence(s) reported in this paper has been submitted to the Gen-BankTM/EBI Data Bank with accession number(s) JF901929.

□S The on-line version of this article (available at http://www.jbc.org) containssupplemental Figs. S1–S4 and Table S1.

1 To whom correspondence should be addressed: Pioneer Valley Life SciencesInstitute, 3601 Main St., Springfield, MA 01107. Tel.: 413-794-0786; Fax:413-794-0857; E-mail: [email protected].

2 The abbreviations used are: OxPhos, oxidative phosphorylation; ETC, elec-tron transport chain; RC, respiratory chain; Gy, gray; MEF, mouse embry-onic fibroblast; FCCP, carbonyl cyanide p-trifluoromethoxyphenylhydra-zone; BHT, butylhydroxytoluene; NAC, N-acetylcysteine; MnTE-2-PyP,Mn(III) meso-tetrakis(N-ethyl-2-pyridyl) porphyrin; Dox, doxycycline; TES,2-{[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]amino}ethanesulfonic acid.

THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 286, NO. 23, pp. 20297–20312, June 10, 2011© 2011 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in the U.S.A.

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and tensin homolog) and activation of genes implicated in sur-vival signaling (e.g. AKT1, HIF�) by ETC/RC deficiencies cancontribute to the process of tumorigenesis (24). PTEN getsinactivated by redox changes because of mitochondrial dys-function (24).Whethermitochondrial dysfunction can regulatetumor suppressor p53 in a way that favors tumorigenesis is notknown. Considering that the tumor suppressor PTEN protectsp53 from its inhibitor Mdm2 (25), it is possible that p53 func-tion could be down-regulated by ETC/RC deficiencies.Impaired p53 signaling is the most common genetic altera-

tion found in cancer and it plays a critical role in apoptosis (26).p53 controls cell fate by transcription-dependent and tran-scription-independent mechanisms following stress signalssuch as DNA damage, oncogene activation, and hypoxia (27–29). p53 has also been implicated in regulating cellular metab-olism (30). It regulates glycolysis and oxidative metabolism inopposing directions (30–32), and therefore it is considered asone of the molecular mediators of the Warburg effect, theswitch from oxidative metabolism toward glycolysis in cancercells (33). Although most studies have focused on the down-stream effects of p53 on cellular metabolism (32), very little isknown about how p53 activity is affected bymitochondrial dys-function (34).In this study, we present evidence that mitochondrial dys-

function suppresses p53 expression and function reversibly.The suppression of p53 by impaired mitochondrial functionrenders cells resistant to �-irradiation (�IR)-induced death.These data demonstrating that the p53-mediated cell deathrequires normal mitochondrial function suggest that impairedmitochondrial function can directly promote tumorigenic cas-cades as well as impair responses to chemotherapy.

EXPERIMENTAL PROCEDURES

Reagents—Rotenone was from Calbiochem. All other reagentswere from Sigma unless otherwise statedCells and Culture Conditions—The respiration-deficient

(Res�) cells used in this study have been described elsewhere(see Table 1 for details) (35–38). As these Res� cells completelyrely on glycolysis for their bioenergetic needs, they must begrown in the presence of plenty of glucosewith frequent changeof the culture medium (39–42). The generation of B2-MWFEi

cells using the pTre-On vector has been described recently (35,38). TheG18-ESSSi cells were also developed by the same strat-egy. Briefly, cells were grown in Dulbecco’s modified Eagle’smedium (DMEM, Invitrogen) supplemented with 10% fetalbovine serum (FBS, Invitrogen), 1% minimal essential mediumnonessential amino acids (Mediatech, Inc., Manassas, VA), and1% antibioticmix (penicillin/streptomycin, Invitrogen) at 37 °Cin a humidified atmosphere of 5% CO2, 95% air. In light of thisstudy, caution must be taken to avoid the chronic presence ofantibiotics such as tetracycline (and its derivatives) that canimpair mitochondrial protein synthesis in cell culture medium.In inducible cells (B2-MWFEi and G18-ESSSi), complex Iassembly was induced using 1 �g/ml doxycycline (Dox) asdescribed previously (35, 38). Cells were harvested after wash-ing once with Ca2�-, Mg2�-free phosphate-buffered saline(PBS), pH 7.4, using trypsin/EDTA (Invitrogen).

Plasmids and Cell Transfection—The p53-GFP fusion pro-tein described byNorris andHaas (43) was a generous gift fromDr. D. Joseph Jerry. The GFP-expressing plasmid (pEGFP-N3)and p53 reporter (p53-Luc) were from Clontech and Strat-agene, respectively. The p53-Luc reporter plasmid expressesfirefly luciferase from a synthetic promoter that contains 14repeats of the p53-enhancer element (TGCCTGGACTTGC-CTGG). Unless otherwise stated, the cells were transfected at�50% confluency with the indicated plasmids using Lipo-fectamine 2000 (Invitrogen) following the manufacturer’sinstructions. The GFP-expressing (pEGFP-N3) and p53reporter (p53-Luc) plasmids were used at 4 and 8 �g/ml con-centrations, respectively, with 10 �l of Lipofectamine 2000 in500 �l of transfection mixture.Cell Death Assays—Propidium iodide and an Alexa Fluor

488-conjugated anti-annexin V antibody were used to mon-itor the cell death by flow cytometry using the FACSCaliburFACScan (BD Biosciences). Cells were �IR-treated with theindicated dose(s) using the Gammator B (RadiationMachineryCorp., Parsippany, NJ). About 1 h prior to �IR, the culturemediumwas replaced with fresh mediumwith or without indi-cated drug(s), and cell death was monitored 24 h post-�IR. Allcells (floating and attached) were collected with minimal han-dling and stained using propidium iodide and annexin V for 15min at room temperature following themanufacturer’s instruc-tions (Vybrant apoptosis assay kit 2, Molecular Probes). Eachexperiment was performed in triplicate and repeated at leastthree times.Protein Analyses—Cells were lysed in RIPA lysis buffer (10

mMTris, pH7.2, 150mMNaCl, 1%TritonX-100, and 0.1%SDS)containing protease inhibitors (P8430, Sigma) and phosphataseinhibitors mixtures (P5726 and P2850, Sigma). A microplate-based BCA protein assay kit (Thermo Scientific) was used todetermine protein concentrations. Western blot analyses wereperformed after separating the proteins on 10–20% SDS-poly-acrylamide gels (Bio-Rad) and transferring them onto PVDFmembrane (0.2 �m) using the iBLOT apparatus (Invitrogen).The following primary antibodies were used at the indicateddilutions: anti-total p53 (DO-1) (1:1000, Santa Cruz Biotech-nology), anti-actin (1:5000, Abcam), and anti-HA epitope(1:1000, Covance Babco). Proteins were visualized using sec-ondary antibody conjugated withHRP (1:4000, Santa Cruz Bio-technology), and the ECLPlus chemiluminescence reagent(Bio-Rad).p53 Activity Assay—p53 activity was assayed using a lucifer-

ase reporter assay as described with a few modifications (44).Cells were seeded in triplicate in 35-mm plates at a density of60,000 cells/ml (G7 and G18) or 50,000 cells/ml (all other celllines) a day before transfection with the pEGFP and p53-Lucplasmids. Nontransfected cells were used as negative controls.24 h post-transfection, the cells were exposed to 10 Gy, and 6 hlater, the cells were harvested for analyses. Unless otherwisestated, all activity assays were performed with 10 Gy �IR tominimize the potential risk associated with DNA damage at ahigher dose (e.g. 40 Gy), which may block the transcription.After aspirating the supernatant, the cell pellet was resus-pended in 300 �l of annexin V binding buffer (Vybrant apopto-sis assay kit 2, Molecular Probes) and then divided into three

Reversible Regulation of p53 by Mitochondrial Dysfunction

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equal aliquots. The first aliquot was used to determine trans-fection efficiency via FACS analysis. The second and third ali-quots were pelleted, and the supernatants were removed. Onepellet was resuspended in 100 �l of RIPA buffer to determineprotein concentration andused forWestern blotting. The otherwas resuspended in the passive lysis buffer to assess the lucifer-ase activity according to the manufacturer’s instructions (Pro-mega) using a TD-20/20 luminometer (Turner Designs). Therelative p53 activity was determined as shown in Equation 1,

luciferase activity (arbitrary units)

(protein concentration) � (transfection efficiency)

(Eq. 1)

The results are reported as the average of averages� S.D. foreach condition from at least three independent experiments.Each experiment had assays performed in quadruplicate foreach condition.Splenocytes and Thymocytes Cultures—Splenocytes and thy-

mocytes were harvested from adult p53�/� and p53�/�

C57BL/6 mice according to previously established protocols(45, 46). Whole spleen and thymus were collected from eutha-nized mice and placed into DMEM. Tissues were minced andgently rubbed between two acid-etched slides to release indi-vidual cells. Cells were transferred to T25 flasks for 2 h in astandard tissue culture CO2 incubator at 37 °C to removeadherent cells. After 2 h, nonadherent cells were collected andplated at a density of 100,000 cells per 60-mm plate. Cell deathfollowing 1 Gy �IR was monitored by flow cytometry asdescribed above. All animal procedures were in compliancewith the IACUC.In Situ Respirometry—The degree of respiratory inhibition

by rotenone was determined using a 24-well extracellular flux(XF24) analyzer from Seahorse Bioscience as described previ-ously (47). HEK293T cells were seeded at 15,000–30,000 cells/well 24–48 h prior to measurements and then gently washedwith and incubated in the respiration buffer (all in mM): 120NaCl, 3.5 KCl, 1.3 CaCl2, 0.4 KH2PO4, 20 Na-TES, 5 NaHCO3,1.2 Na2SO4, 2 MgCl2, and 15 D-glucose, containing 0.4% fattyacid-free BSA. The determination of the rates of oxygen con-sumption by the XF24 analyzer is based on oxygen-dependentfluorescence quenching of a fluorophore and the correctionsfor back diffusion of oxygen in measuring wells (see Ref. 47 fordetails). The XF24 respirometer allows up to four sequentialadditions of desired drugs using injection ports A–D. Respira-tion assays were performed with repeated cycles of 1 min ofmixing, 1min of waiting, and 3min ofmeasurement before andafter the additions of different drugs. Full correction algorithm(Akos) was applied to determine the respiration rates (47).The maximal respiratory capacity was determined in the pres-ence of 3 �M carbonyl cyanide p-trifluoromethoxyphenylhy-drazone (FCCP), which was determined after careful titrationsin the presence and absence of 1 �g/ml oligomycin, a complexV inhibitor. In our experience although the presence of oligo-mycin does not change the required FCCP concentration formaximal respiratory stimulation (data not shown), it can impairrespiratory capacity up to �45% (see supplemental Fig. S1D).Similar effect is observed in almost all other cellswehave tested,

including primary neurons (data not shown). Therefore, theoligomycin was excluded from the experiments toward deter-mining the maximal respiratory capacity of cells. This effect isobserved irrespective of the substrate used such as glucose ver-sus pyruvate.Induction of �IR Resistance in Respiration-competent (Res�)

