Parkin, a p53 target gene, mediates the role of p53 inglucose metabolism and the Warburg effectCen Zhanga, Meihua Lina, Rui Wua, Xiaowen Wanga, Bo Yangb, Arnold J. Levinec,d,1, Wenwei Huc, and Zhaohui Fenga,1

aDepartment of Radiation Oncology and cDepartment of Pediatrics, Cancer Institute of New Jersey, University of Medicine and Dentistry of New Jersey,New Brunswick, NJ 08903; bCollege of Pharmaceutical Sciences, Zhejiang University, Hangzhou 310058, China; and dInstitute for Advanced Study,Princeton, NJ 08540

Contributed by Arnold J. Levine, August 24, 2011 (sent for review June 8, 2011)

Regulation of energy metabolism is a novel function of p53 intumor suppression. Parkin (PARK2), a Parkinson disease-associatedgene, is a potential tumor suppressor whose expression is fre-quently diminished in tumors. Here Parkin was identified as ap53 target gene that is an important mediator of p53’s functionin regulating energy metabolism. The human and mouse Parkingenes contain functional p53 responsive elements, and p53increases the transcription of Parkin in both humans and mice.Parkin contributes to the function of p53 in glucose metabolism;Parkin deficiency activates glycolysis and reduces mitochondrialrespiration, leading to the Warburg effect. Restoration of Parkinexpression reverses the Warburg effect in cells. Thus, Parkin de-ficiency is a novel mechanism for the Warburg effect in tumors.Parkin also contributes to the function of p53 in antioxidantdefense. Furthermore, Parkin deficiency sensitizes mice to γ-irra-diation-induced tumorigenesis, which provides further direct evi-dence to support a role of Parkin in tumor suppression. Our resultssuggest that as a novel component in the p53 pathway, Parkincontributes to the functions of p53 in regulating energy metabo-lism, especially the Warburg effect, and antioxidant defense, andthus the function of p53 in tumor suppression.

Metabolic alterations are a hallmark of tumor cells (1, 2).Whereas normal cells use mitochondrial respiration to

provide energy, the majority of tumor cells preferentially useaerobic glycolysis, a switch known as the Warburg effect (3).Because glycolysis produces ATP much less efficiently thanmitochondrial respiration, tumor cells compensate by having amuch higher rate of glucose uptake and utilization than normalcells (1, 2). Recent studies have strongly suggested that theWarburg effect is a key contributor to malignant progression (1,2), and reversing the Warburg effect inhibits the tumorigenicityof cancer cells (4, 5). However, the underlying mechanisms forthe Warburg effect are not well-understood (1, 2).p53 plays a central role in tumor prevention. As a transcription

factor, in response to stress, p53 transcribes its target genes tostart various cellular responses, including cell-cycle arrest, apo-ptosis, and/or senescence, to prevent tumor formation (6, 7).Recent studies have revealed that regulating energy metabolismand the Warburg effect is a novel function of p53 in tumorsuppression (2, 8). p53 induces TIGAR (TP53-induced glycolysisand apoptosis regulator) to reduce glycolysis (9), and inducesSCO2 (10) and GLS2 (11, 12) to promote mitochondrial respi-ration. Loss of p53 results in decreased mitochondrial respirationand enhanced glycolysis, leading to the Warburg effect. Fur-thermore, regulating antioxidant defense has recently beenrevealed as another novel function for p53 (8, 13). p53 inducesseveral antioxidant genes, including Sestrins (14), TIGAR (9),ALDH4 (15), and GLS2 (11, 12), to reduce the levels of reactiveoxygen species (ROS) and DNA damage in cells, which con-tributes greatly to the role of p53 as a tumor suppressor.Parkin (PARK2) was first identified as a gene associated with

Parkinson disease (PD), a neurodegenerative disease. Mutations ofParkin account for most autosomal recessive forms of juvenileParkinson disease. Parkin deficiency leads to mitochondrial dys-function and enhanced oxidative stress in neuronal cells in Dro-sophila andmice, which are believed to contribute greatly to PD (16,

17). Recently, Parkin has been suggested to be a potential tumorsuppressor, and diminished expression and mutations of the Parkingene have been frequently observed in various tumors (18–22).However, the mechanisms by which Parkin contributes to tumorsuppression and the regulation of Parkin are not well-understood.Here we identified Parkin as a p53 target gene. p53 increases

the transcription of Parkin gene both in vitro and in vivo. Parkincontributes to p53’s role in regulating glucose metabolism andthe Warburg effect; Parkin deficiency results in the Warburgeffect, whereas restoration of Parkin expression reverses theWarburg effect in cells. Parkin also mediates p53’s role in anti-oxidant defense. These results suggest that the functions ofParkin in regulating energy metabolism and antioxidant defenseshould contribute greatly to Parkin’s role in tumor suppressionand, furthermore, contribute to p53’s role as a tumor suppressor.