CellsUsingDrugsAffectingMitochondrial Function—Res� cellswere treated with either the complex I inhibitor rotenone ormitochondrial protein synthesis inhibitor chloramphenicolprior to �IR to recapitulate the effects of complex I deficiencies(as in B2 andG18 cells) or impairedmitochondrial protein syn-thesis (as in G7 cells), respectively, on �IR sensitivity. The rote-none was added at the indicated concentration 1 h prior to orafter �IR (1–20 h), whereas the chloramphenicol (50 �g/ml)was added 24–48 h prior to �IR, and then relative cell deathwas monitored 24 h later as described above.Measurements ofMitochondrial Superoxide—Mitochondrial

superoxide production was monitored by using a superoxide-sensitive probe, the MitoSox (48, 49). Cells were treated with0.2 �M MitoSox that showed detectable linear increase in fluo-rescence following rotenone treatment (see supplemental Fig.S3F). Live cells were imaged at 37 °C using Nikon EclipseTE2000-U microscope equipped with a temperature enclosureandYFP/HQ (excitation), 590LP (emission), and 565LP (dichroic)filters. The relative levels of superoxide production in differentcellswere determined by flow cytometry (FACSCalibur FACScan,BD Biosciences). �IR-treated cells in the presence and absence ofsuperoxide dismutase mimetic, the Mn(III)meso-tetrakis(N-eth-yl-2-pyridyl) porphyrin (MnTE-2-PyP) (48)were compared.Vehi-cle (DMSO) or 25 �MMnTE-2-PyP (1 h)-treated cells were incu-bated with 0.2 �MMitoSox for at least 30 min prior to irradiationand thenanalyzedafter�45min.All experimentswereperformedin the respiration buffer.Real Time PCR—RNA was isolated using TRIzol reagent

(Invitrogen) according to the manufacturer’s description, andthe PCR was performed as described previously (44, 50). Iso-lated RNAs were reverse-transcribed using SuperScript III(Invitrogen) and analyzed for relative gene expression levels byquantitative PCRwithBrilliant II SYBRGreenquantitative PCRmastermix (Stratagene). As the sequence information for ham-ster p53 targets is unavailable, we designed primers for hamster�-actin and p21 cDNA amplification and confirmed the speci-ficity by sequencing. The primers used were as follows: 5�-AGGTATTCTGACCCTGAAGTACC-3� (forward, hamster�-actin) and 5�-CAGCTCATAGCTCTTCTCCAG-3� (re-verse, hamster �-actin); 5�-AGAACGGTGGAACTTTGACT-TCG-3� (forward, hamster p21) and 5�-GCGCTTGGAGTGA-TAGAAATCTG-3� (reverse, hamster p21). The primers formouse genes were based on primer design data base (RocheApplied Science). These were as follows: 5�-GTGAGCGGCT-GCTTGTCT-3� (forward, Bax) and 5�-GGTCCCGAAGTAG-GAGAGGA-3� (reverse, Bax); 5�-TTCTCCGGAGTGTTCA-TGC-3� (forward, p53 up-regulated modulator of apoptosis,Puma) and 5�-TACAGCGGAGGGCATCAG-3� (reverse,Puma); 5�-CAGATCCACAGCGATATCCA-3� (forward, p21)and 5�-GGCACACTTTGCTCCTGTG-3� (reverse, p21). Thereal time PCRs were performed using Mx3005P (Stratagene),




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and the results were analyzed relative to actin expression asdescribed previously (51).ATP Assays—ATP concentrations in different samples were

determined using the luciferin-luciferase assay (52). ATP wasextracted with either boiling water or the extraction reagentprovided with the ATP bioluminescent somatic cell assay kit(FLASC) from Sigma. Cells were plated at 35,000 in 30-mmplates. 24 h later they were treated with 40 Gy �IR and thenharvested at the indicated times for ATP assays. Harvested cellswere diluted to a final concentration of 2.5 � 105/ml, and ATPwas assayed following the manufacturer’s instructions forFLASC kit using a TD-20/20 luminometer (Turner Designs).An ATP standard curve was used to determine relative ATPlevels in the samples.

RESULTS

Mitochondrial Dysfunction Protects against �IR-induced CellDeath—Todeterminewhethermitochondrial dysfunction pro-tects cells from p53-mediated cell death, we used several Res�mutants that have been previously characterized (see Table 1)(35–37, 42). Because �IR-induced death is primarily mediatedby the p53 pathway (45), the �IR was used as genotoxic stress tomeasure the level of protection offered by mitochondrial dys-function. All experiments were done in the presence of plentyof glucose (4.5 mg/ml) as Res� cells rely on glycolysis for theirbioenergetic needs. Dose-response curves for �IR-induceddeath in Res� (B2-MWFE, G3) and Res� (B2, B2-S55A, G7, andG18) cells are shown in Fig. 1. Clearly, the Res� (B2, B2-S55A,G7, and G18) cells were protected from �IR-induced cell deathat all doses tested. B2-S55A cells, which have partial (�50%)complex I deficiency (35), were partially protected (Fig. 1A)compared with B2 cells in which complex I assembly is com-pletely lacking due to the absence of the MWFE subunit (42).Serine 55 in the MWFE protein is an evolutionarily conservedpotential complex I regulatory site for protein kinase A-medi-ated phosphorylation (35, 53). However, in the cells used here,we have not found any evidence of acute regulation of cellularrespiration by this residue in the presence of cAMP agonists.3Like B2 cells, the G18 cells, with complete complex I deficiencybecause of the absence of a different subunit, the ESSS (37),were also protected from�IR-induced death (Fig. 1B). Likewise,the G7 cells with severe OxPhos defects resulting from the

complete absence of mitochondrial protein synthesis were alsoprotected (Fig. 1, B, E, and F) (54).To determine whether the protection from �IR-induced cell

death in Res� cells is a consequence of the loss of ETC/RCassembly or activity, we measured �IR-induced death in thepresence of rotenone, which specifically inhibits complex Iactivity when used in the low nanomolar (�100 nM) range (55).Higher rotenone concentrations affect the microtubule assem-bly. Our studies with primary neurons have shown that signif-icant respiratory inhibition can be detected with rotenone con-centration as little as 10 nM (48). Therefore, we decided to testthe effect of 0–50 nM rotenone on cellular respiration using amicroplate-based respirometry assay (47). To determine therelevance of our findings in relation to human pathophysiology,we wanted to test the effect of mitochondrial impairments inHEK293T cells first. As expected, therewas a clear dose-depen-dent decline of basal respiration (Fig. 2A) as we have observedwith primary rat neurons (48). Because the FCCP-stimulatedrespiration is not very stable despite careful FCCP titrations, webelieve that accurate determination of the extent by which therespiratory capacity (FCCP-stimulated maximal respiration) isrestricted is only possible by treating cells with rotenone beforeFCCP stimulation as shown in Fig. 2A. As expected, a dose-de-pendent decline of the respiratory capacity of HEK293T cellswas seen. About 42.4 � 4.8% decline in the respiratory capacitycould be observed even with as little as 5 nM rotenone (Fig. 2A).50 nM rotenone could inhibit 68.0 � 5.3% of the basal and 72 �6.9% of the maximal HEK293T cell respiration. The B2-MWFEand G3 cells also showed similar rotenone sensitivity (data notshown). A side-by-side comparison of rotenone effects onFCCP-stimulated respiration showed relatively more inhibi-tion when the rotenone was added before FCCP stimulation(supplemental Fig. S1, A–C). Our estimates suggest that 50 nMrotenone could inhibit 79.4� 4.3% of the complex I-dependentrespiration in B2-MWFE, G3, and HEK293T cells.To determine the effect of rotenone on �IR-induced death,

the HEK293T cells were treated with 0–50 nM rotenone 1 hprior to�IR, and the extent of deathwas quantified 24 h later viaflow cytometry as described under “Experimental Procedures.”The relationship between the rotenone concentration and �IRdoses onHEK293T cell sensitivity is shown in Fig. 2B. Rotenoneprotected cells from �IR-induced death in a dose-dependentmanner compared with controls. The dose-dependent protec-tion was clearer at lower radiation doses. 50 nM rotenone gave3 N. Yadava, unpublished data.

TABLE 1Description of the Res� cells used in this study

Cell line DescriptionRespiratorycompetence Ref.

B2 Complex I-deficient CCL16-B2 Chinese hamster cells. Null for the MWFEsubunit (Ndufa1 gene encoded)

Res� 63

B2-MWFE B2 cells complemented with HA epitope-tagged wild type hamster MWFEprotein (MWFE-HA)

Res� 42

B2-S55A B2 cells complemented with HA epitope-tagged MWFE bearing point mutationS55A

Res� �50% 35

B2-MWFEi B2 cells with Dox-inducible expression of MWFE HA Conditionally Res� 35, 38G7 V79-G7 Chinese hamster cells with impaired mitochondrial protein synthesis,

gene unknownRes� 54

G18 Complex I-deficient V79-G18 Chinese hamster cells. Null for the ESSSsubunit (Ndufb11 gene encoded)

Res� 37

G18-ESSSi G18 cells with Dox-inducible expression of ESSS-HA Conditionally Res� P. Potluri, unpublished dataG3 The parental V79 cells from which the G7 and G18 mutants were derived Res� 101




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maximal protection. Similar dose-dependent rotenone-medi-ated protection was also observed in G3 (Fig. 2D) andB2-MWFE (data not shown) cells. These data support theobservations made from complex I-deficient Res� (B2,B2-S55A, and G18) cells in which the OxPhos machinery isimpaired by genetic mutations (Fig. 1) (35–37, 42). Further-more, to determine the temporal effects of mitochondrial dys-function on �IR-induced death, we treatedHEK293T cells withrotenone at different time points after �IR, and we then deter-mined the relative level of protection. In cells treated at or after1 h post-�IR, the protective effect of rotenone declined drasti-cally (Fig. 2C). A similar protection profile was also observedwith G3 (Fig. 2E) and B2-MWFE (data not shown) cells. Thesedata clearly suggest that only the pre-existing respiratory inhi-bition is protective. The degree of protection with 50 nM rote-none was comparable with that offered by genetic impairmentsin Res� (B2, G7, and G18) cells with severe ETC/RC deficien-cies (Fig. 2D).

The p53 is a tumor suppressor, which directs radiation-in-duced cell death by both transcription-dependent and -inde-pendent mechanisms (28, 29, 45). To investigate whether �IR-induced death is indeed p53-dependent, the mouse embryonicfibroblasts (MEFs) derived from p53 wild type (p53�/�) andnull (p53�/�) C57BL/6 mice were irradiated and assayed for celldeath (46). As expected, the p53�/� MEFs were completely pro-tected from the �IR-induced death, although the p53�/�

MEFs were highly susceptible (Fig. 3A). Furthermore, the levelof protection by 50 nM rotenone was comparable with thatoffered by the complete loss of p53 protein in p53�/� MEFs(Fig. 3A).Recent studies have suggested that the p53 can quickly trans-

locate to mitochondria following irradiation (56). Thus, it isconceivable that in our fibroblast lines the high dose �IR (40Gy) is inducing a direct mitochondrial interaction with p53,which is responsible for efficient cell death in control cells,whereas the loss of mitochondrial function by pre-existing

FIGURE 1. Protection of Res� cells from �IR-induced death. Cells were seeded 24 h prior �IR to reach �70% confluency, and the level of cell death wasmonitored by flow cytometry (see “Experimental Procedures”). A, relative �IR sensitivities of Res� cells due to mutations in the MWFE subunit of complex I.B2-MWFE cells, Res� cells; B2-S55A cells, Res�, �50% less complex I; and B2 cells, Res�, lack complex I. B, relative �IR sensitivity of other Res� cells. G3 cells, Res�

cells; G18 cells, Res�, lack complex I due to the absence of the ESSS subunit; and G7 cells, Res� due to the absence of all mtDNA-encoded proteins. See Table1 for details of each cell line. The percent live cells 24 h post-�IR with different doses (0, 5, 10, 20, and 40 Gy) are reported. C–F, representative flow cytometrydata showing the propidium iodide (FL2-H) and annexin V (FL1-H) signals for the Res� G3 (C, control; D, 40 Gy �IR-treated) and Res� G7 (E, control; F, 40 Gy�IR-treated) cells.




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ETC/RC defects in Res� (B2, G18, and G7) and rotenone-treated cells may prevent this direct action.To test whether mitochondrial dysfunction indeed impairs

the direct actions of p53 on mitochondria, we determined theeffect of ETC/RC inhibition in murine thymocytes and spleno-cytes in which the direct effects of p53 on mitochondria havebeen established (56). The thymocytes and splenocytes wereisolated from age-matched p53�/� and p53�/� C57BL/6 mice,and the cell death was monitored 24 h post-�IR (46). The datafrom thymocytes (Fig. 3B) and splenocytes (Fig. 3C) werealso in agreement with the data from MEFs showing compa-rable protection by the complete loss of p53 and rotenone

pretreatment (Fig. 3A). These data clearly suggest that com-plex I inhibition suppresses p53 function that results in pro-tection against �IR-induced death. Furthermore, this effectwas observed across different cell types (fibroblasts, spleno-cytes, and thymocytes) and species (mouse: MEFs, spleno-cytes, and thymocytes; hamster: lung fibroblasts-B and Gseries cells; and human: HEK293T cells).Mitochondrial Dysfunction Alters p53 Expression and

Function—Our experimental results demonstrated that therole of p53was critical in determining the�IR sensitivity of cells(Fig. 3) as expected (45). Thus, it remained to determinewhether mitochondrial dysfunction affected p53 expression

FIGURE 2. Effect of complex I inhibition with rotenone on the �IR sensitivity of Res� cells. A, rotenone titration of the basal and maximal (FCCP-stimulated)respiration rates of HEK293T cells using in situ respirometry (see “Experimental Procedures”). Sequential additions (to block mitochondrial respiration com-pletely) were made as follows: arrow A, rotenone (0, 5, 10, 20, or 50 nM); arrow B, 3 �M FCCP; and arrow C, 1 �M rotenone with 2 �M myxothiazol. The respirationrate just before rotenone addition was set to “0” for comparing % modulation following each addition. B and D, dose-dependent protection of HEK293T (B) andG3 (D) cells by rotenone added 1 h prior to 40 Gy �IR. C and E, loss of protection offered by rotenone to HEK293T (C) and G3 (E) cells when added after �IRtreatment. Rotenone was added either before (�1, 1 h after) or 1–20 after (�1, 1 h; �2, 2 h; �4, 4 h; �8, 8 h; �20, 200 h after) 40 Gy �IR treatment. Cell deathwas monitored at 24 h post-IR. F, relative level of rotenone-mediated protection in Res� cell (B2-MWFE, G3) compared with that observed by genetic mutationsin Res� (B2, G7, and G18) cells following 40 Gy �IR.




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(protein levels) or function in Res� cells. This distinction wasexplored by monitoring the expression of p53 protein in Res�cells byWestern blot analyses. Toward this goal, cells with andwithout �IR (10 Gy, 6 h) were analyzed for the relative levels ofthe p53 protein. Although p53 protein could be detected inB2-MWFE (Res�) and B2-S55A (Res�, �50% complex I-defi-cient) cells, it was undetectable in B2 (Res�) cells with completecomplex I deficiency (Fig. 4A, top panel) under identical condi-tions. As in B2 cells (Fig. 4A, top panel), the p53 proteinwas alsonot detected in G7 cells that have impaired mitochondrial pro-teins synthesis (Fig. 4B, top panel). In contrast to B2 cells, thep53was detectable at near normal level inG18 cells due to a nullmutation in a different subunit, the ESSS (Fig. 4B, top panel)(37). At this stage, we do not know the underlying basis for whythe complex I deficiency in B2 versusG18 cells results in differ-ent fates of p53. When Res� (MEFs and HEK293T) cells weretreated with 50 nM rotenone, the induction of p53 protein fol-lowing �IRwas also blocked (Figs. 3A and 4D). Because theG18Res� cells expressing normal levels of p53 were also protectedfrom �IR-induced death (Fig. 1B) and rotenone treatmentcompromised �IR sensitivity in Res� cells (G3, B2-MWFE,HEK293T, and p53�/� MEFs, thymocytes, and splenocytes)(Figs. 2 and 3), we concluded that p53 activity may be sup-pressed in the presence of mitochondrial dysfunction.To determine the effect ofmitochondrial dysfunction on p53

transactivation activity, we used a luciferase-based p53 reporterassay as described under “Experimental Procedures.” In thisassay, the luciferase expressionwas driven bymultiple copies ofa p53 response element isolated from the human p21 promoter.The p21 gene is a well established transcriptional target of p53.The normal and Res� cells were transfected with the reporterplasmid, and 24 h post-transfection they were 10 Gy �IR-treated. The relative luciferase activity was measured 6 h post-�IR. At 6 h after �IR, maximal p53 activity was found (data notshown).Although inRes� cells (B2-MWFE,G3) the p53 activitywas up-regulated over 6-fold following �IR, the induction wasnot observed in Res� cells (B2, G7, and G18) with severeETC/RC deficiencies (Fig. 4, A and B, bottom panels; supple-mental Table S1). The induction of p53 activity inB2-S55Acellswith partial complex I deficiency was significantly lower (�2-fold) compared with B2-MWFE cells (6-fold). This is in agree-ment with the partial �IR sensitivity of B2-S55A cells (Fig. 1A).Because Ser-15 phosphorylation status did not correlate withp53 transactivation activity across different cells in our hands, itis not shown. To determine whether rotenone also impairs p53activity in Res� cells (B2-MWFE, G3, HEK293T), we measuredthe p53 activity in cells pretreated with rotenone. As expected,rotenone pretreatment abolished the �IR-dependent inductionof p53 activity in Res� cells (Fig. 4C; supplemental Table S1).Also, the presence of rotenone lowered the �IR-inducedincrease in p53 protein level in HEK293T cells (Fig. 4D) andMEFs (Fig. 3A).To determine whether complex I deficiencies can influence

endogenous expression of p53 target genes, we monitored p21expression in Res� versus Res� cells by real time PCR asdescribed under “Experimental Procedures.” Our data in Fig. 4(E and F) suggest that complex I deficiency can influence theexpression of endogenous p53 targets. As expected, based on

FIGURE 3. p53 dependence of �IR sensitivity and the relative protectionby rotenone compared with complete p53 loss. Cell death was assayed24 h post-�IR in the wild type (p53�/�) and null (p53�/�) cells derived fromC57BL/6 mice. A, MEFs. Top panel, Western blot analysis of the total p53 pro-tein. Actin was monitored as loading control. Bottom panel, relative levels ofdeath in p53�/� and p53�/� MEFs induced by 40 Gy �IR. B, p53-dependent 1Gy �IR-induced death in thymocytes. C, p53-dependent 1 Gy �IR-induceddeath in splenocytes. Because of the differences in survival between p53�/�

and p53�/� thymocytes and lymphocytes following isolation, their controlvalues were set to 100%. Note that thymocytes and splenocytes are verysensitive to radiation. Thus, they were treated with 1 Gy �IR. Control,untreated cells; IR, �-irradiated; Rot, 50 nM rotenone treated, Rot�IR, rotenone(50 nM) plus IR-treated.




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basal p53 activities inB2 andG18Res� cells, p21 expressionwassignificantly lower in B2 cells comparedwith their controls (B2-MWFE), although there was no significant difference in G18(Res�) versusG3 (Res�) cells. Furthermore, �IR did not inducep21 expression in Res� (B2 andG18) cells, although there was alow level induction in normal cells (B2-MWFE and G3).Because of the lack of sequence information for hamster p53targets, we could notmonitor expression of other genes in thesecells. Alternatively, wemonitored the effect of rotenone on�IR-induced expression of selected p53 genes in mouse splenocytesand thymocytes, which are also protected in the presence ofrotenone (Fig. 2, B and C). Splenocytes and thymocytes are

highly radiation-sensitive cells. The �IR-induced p53 responseiswell characterized in these cells (45, 57). The inhibition of p21induction by �IR in B2-MWFE cells in the presence of rotenonesuggested that rotenone could affect the expression of p53 tar-gets. To test this, we used splenocytes and thymocytes from thep53 wild type (p53�/�) and null (p53�/�) C57BL/6 mice andmonitored the expression of p21, Bax, andPuma,which arewellknown p53 targets induced by �IR (58). A time course analysissuggested that the maximal induction of all three genes wasachieved 6 h post-�IR (1Gy) in both cell types (data not shown).Therefore, 6 h was selected for comparative analyses in thepresence and absence of 50 nM rotenone. The p53�/� C57BL/6

FIGURE 4. Relative levels of p53 protein expression and its activity in Res� cells. A, effects of complex I deficiencies because of mutations in the MWFEsubunit. Top panel, Western blot analysis of the total p53 protein using the DO1 antibody. Actin was monitored for equal protein loading. Bottom panel, relativep53 activity. B, effects of blocked mitochondrial protein in G7 cells or another complex I deficiency because of the absence of ESSS subunit in G18 cells. G3 cellsare wild type controls. Top panel, Western blot analysis of the total p53 protein. Bottom panel, relative p53 activity. C, rotenone addition 1 h before 10 Gy�IR-blocked p53 activity induction in Res� cells. D, effect of rotenone on p53 protein level in HEK293T cells. E and F, real time quantitative PCR-based analysisof the p21 expression in B2-MWFE versus B2 cells (E) and in G3 versus G18 (F) cells following 40 Gy �IR. Lower �Ct means higher expression and vice versa. Cellswere harvested 6 h post-�IR for the assays as described under “Experimental Procedures.” Note that for activity assays 10 Gy (instead of 40 Gy) �IR was used toavoid any detrimental effects on the transcription due to DNA damage. The values for B2-MWFE, G3, and HEK293T controls were set to “1” for determining therelative activities of the corresponding Res� and rotenone-treated cells. See supplemental Table S1 for the actual values. Ctr, untreated cells; IR, �IR-treated (10Gy) cells; Rot, 50 nM rotenone treated; Rot�IR, rotenone plus �IR-treated. Standard deviations are shown. *, significant from the untreated B2-MWFE control(Ctr) at p � 0.001.




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splenocytes and thymocytes served as negative controls. Data inFig. 5 show a significant inhibition of all three genes following�IR in cells treated with 50 nM rotenone. However, in contrastto the inhibition of �IR-induced response, the basal expressionof p21, Bax, and Puma in splenocytes was significantlyincreased (Fig. 5,A–C). This level of increase by rotenone alonedid not result in increased cell death in splenocytes, althoughthe impaired �IR response was sufficient to protect the cells(Fig. 3C). There was no such increase in basal expressions ofp21, Bax, and Puma in thymocytes (Fig. 5, D–F). As expectedin p53�/� cells, no significant inductions in these genes werefound (Fig. 5). Taken together, our data clearly suggest thatthe respiratory inhibition either because of genetic muta-tions or pharmacological impairments can suppress p53expression/function.

Effect of Complex I Deficiency on p53 Expression/Function IsReversible—To eliminate the possibility that the p53 gene inRes� cells may be mutated, we used a conditional complex Iassembly system (B2-MWFEi) recently described by us todetermine whether restoring complex I assembly would resultin gain-of-p53 function and �IR sensitivity (35, 38). In B2-MW-FEi cells, complex I assembly was induced with 1 �g/ml Dox inthe medium for 48 h. Then cells were allowed to recover with-out Dox for 24 h and treated with 40 Gy �IR. Data in Fig. 6Ashow the induction of the MWFE protein as indicator of com-plex I assembly, which led to the recovery of p53 protein expres-sion, �IR sensitivity (Fig. 6B), and activity (Fig. 6C). The assem-bly of complex I under these conditions has been established inprevious studies and confirmed in this study (data not shown).Under this paradigm, the cellular respiration of stably trans-

FIGURE 5. Effect of rotenone on p53 transcriptional activity. The activity of p53 was monitored by measuring the mRNA expression of p53 target genes p21,Bax, and Puma following 1 Gy �IR in the presence and absence of rotenone. Splenocytes (A–C) and thymocytes (D–F) isolated from p53�/� (WT) and p53�/�

(Null) C57BL/6 mice were harvested 6 h post-�IR to determine the relative expression of p21 (A and D), Bax (B and E) and Puma (C and F) by real time PCR asdescribed under “Experimental Procedures.” Statistical significance was determined by one way analysis of variance. Significant differences (p � 0.001)between the controls versus treated (rotenone, IR), and IR versus IR � rotenone are denoted with a and b, respectively.




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fected cells (B2-MWFE) and Dox-induced cells (B2-MWFEi)were comparable (data not shown). A similar observation wasmade with another conditional system, the G18-ESSSi (Fig. 6,D–F), which expresses the ESSS protein using the same induc-ible strategy in Res� G18 cells (35). It should be noted that inG18 cells, the p53 protein level was not reduced (Fig. 4B, toppanel), although the �IR-induced p53 activity was missing (Fig.4B, bottom panel), which correlated with �IR resistance (Fig.1B). In G18-ESSSi cells as well the restoration of complex Iassembly, which is indicated by the ESSS protein expression(Fig. 6D) by Dox treatment, restored �IR sensitivity (Fig. 6E),and p53 activity (Fig. 6F). As expected, the restored p53 activityfollowing the induction of MWFE and ESSS proteins inB2-MWFEi and G18-ESSSi cells is rotenone sensitive (supple-mental Fig. S2). The gain-of-p53 activity (�6-fold post-�IR) inboth inducible systems (B2-MWFEi and G18-ESSSi) confirmsthat normal ETC/RC function is required for p53-expression/function (Fig. 6, C and F).Chloramphenicol Treatment Recapitulates the Protective

Effect of Impaired Mitochondrial Protein Synthesis Observed inG7 Cells—To determine whether G7 cells are protectedbecause of the impaired mitochondrial protein synthesis orbecause of an unknown mutation in the p53 gene, we treated

the parental Res�, the G3 cells, with a mitochondrial proteinsynthesis inhibitor, the chloramphenicol. Cells were pretreatedwith 50 �g/ml chloramphenicol for 48–72 h prior to �IRbecause we hypothesized that the impaired mitochondrial pro-tein synthesis would affect the p53 expression/function viablocking the assembly of OxPhosmachinery. This should showan effect on the p53 protein expression and �IR sensitivity sim-ilar to the effects observed in G7 cells (Figs. 1B and 4B). Celldeath was monitored 24 h post-�IR as described under “Exper-imental Procedures.” As expected, there was a progressiveincrease in cellular protection by chloramphenicol treatmentfor 24–48 h (Fig. 7A). This could be due to slow turnover/disappearance of ETC/RCcomplexes as shownby us and others(38, 59). The resistance to �IR-induced death in 48-h chloram-phenicol-treated cells could be correlated with the correspondingdeclineof thep53protein levels (Fig. 7B) similar to thatobserved inG7 cells (Fig. 4B, top panel). The humanHEK293T cells also showa similar decline in p53 levels following chloramphenicol treat-ment,whichresult in theirprotection from�IR-induceddeathandthe suppression of p53 activity (data not shown).Ectopic Expression of p53 in Res� Cells Does Not Restore �IR

Sensitivity—One of the therapeutic strategies currently pro-posed for treating some cancers involves the restoration of nor-

FIGURE 6. Effects of complex I deficiencies on p53 expression/function are reversible. A–C, restoration of the p53 protein expression (A), �IR-sensitivity (B),and activity (C) in B2-MWFEi cells following the induction of MWFE subunit. D–F, restoration of the p53 protein expression (D), �IR sensitivity (E), and activity (F)in G18-ESSSi cells following the induction of ESSS subunit. The expressions of the MWFE and ESSS were induced by treating the B2-MWFEi and G18-ESSSi cells,respectively, with 1 �g/ml Dox for 48 h. Then the Dox was removed for 24 h prior to �IR treatment to eliminate its potential inhibitory effects on themitochondrial protein synthesis. The MWFE and ESSS proteins were detected using the anti-HA epitope antibody. The p53 was detected with the DO1antibody, and the actin served as the loading control. As expected, the p53 protein in G18-ESSSi cells was present in both the absence and presence of Dox.Relative p53 activity is shown in the absence (�Dox) and presence (�Dox) of the MWFE and ESSS protein expression. The assays were performed in quadru-plicate per condition within each experiment, and the values for Dox-treated controls were set to 1. Ctr, controls; IR, �IR-treated. The Western blots, cell death,and activity data were obtained from the same set of cells. See “Experimental Procedures” for details of the assays.




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mal p53 protein function by ectopic expression or using re-ac-tivating drugs (60). Such a strategy may face problems, if thetumors have pre-existing mitochondrial dysfunction, whichoften is the case as suggested by the prevalence of mtDNAmutations in almost all types of cancers (1, 3). Thus, it wasessential to testwhether ectopic expression of p53 could restore�IR sensitivity in Res� cells. Therefore, the Res� B2 and G18

cells were transfected with wild type human p53-GFP, and thesensitivity to �IR-induced death was examined. The p53�/�

MEFswere used as positive controls for the rescue experiments.Although p53-GFP rescued the �IR sensitivity in p53�/�MEFs,no rescue was evident in p53-GFP transfected complex I-defi-cient B2 andG18 cells (Fig. 8A). As expected, the �IR sensitivityin MEFs correlated with the restoration of p53 activity,although no activity was detected in the p53-transfected Res�

(B2 and G18) cells (Fig. 8B). These data along with those in Fig.6 confirm that without restoring the ETC/RC function, it is notpossible to rescue p53 function in the presence of mitochon-drial dysfunction. These results also support our hypothesisthat mitochondrial ETC/RC activity is an important upstreamregulator of the tumor suppressor protein p53.Effect of Antioxidants on p53 Activity and Radiation Sensitiv-

ity of Res� Cells—The protection by rotenone, which increasesmitochondrial superoxide production in cells (supplementalFig. S3F) (48) suggested that pre-existing oxidative stress mayplay a role in the protection against �IR-induced death. Todetermine the role of oxidative/redox stress in p53 suppressionand �IR sensitivity, we monitored the ability of N-acetylcys-teine (NAC) to alleviate glutathione redox stress, and butylhy-droxytoluene (BHT) andMnTE-2-PyP to scavenge peroxyradi-cals and superoxide, respectively (48, 61, 62). Cells were treatedwith the indicated antioxidants at least 1 h prior to �IR, andtheir effects on cell death and p53 activity were determined asdescribed under “Experimental Procedures.” Data in Fig. 9clearly show that these treatments did not make any significantdifference in �IR sensitivity and p53 activity in Res� B2 and G7cells. Both the B2 and G7 cells have reduced p53 levels/activity(Fig. 4,A and B). The outcome was not any different in the G18cells, which show normal basal level of p53 (data not shown andsee Fig. 4B). If the oxidative/redox stresses were the underlyingcause for p53 suppression in Res� cells, then treatments withNAC, BHT, and MnTE-2-PyP could have restored the p53activity and �IR-sensitivity of Res� cells. The lack of reversal of

FIGURE 7. Effect of chloramphenicol on �IR sensitivity of cells. A, protec-tion of the �IR-sensitive G3 (Res�) cells by the chloramphenicol, a mitochon-drial protein synthesis inhibitor. Cells were pretreated with 50 �g/ml chlor-amphenicol for 24 and 48 h and then �IR-treated (40 Gy) with thechloramphenicol present in the medium. Cell death was assayed 24 h later.B, chloramphenicol addition (48 h prior to �IR) reduced the p53 protein levelin G3 cells comparable with that seen in G7 cells. The total p53 level wasmonitored using the DO1 antibody, while using actin as the loading control.Western blot for G3 cells corresponding to the 48 h data set in A are shown.Ctr, untreated controls; IR, �IR-treated; Chl, chloramphenicol treated; Chl�IR,chloramphenicol plus �IR-treated.

FIGURE 8. Ectopic expression of p53 does not restore the �IR sensitivity in Res� cells. A, relative rescues of the �IR sensitivity in Res� (B2 and G18) cells andp53�/� MEFs. GFP positive % live cells are reported with nonirradiated controls set to 100%. B, relative level of p53 activity rescue in Res� (B2 and G18) cells andp53�/� MEFs. Transiently (24 h) co-transfected cells with the p53-GFP and p53 activity reporter were �IR-treated and analyzed for relative levels of death asdescribed under “Experimental Procedures.” Cells were treated in triplicate from which the two sets were used for the determination of cell death (24 h post-�IR(40 Gy), A), and the remaining set was used for the p53 activity assay (6 h post-�IR (10 Gy), B). The p53�/� MEFs from C57BL/6 mice were used as positive controlsto demonstrate the rescue of the �IR sensitivity and p53 activity. Ctr, non-�IR-treated transfected cells; IR, �IR-treated transfected cells.




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�IR sensitivity correlatedwith the lack p53 activity (Fig. 9,C andF) and protein recovery (supplemental Fig. S3G) by NAC, BHT,andMnTE-2-PyP treatments. At lower doses (10 Gy), althoughthe B2-MWFE cells were not protected by antioxidants, the G3cells were partially protected (Fig. 9D). The reason for this dif-ference between the B2-MWFE and G3 cells is not clear. Bothcells show increased superoxide production following �IRtreatment that can be quenched by 25 �M MnTE-2-PyP (sup-plemental Fig. S3, A and D). The superoxide production wasmonitored using flow cytometry withMitoSox that reports mito-chondrial superoxide (48, 49). There were expected differencesbetween theRes� cells. Although inG7 cells no increase in super-oxide production following �IR was noticed, there was a signifi-

cant increase in B2 and G18 cells (supplemental Fig. S3, B, C, andE). However, this increase was relatively modest compared withcontrol cells. Because G7 cells have combined defects of com-plexes I and III–V, theywere not expected to produce superoxide.However, in B2 andG18 cells with isolated complex I deficiencies,superoxide generation may be possible by the ETC/RC down-stream to complex II or by the partial assembly of a complex Iperipheral arm (63). At this stage, it is unclear whether theincreased superoxide production in B2 and G18 cells follow-ing �IR is coming from partial complex I assemblies or theETC/RC downstream to complex II. The failure of MnTE-2-PyP to protect the control cells while successfully quenchingthe superoxide production suggests that lower mitochon-

FIGURE 9. Effect of antioxidants on �IR sensitivity and p53 activity. A–C, antioxidant effects on �IR sensitivity (A and B) and p53 activity (C) of B2-MWFEversus B2 cells. D–F, antioxidant effects on �IR sensitivity (D and E) and p53 activity (F) of G3 versus G18 cells. Cells were treated with 30 mM NAC, 200 �M BHT,or 25 �M MnTE-2-PyP 2.5 h before �IR. Cell death was monitored 24 h post-IR at 10 Gy (A and D) or 40 Gy (B and E). p53 activity was monitored 6 h post-IR asdescribed under “Experimental Procedures.”




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drial superoxide production in Res� cells following �IR orpre-existing oxidative stress is not the underlying cause fortheir resistance.

DISCUSSION

Mitochondrial Dysfunction Impairs p53-mediated CellDeath—Mitochondrial dysfunction confers opposing cell fatesin cancer versus degenerative diseases. Althoughmitochondrialdysfunction predisposes cells to death in degenerative diseases,it provides survival advantages to cancer cells. This suggeststhat mitochondrial dysfunction elicits different responsesunder different pathophysiological conditions. We haverecently shown that even minimal impairments of complex Ican lead to neuronal death under excitotoxic stress with highbioenergetic demand by causing bioenergetic deficit (48). Inthis study we have tested how mitochondrial dysfunction canaffect cell fate under a genotoxic stress, such as �IR in mitoticcells. Our data suggest that pre-existing mitochondrial dys-function protects cells from �IR-induced death by suppressingp53 expression/function (Figs. 1 and 2). The cellular protectionin the presence of suppressed p53 is not surprising as p53 is akey player in radiation-induced cell death (Fig. 3) (45) and cellscan meet their bioenergetic demands solely via glycolysis (39,40). The level of cellular protection is increased with the degreeof ETC/RC impairment. It is mediated either by the down-reg-ulation of p53 protein expression (B2 and G7 cells) or function(G18 and rotenone-treated Res� cells) (Fig. 4). To our knowl-edge this is the first study describing reversible suppression ofp53 expression/function by complex I deficiencies (Fig. 6). Thisstudy provides compelling evidence that mitochondrial metab-olism can indeed regulate p53, and it completes a regulatoryfeedback loop in associationwith positive regulation ofOxPhosby p53 via complex IV assembly factor SCO2 (30). This may bethe reason why p53 has to make sure that mitochondria func-tion normally by its recently describedmtDNA repair activities(64–66).Our data showing resistance to �IR-induced death is in

agreement with the decreased susceptibility to staurosporine-induced death in cells without mtDNA as shown by Dey andMoraes (67). Impaired ETC, particularly at complexes I and II,is also found to protect against the tumor necrosis factor-in-duced cell death (61, 68). However, the protection offered byrotenone is in disagreement with a few studies showingincreased cell death (autophagy or apoptosis) (55, 69, 70). Thesedifferences could be due to the experimental conditions used.Although in our studywe have used rotenone in lownanomolarconcentrations with a secondary genotoxic stress, others havetreated cells chronically with rotenone without a secondarystress. Someof these studies have used higher doses of rotenonethat affect cell death (non-neuronal) by impairing microtubuleassembly (55, 70) and others have used neuronal cells that maydiffer in cellular physiology (69). This is supported by the obser-vation that chronic rotenone exposure still potentiates death indopaminergic neurons in the absence of a functional complex Ieven in low nanomolar range (71). It is noteworthy that none ofthese studies examined p53 expression/function followingrotenone treatments. Thus, our studies described here provide

a novel insight into the mechanisms by which mitochondrialdysfunction can induce pathophysiology.Our knowledge of how ETC/RC impairments regulate p53

expression/function is limited (34). As the p53 is considered animportant player in regulating cellular oxidative/redox stressand itself being a redox-regulated protein (72), it is expectedthat ETC/RC impairmentsmay affect the p53 expression/func-tion. A few studies have shown the opposite effect of ETC/RCinhibition on p53 expression/function (73, 74). It is found thatcomplex I inhibition up-regulates p53 in dopaminergic neurons(73), although in MCF-7 cancer cells, it blocks p53 up-regula-tion in response to hypoxia (74). However, a recent study foundthat only complex III inhibitors could induce p53 via impairingpyrimidine biosynthesis (in the absence of secondary stress),which does not depend on the functional status of the ETC/RC(75). In our study, the complex I deficiencies induced either byrotenone treatment or by genetic mutations clearly suppressthe p53 induction following a genotoxic stress (Figs. 4 and 5).These studies clearly establish that ETC/RC function is animportant regulator of the p53 expression/function thatmay berelevant to tumorigenesis. This is also supported by the down-regulation of p53 protein in mammary epithelial cells lackingmtDNA (MCF12A�°) with mitochondrial dysfunction (76).The comparable level of protection by mitochondrial dys-

function and p53 loss suggests that all apoptotic functions ofp53 (transcription-dependent and -independent) could becompromised bymitochondrial ETC/RC defects (Figs. 3 and 5)(27, 29, 77). Although at this stage we do not know the exactmechanisms by which p53 is inactivated by mitochondrial dys-function, a multitude of possibilities exists (see below). At leastin B2 cells, the reduced level of p53 protein is at the post-tran-scriptional level as we did not find any reduction in p53 mRNAexpression (data not shown). However, in G7 (Res�) or chlor-amphenicol-treated (Res�) cells this possibility is not excluded.How the loss of the MWFE and ESSS subunits differentiallyregulates p53 protein expression is an interesting observationthat will be followed up in future studies using moleculargenetic approaches.The failure ofG18 cells to activate p53 following�IR suggests

that one or more steps involved in p53 activation are impaireddue to ETC/RC deficiency. This seems plausible as p53 activa-tion is regulated by a number of interdependent post-transla-tional modifications (78, 79). The analysis of differential andreversible post-translation modifications of p53 in response toETC/RC deficiencies is an interesting topic, which we believeshould be followed in detail in a separate investigation. Thesemodifications are expected to be different from the Ser-15phosphorylation, which did not correlate well with p53 activityin conjunction with mitochondrial impairments (data notshown).As ETC/RC deficiencies are often found associated with

increased oxidative/redox stresses, inRes� cells they could havebeen the underlying cause for p53 suppression and �IR resis-tance. However, our data suggest that the oxidative/redoxstress is not associated with the radioresistance of Res� cellsand p53 suppression (Fig. 9). This leaves open two other majorpossibilities that may influence the p53 expression/function.First, after �IR an acute bioenergetic deficit may affect the p53




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function. A role for the ATP/ADP switch in regulating the p53function has been suggested by an in vitro study (80). Second,mitochondrial membrane potential (��m) may play a role inregulating the intracellular ionic (e.g.Ca2�) homeostasis that inturn could affect p53 expression/function (81–83). Becausep53 is a Zn2�-binding protein (84), it would be interesting toinvestigate whether ETC/RC deficiencies can affect p53 func-tion by deregulating Zn2� homeostasis. Intracellular regulationof another ion, Cu�, has been shown to affect the p53 function(85). Furthermore, it has been recently observed that ��m canaffect the p53 function at themitochondria (86). Irrespective ofhow p53 function is regulated, it is important to note that tran-scriptional activity of p53 is an important read-out for overallapoptotic functions of p53, because the DNA-binding domainis implicated in its direct action on mitochondria (28). This isprobably via controlling p53 conformation (84, 85). Becausethese possibilities (ATP deficit and ionic homeostasis) areinterlinked with ��m (87), it requires a separate investigationwith real time measurements. We plan to address these issuesby testing cell-permeable substrates to bypass complex I forgenerating ��m and then determining the recovery of p53expression/function. Toward this end, we have promisingresults with monomethyl succinate that rescues the �IR sensi-tivity in complex I-deficient B2 andG18 cells but not inG7 cells,which have defects in complex I and III–V (data not shown).Monomethyl succinate feeds electrons to complex II in intactcells by elevating intracellular succinate level.Contrary to what one would simply expect, a transient

decline in ATP level is observed only in Res� cells (supplemen-tal Fig. S4). This decline could be either a consequence of theactivation of the apoptotic program or perturbed ionic home-ostasis in the presence of functional ETC/RC (81, 88). Althoughin Res� cells the ATP level declines by 60–70%, no decline isobserved inRes� cells suggesting that glycolysis alone can com-pensate for ATP demand. Moreover, in Res� cells the ATPdemandmay be lower as they are protected because of suppres-sion of the p53 (this study) and dysfunctional ETC/RC, whichmay prevent major changes in ionic homeostasis following �IRand thus cost less bioenergetically (81). The transient nucleo-tide (ATP) depletion may be a key requirement in the progres-sion of apoptosis as suggested previously (88). Our data pointtoward the possibility that functional ETC/RC may play a sig-nificant role in this phenomenon apart from regulating p53expression/function via a dynamic interplay between cellularbioenergetics and Ca2� or other ions homeostasis as suggestedabove.Cancer, Aging, and Mitochondrial Dysfunction—The data

presented in this study show how mitochondrial dysfunctioncan affect p53 expression/function and its effect on �IR-in-duced cell death. These findings may have serious implicationsas the p53 pathway is most frequently impaired in cancerseither due to mutations in the p53 gene itself or indirectlythrough proteins that modulate its activity (27). In relation tocancer development, it is expected thatmitochondrial dysfunc-tion would down-regulate p53 expression/function to relievethe suppression of glycolysis by its target genes, e.g.TIGAR (31,32). Although we have not measured the expression of TIGARin Res� cells, its up-regulation is plausible. Without negative

feedback regulation of p53, the cells with severe mitochondrialdysfunction would experience bioenergetic deficits because ofthe repression of glycolysis by p53. The metabolic switch fromOxPhos to glycolysis in the presence of reduced p53 expressionsupports the assumption that the p53 suppression is a viableoption tomeet the bioenergetic needs of cells (89). Thus, de-re-pression of glycolysis may be essential in mitotic cells, whichmay have limited OxPhos capacities (the ability to make ATP,as measured by ADP-stimulated respiration). Our studies sug-gest that in many fibroblasts, there is a very little spare OxPhoscapacity compared with ETC/RC capacity (measured by proto-nophore FCCP stimulation).4 This observation in associationwith the acute decline of ATP in normal cells (supplementalFig. S4) suggests that ETC/RC activity may transiently increasethe ATP demand following �IR by altering the ionic homeosta-sis as suggested above.As tumorigenesis is a multistep process involving four to

seven rate-limiting, stochastic events that determine age-de-pendent incidence (90), it is possible that functional mitochon-dria can play an active role in suppressing tumorigenesis bykeeping p53 functional (this study). This view is supported bystudies that demonstrate that mitochondrial dysfunction caninitiate molecular and biochemical changes leading to gain ofseveral phenotypic hallmarks of cancer as follows: specificallyby (i) inhibiting tumor suppressors PTEN (24) and p53 (thisstudy); (ii) activating oncogenic pathways such as AKT1 andHIF1� (24, 91–93); (iii) inducing extracellular matrix remodel-ing to allow invasiveness (94); (iv) conferring resistance to celldeath (67); and (v) forcing metabolic shift toward glycolysis(95). Thus, in light of perturbations in cellular physiology/sig-naling by ETC/RC defects, it is anticipated that mitochondrialdysfunction could be tumorigenic, whereas normal mitochon-drial function would maintain the regulatory surveillance byp53 (this study) in response to oncogenic stress and preventadaptive changes that could favor cancer development. Thus,this study supports the current view that functional mitochon-dria could act as tumor suppressors via regulating p53 expres-sion/function. Conversely, the defective ETC/RC can promotetumorigenesis as suggested by a few recent studies (76, 96). Theimpairment of p53 (this study) along with the activation ofAKT1 by mitochondrial dysfunction may also be relevant tochemoresistance that is very often observed during cancer ther-apy (97, 98).Considering that age is a major risk factor for tumorigenesis

(99) and mitochondrial function declines with age (100), it ispossible that mitochondrial dysfunction plays a key role intumor initiation as originally suggested by Otto Warburg in1956 (33).Warburg proposed that an “irreversible injury” to theETC/RC was an initiating event in cancer development. It isnoteworthy that like mitochondrial function, p53 function alsodeclines with age by an unknown mechanism (58). From thisperspective it would be interesting to explore the role of mito-chondrial dysfunction preceding p53 functional decline as theunderlying cause to explain the increased incidence of tumori-genesis with age.

4 C. Kim, unpublished results.




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Acknowledgments—We thank Amir Misaghi, Arturo Orjalo, JustyneOgdahl, Matthew Carter, Pinal Patel, Maureen Lahti, and Dr. KellyGauger for their technical assistance and help.

REFERENCES1. Brandon, M., Baldi, P., and Wallace, D. C. (2006) Oncogene 25,

4647–46622. Wallace, D. C. (2005) Annu. Rev. Genet. 39, 359–4073. Modica-Napolitano, J. S., Kulawiec, M., and Singh, K. K. (2007) Curr.

Mol. Med. 7, 121–1314. Tucker, E. J., Compton, A. G., and Thorburn, D. R. (2010) Curr. Neurol.

Neurosci. Rep. 10, 277–2855. Gasparre, G., Porcelli, A. M., Bonora, E., Pennisi, L. F., Toller, M., Iom-

marini, L., Ghelli, A., Moretti, M., Betts, C. M., Martinelli, G. N., Ceroni,A. R., Curcio, F., Carelli, V., Rugolo,M., Tallini, G., and Romeo, G. (2007)Proc. Natl. Acad. Sci. U.S.A. 104, 9001–9006

6. Mayr, J. A., Meierhofer, D., Zimmermann, F., Feichtinger, R., Kogler, C.,Ratschek,M., Schmeller, N., Sperl,W., and Kofler, B. (2008)Clin. CancerRes. 14, 2270–2275

7. Zhou, S., Kachhap, S., Sun,W.,Wu, G., Chuang, A., Poeta, L., Grumbine,L., Mithani, S. K., Chatterjee, A., Koch, W., Westra, W. H., Maitra, A.,Glazer, C., Carducci, M., Sidransky, D., McFate, T., Verma, A., and Cali-fano, J. A. (2007) Proc. Natl. Acad. Sci. U.S.A. 104, 7540–7545

8. Kulawiec, M., Owens, K. M., and Singh, K. K. (2009) J. Hum. Genet. 54,647–654

9. Theodoratou, E., Din, F. V., Farrington, S. M., Cetnarskyj, R., Barnetson,R. A., Porteous, M. E., Dunlop, M. G., Campbell, H., and Tenesa, A.(2010) Carcinogenesis 31, 296–301

10. Czarnecka, A. M., Klemba, A., Krawczyk, T., Zdrozny, M., Arnold, R. S.,Bartnik, E., and Petros, J. A. (2010) Oncol. Rep. 23, 531–535

11. Czarnecka, A. M., Krawczyk, T., Zdrozny, M., Lubinski, J., Arnold, R. S.,Kukwa, W., Scinska, A., Golik, P., Bartnik, E., and Petros, J. A. (2010)Breast Cancer Res. Treat. 121, 511–518

12. Ray, A. M., Zuhlke, K. A., Levin, A. M., Douglas, J. A., Cooney, K. A., andPetros, J. A. (2009) Prostate 69, 956–960

13. Baysal, B. E.,Willett-Brozick, J. E., Lawrence, E. C., Drovdlic, C.M., Savul,S. A., McLeod, D. R., Yee, H. A., Brackmann, D. E., Slattery, W. H., 3rd,Myers, E. N., Ferrell, R. E., and Rubinstein,W. S. (2002) J.Med. Genet. 39,178–183

14. Baysal, B. E., Ferrell, R. E., Willett-Brozick, J. E., Lawrence, E. C., Myssi-orek, D., Bosch, A., van der Mey, A., Taschner, P. E., Rubinstein, W. S.,Myers, E. N., Richard, C.W., 3rd, Cornelisse, C. J., Devilee, P., andDevlin,B. (2000) Science 287, 848–851

15. Mamelak, A. J., Kowalski, J.,Murphy, K., Yadava,N., Zahurak,M., Kouba,D. J., Howell, B. G., Tzu, J., Cummins, D. L., Liegeois, N. J., Berg, K., andSauder, D. N. (2005) Exp. Dermatol. 14, 336–348

16. Cuezva, J.M., Sanchez-Arago,M., Sala, S., Blanco-Rivero, A., andOrtega,A. D. (2007) J. Bioenerg. Biomembr. 39, 259–265

17. Gottlieb, E., and Tomlinson, I. P. (2005) Nat. Rev. Cancer 5, 857–86618. Cuezva, J. M., Ortega, A. D., Willers, I., Sanchez-Cenizo, L., Aldea, M.,

and Sanchez-Arago, M. (2009) Biochim. Biophys. Acta 1792, 1145–115819. Zanssen, S., and Schon, E. A. (2005) PLoS. Med. 2, e40120. Selak, M. A., Armour, S. M., MacKenzie, E. D., Boulahbel, H., Watson,

D. G., Mansfield, K. D., Pan, Y., Simon, M. C., Thompson, C. B., andGottlieb, E. (2005) Cancer Cell 7, 77–85

21. King, A., Selak, M. A., and Gottlieb, E. (2006) Oncogene 25, 4675–468222. Pollard, P. J., Briere, J. J., Alam, N. A., Barwell, J., Barclay, E., Wortham,

N. C., Hunt, T., Mitchell, M., Olpin, S., Moat, S. J., Hargreaves, I. P.,Heales, S. J., Chung, Y. L., Griffiths, J. R., Dalgleish, A., McGrath, J. A.,Gleeson, M. J., Hodgson, S. V., Poulsom, R., Rustin, P., and Tomlinson,I. P. (2005) Hum. Mol. Genet. 14, 2231–2239

23. Tomlinson, I. P., Alam, N. A., Rowan, A. J., Barclay, E., Jaeger, E. E.,Kelsell, D., Leigh, I., Gorman, P., Lamlum,H., Rahman, S., Roylance, R. R.,Olpin, S., Bevan, S., Barker, K., Hearle, N., Houlston, R. S., Kiuru, M.,Lehtonen, R., Karhu, A., Vilkki, S., Laiho, P., Eklund, C., Vierimaa, O.,Aittomaki, K., Hietala, M., Sistonen, P., Paetau, A., Salovaara, R., Herva,

R., Launonen, V., and Aaltonen, L. A. (2002) Nat. Genet. 30, 406–41024. Pelicano, H., Xu, R. H., Du, M., Feng, L., Sasaki, R., Carew, J. S., Hu, Y.,

Ramdas, L., Hu, L., Keating,M. J., Zhang,W., Plunkett,W., andHuang, P.(2006) J. Cell Biol. 175, 913–923

25. Mayo, L. D., Dixon, J. E., Durden, D. L., Tonks, N. K., and Donner, D. B.(2002) J. Biol. Chem. 277, 5484–5489

26. Joerger, A. C., and Fersht, A. R. (2008) Annu. Rev. Biochem. 77, 557–58227. Green, D. R., and Kroemer, G. (2009) Nature 458, 1127–113028. Mihara, M., Erster, S., Zaika, A., Petrenko, O., Chittenden, T., Pancoska,

P., and Moll, U. M. (2003)Mol. Cell 11, 577–59029. Chipuk, J. E., Kuwana, T., Bouchier-Hayes, L., Droin, N. M., Newmeyer,

D. D., Schuler, M., and Green, D. R. (2004) Science 303, 1010–101430. Matoba, S., Kang, J. G., Patino, W. D., Wragg, A., Boehm, M., Gavrilova,

O., Hurley, P. J., Bunz, F., and Hwang, P. M. (2006) Science 312,1650–1653

31. Bensaad, K., Tsuruta, A., Selak,M. A., Vidal,M. N., Nakano, K., Bartrons,R., Gottlieb, E., and Vousden, K. H. (2006) Cell 126, 107–120

32. Vousden, K. H., and Ryan, K. M. (2009) Nat. Rev. Cancer 9, 691–70033. Warburg, O. (1956) Science 123, 309–31434. Holley, A. K., and St Clair, D. K. (2009) Future Oncol. 5, 117–13035. Yadava, N., Potluri, P., and Scheffler, I. E. (2008) Int. J. Biochem. Cell Biol.

40, 447–46036. Scheffler, I. E., Yadava, N., and Potluri, P. (2004) Biochim. Biophys. Acta

1659, 160–17137. Potluri, P., Yadava, N., and Scheffler, I. E. (2004) Eur. J. Biochem. 271,

3265–327338. Yadava, N., Houchens, T., Potluri, P., and Scheffler, I. E. (2004) J. Biol.

Chem. 279, 12406–1241339. DeFrancesco, L.,Werntz, D., and Scheffler, I. E. (1975) J. Cell. Physiol. 85,

293–30540. Donnelly, M., and Scheffler, I. E. (1976) J. Cell. Physiol. 89, 39–5141. Scheffler, I. E., and Yadava, N. (2001) J. Bioenerg. Biomembr. 33, 243–25042. Yadava, N., Potluri, P., Smith, E. N., Bisevac, A., and Scheffler, I. E. (2002)

J. Biol. Chem. 277, 21221–2123043. Norris, P. S., and Haas, M. (1997) Oncogene 15, 2241–224744. Lu, S., Becker, K. A., Hagen,M. J., Yan, H., Roberts, A. L., Mathews, L. A.,

Schneider, S. S., Siegelmann, H. T., MacBeth, K. J., Tirrell, S. M., Blan-chard, J. L., and Jerry, D. J. (2008) Endocrinology 149, 4809–4820

45. Lowe, S. W., Schmitt, E. M., Smith, S. W., Osborne, B. A., and Jacks, T.(1993) Nature 362, 847–849

46. Blackburn, A. C., McLary, S. C., Naeem, R., Luszcz, J., Stockton, D. W.,Donehower, L. A., Mohammed,M.,Mailhes, J. B., Soferr, T., Naber, S. P.,Otis, C. N., and Jerry, D. J. (2004) Cancer Res. 64, 5140–5147

47. Gerencser, A. A., Neilson, A., Choi, S. W., Edman, U., Yadava, N., Oh,R. J., Ferrick, D. A., Nicholls, D. G., and Brand, M. D. (2009) Anal. Chem.81, 6868–6878

48. Yadava, N., and Nicholls, D. G. (2007) J. Neurosci. 27, 7310–731749. Johnson-Cadwell, L. I., Jekabsons, M. B., Wang, A., Polster, B. M., and

Nicholls, D. G. (2007) J. Neurochem. 101, 1619–163150. Gauger, K. J., Hugh, J. M., Troester, M. A., and Schneider, S. S. (2009)

Cancer Cell Int. 9, 1151. Schmittgen, T. D., and Livak, K. J. (2008) Nat. Protoc. 3, 1101–110852. Yang, N. C., Ho,W.M., Chen, Y. H., andHu,M. L. (2002)Anal. Biochem.

306, 323–32753. Chen, R., Fearnley, I.M., Peak-Chew, S. Y., andWalker, J. E. (2004) J. Biol.

Chem. 279, 26036–2604554. Ditta, G., Soderberg, K., and Scheffler, I. E. (1977) Nature 268, 64–6755. Barrientos, A., andMoraes, C. T. (1999) J. Biol. Chem. 274, 16188–1619756. Erster, S., Mihara, M., Kim, R. H., Petrenko, O., and Moll, U. M. (2004)

Mol. Cell. Biol. 24, 6728–674157. Christophorou, M. A., Martin-Zanca, D., Soucek, L., Lawlor, E. R.,

Brown-Swigart, L., Verschuren, E. W., and Evan, G. I. (2005)Nat. Genet.37, 718–726

58. Feng, Z., Hu, W., Teresky, A. K., Hernando, E., Cordon-Cardo, C., andLevine, A. J. (2007) Proc. Natl. Acad. Sci. U.S.A. 104, 16633–16638

59. Ugalde, C., Vogel, R., Huijbens, R., Van Den Heuvel, B., Smeitink, J., andNijtmans, L. (2004) Hum. Mol. Genet. 13, 2461–2472

60. Meulmeester, E., and Jochemsen, A. G. (2008)Curr. Cancer Drug Targets




http://ww

w.jbc.org/

Dow

nloaded from

http://www.jbc.org/

8, 87–9761. Schulze-Osthoff, K., Bakker, A. C., Vanhaesebroeck, B., Beyaert, R., Ja-

cob, W. A., and Fiers, W. (1992) J. Biol. Chem. 267, 5317–532362. Hockenbery, D. M., Oltvai, Z. N., Yin, X. M., Milliman, C. L., and Kors-

meyer, S. J. (1993) Cell 75, 241–25163. Au, H. C., Seo, B. B.,Matsuno-Yagi, A., Yagi, T., and Scheffler, I. E. (1999)

Proc. Natl. Acad. Sci. U.S.A. 96, 4354–435964. Achanta, G., Sasaki, R., Feng, L., Carew, J. S., Lu, W., Pelicano, H., Keat-

ing, M. J., and Huang, P. (2005) EMBO J. 24, 3482–349265. de Souza-Pinto, N. C., Harris, C. C., and Bohr, V. A. (2004)Oncogene 23,

6559–656866. Lebedeva, M. A., Eaton, J. S., and Shadel, G. S. (2009) Biochim. Biophys.

Acta 1787, 328–33467. Dey, R., and Moraes, C. T. (2000) J. Biol. Chem. 275, 7087–709468. Schulze-Osthoff, K., Beyaert, R., Vandevoorde, V., Haegeman, G., and

Fiers, W. (1993) EMBO J. 12, 3095–310469. Sherer, T. B., Betarbet, R., Testa, C. M., Seo, B. B., Richardson, J. R., Kim,

J. H., Miller, G. W., Yagi, T., Matsuno-Yagi, A., and Greenamyre, J. T.(2003) J. Neurosci. 23, 10756–10764

70. Chen, Y., McMillan-Ward, E., Kong, J., Israels, S. J., and Gibson, S. B.(2007) J. Cell Sci. 120, 4155–4166

71. Choi, W. S., Kruse, S. E., Palmiter, R. D., and Xia, Z. (2008) Proc. Natl.Acad. Sci. U.S.A. 105, 15136–15141

72. Liu, B., Chen, Y., and St Clair, D. K. (2008) Free Radic. Biol. Med. 44,1529–1535

73. Perier, C., Tieu, K., Guegan, C., Caspersen, C., Jackson-Lewis, V., Carelli,V., Martinuzzi, A., Hirano, M., Przedborski, S., and Vila, M. (2005) Proc.Natl. Acad. Sci. U.S.A. 102, 19126–19131

74. Chandel, N. S., Vander Heiden, M. G., Thompson, C. B., and Schu-macker, P. T. (2000) Oncogene 19, 3840–3848

75. Khutornenko, A. A., Roudko, V. V., Chernyak, B. V., Vartapetian, A. B.,Chumakov, P. M., and Evstafieva, A. G. (2010) Proc. Natl. Acad. Sci.U.S.A. 107, 12828–12833

76. Kulawiec,M., Safina, A., Desouki,M.M., Still, I.,Matsui, S., Bakin, A., andSingh, K. K. (2008) Cancer Biol. Ther. 7, 1732–1743

77. Mihara, M., and Moll, U. M. (2003)Methods Mol. Biol. 234, 203–20978. Kenzelmann Broz, D., and Attardi, L. D. (2010) Carcinogenesis 31,

1311–131879. Saito, S., Yamaguchi, H., Higashimoto, Y., Chao, C., Xu, Y., Fornace, A. J.,

Jr., Appella, E., and Anderson, C. W. (2003) J. Biol. Chem. 278,37536–37544

80. Okorokov, A. L., and Milner, J. (1999)Mol. Cell. Biol. 19, 7501–751081. Distelmaier, F., Koopman, W. J., van den Heuvel, L. P., Rodenburg, R. J.,

Mayatepek, E., Willems, P. H., and Smeitink, J. A. (2009) Brain 132,

833–84282. Metcalfe, S., Weeds, A., Okorokov, A. L., Milner, J., Cockman, M., and

Pope, B. (1999) Oncogene 18, 2351–235583. Lau, D., and Bading, H. (2009) J. Neurosci. 29, 4420–442984. Verhaegh, G.W., Parat,M.O., Richard,M. J., andHainaut, P. (1998)Mol.

Carcinog. 21, 205–21485. Verhaegh, G. W., Richard, M. J., and Hainaut, P. (1997) Mol. Cell. Biol.

17, 5699–570686. Wang, F., Fu, X., Chen, X., Chen, X., and Zhao, Y. (2010) PLoS. ONE 5,

e1345987. Nicholls, D. G. (2010) Essays Biochem. 47, 25–3588. Chandra, D., Bratton, S. B., Person, M. D., Tian, Y., Martin, A. G., Ayres,

M., Fearnhead, H. O., Gandhi, V., and Tang, D. G. (2006) Cell 125,1333–1346

89. Burns, D. M., and Richter, J. D. (2008) Genes Dev. 22, 3449–346090. Hanahan, D., and Weinberg, R. A. (2000) Cell 100, 57–7091. Porcelli, A.M.,Ghelli, A., Ceccarelli, C., Lang,M., Cenacchi, G., Capristo,

M., Pennisi, L. F., Morra, I., Ciccarelli, E., Melcarne, A., Bartoletti-Stella,A., Salfi, N., Tallini, G., Martinuzzi, A., Carelli, V., Attimonelli, M., Ru-golo, M., Romeo, G., and Gasparre, G. (2010) Hum. Mol. Genet. 19,1019–1032

92. Sun,W., Zhou, S., Chang, S. S., McFate, T., Verma, A., and Califano, J. A.(2009) Clin. Cancer Res. 15, 476–484

93. Briere, J. J., Favier, J., Benit, P., El Ghouzzi, V., Lorenzato, A., Rabier, D.,Di Renzo, M. F., Gimenez-Roqueplo, A. P., and Rustin, P. (2005) Hum.Mol. Genet. 14, 3263–3269

94. van Waveren, C., Sun, Y., Cheung, H. S., and Moraes, C. T. (2006) Car-cinogenesis 27, 409–418

95. Acebo, P., Giner, D., Calvo, P., Blanco-Rivero, A., Ortega, A. D., Fernan-dez, P. L., Roncador, G., Fernandez-Malave, E., Chamorro, M., andCuezva, J. M. (2009) Transl. Oncol. 2, 138–145

96. Petros, J. A., Baumann, A. K., Ruiz-Pesini, E., Amin, M. B., Sun, C. Q.,Hall, J., Lim, S., Issa, M. M., Flanders, W. D., Hosseini, S. H., Marshall,F. F., andWallace, D. C. (2005)Proc. Natl. Acad. Sci. U.S.A. 102, 719–724

97. Abedini, M. R., Muller, E. J., Bergeron, R., Gray, D. A., and Tsang, B. K.(2010) Oncogene 29, 11–25

98. Roberts, A. M., Watson, I. R., Evans, A. J., Foster, D. A., Irwin, M. S., andOhh, M. (2009) Cancer Res. 69, 9056–9064

99. Benz, C. C., and Yau, C. (2008) Nat. Rev. Cancer 8, 875–879100. Loeb, L. A.,Wallace, D. C., andMartin, G.M. (2005)Proc. Natl. Acad. Sci.

U.S.A. 102, 18769–18770101. Ditta, G., Soderberg, K., Landy, F., and Scheffler, I. E. (1976) Somatic Cell

Genet. 2, 331–344




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Sabyasachi Sen, D. Joseph Jerry, Sallie Schneider and Nagendra YadavaShannon Compton, Chul Kim, Nicholas B. Griner, Prasanth Potluri, Immo E. Scheffler,Mitochondrial Dysfunction Impairs Tumor Suppressor p53 Expression/Function

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MitochondrialDysfunctionImpairsTumorSuppressorp53 ... · PDF fileMitochondrialDysfunctionImpairsTumorSuppressorp53 Expression/Function* S Receivedforpublication,July23,2010,andinrevisedform,March29,2011