ResultsHuman Parkin Gene Is a p53 Target Gene. As a transcription factor,p53 binds to the p53 responsive elements (REs) in its target genesto transcriptionally regulate their expression in response to stress(23). The p53MHalgorithm is a computational programdevelopedfor genome-wide scanning for potential p53 targets by identifyingputative p53REs in genes (24), which has been successfully appliedto identify new p53 targets (11, 25, 26). Using this algorithm, weidentified human Parkin gene as a potential p53 target.To investigate whether p53 transcriptionally regulates the

Parkin gene, Parkin expression was examined in various cell linesexposed to different stress signals. A pair of isogenic p53 wild-type and p53-deficient human lung cell lines, H460-con andH460-p53siRNA, which stably express a control vector and a p53shRNA vector in p53 wild-type H460 cells, respectively, were used.H460-p53siRNA cells displayed greatly decreased p53 proteinlevels and decreased induction of p53 target MDM2 in response toEtoposide and H2O2 compared with H460-con cells (Fig. 1A).Etoposide and H2O2 clearly induced Parkin expression in H460-con cells at both protein and mRNA levels (Fig. 1 A and B). Thisinduction is p53-dependent because no clear induction of Parkinwas observed inH460-p53siRNA cells. Furthermore, p53 increasedthe basal levels of Parkin under nonstressed conditions. In additionto these stress signals, Nutlin-3a, a nongenotoxic small moleculethat activates p53 through disruption of p53-MDM2 interaction(27), clearly induced Parkin expression in a p53-dependentmanner inH460 cells (Fig. 1C andD). The regulation of Parkin byp53 activation was also confirmed by immunofluorescence (IF)staining (Fig. 1E). Results in Fig. 1E further show that Parkin ismainly localized inmitochondria and cytoplasm, and not in nuclei.Similar results were observed in p53 wild-type HCT116-con andp53-deficient HCT116-p53siRNA (Fig. S1). These results to-

Author contributions: A.J.L., W.H., and Z.F. designed research; C.Z., M.L., R.W., and X.W.performed research; C.Z., M.L., R.W., X.W., B.Y., A.J.L., W.H., and Z.F. analyzed data; andA.J.L., W.H., and Z.F. wrote the paper.

The authors declare no conflict of interest.1To whom correspondence may be addressed. E-mail: alevine@ias.edu or fengzh@umdnj.edu.

This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1113884108/-/DCSupplemental.

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gether demonstrate that p53 increases Parkin expression underboth stressed and nonstressed conditions.

Human Parkin Gene Contains a Functional p53 Responsive Element.To investigate whether p53 regulates Parkin expression throughits direct binding to the two putative p53 REs in human Parkinpromoter region and intron 1 predicated by the p53MH algo-rithm (Fig. 2A), H460-con and H460-p53siRNA cells weretreated with Etoposide to activate p53, and chromatin immu-noprecipitation (ChIP) assays were performed. The anti-p53antibody specifically pulled down the DNA fragment containingthe putative p53 RE in Parkin intron 1 in H460-con cells treatedwith Etoposide, but not in H460-p53siRNA cells (Fig. 2B). Theseresults demonstrate that p53 protein physically interacts with theputative p53 RE in human Parkin intron 1 in vivo.To further investigate whether these two putative p53 REs

confer p53-dependent transcriptional activities, the DNA frag-

ments containing one copy of these two putative p53 REs wereinserted into the promoter of PGL2 luciferase reporter vector.p53 null human lung H1299 cells were cotransfected with thereporter vectors and a vector expressing either wild-type orR273H mutant p53. Compared with mutant p53, the expression ofwild-type p53 greatly enhanced luciferase activities of the reportervector containing the putative p53 RE in the Parkin intron 1 (by>20-fold), but not in the promoter region (by less than twofold)(Fig. 2C). Taken together, our data demonstrate that humanParkin gene is a p53 target gene; p53 binds to the p53 RE inParkin intron 1 and increases Parkin transcription in cells.

Mouse Parkin Gene Is a p53 Target Gene. The p53MH algorithmalso showed that mouse Parkin gene contains three putative p53REs in its promoter region, including RE A and two overlappedREs, B and C (Fig. 3A). This suggested that mouse Parkin genewas a potential p53 target. Two luciferase reporter vectors wereconstructed that contained a copy of RE A and REs B+C. Lu-ciferase reporter assays showed that the expression of wild-typep53 in p53 null H1299 cells and mouse embryonic fibroblasts(p53−/− MEF) clearly enhanced the luciferase activities of bothreporter vectors by approximately four- to sixfold compared withmutant p53 (Fig. 3B). Furthermore, H2O2 clearly induced Parkinexpression at both mRNA and protein levels in p53+/+ MEF butnot p53−/− MEF cells (Fig. 3 C and D). p53+/+ MEF cells alsodisplayed a higher level of basal Parkin expression than p53−/−MEF cells. These results demonstrate that mouse Parkin is a p53target gene; p53 increases Parkin transcription through the reg-ulation of p53 REs in Parkin promoter region.To investigate whether p53 activation induces Parkin expres-

sion in vivo, p53+/+ and p53−/− C57BL6/J mice were subjected towhole-body γ-irradiation (IR) (4 Gy). Parkin was clearly inducedat both mRNA and protein levels (by approximately four- tosixfold at 20 h after IR) in the spleen and thymus, two highly radio-sensitive tissues that display the clearest p53 responses to IR, inp53+/+ but not p53−/− mice (Fig. 3 E and F). The p53-dependentinduction of Parkin by IR appears to be tissue-specific becauseParkin was not induced in the cortex of brain, liver, or kidney.Together, these results demonstrate that the regulation of Parkinby p53 is evolutionarily conserved from mice to humans.

Parkin Contributes to the Function of p53 in Regulating GlucoseMetabolism and the Warburg Effect. p53 has been reported to re-duce glycolysis and promote mitochondrial respiration in cells.

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Fig. 1. p53 regulates the expression of human Parkin gene. H460-con andH460-p53siRNA cells were treated with Etoposide (Etp, 10 μM) or H2O2 (200μM) for different amounts of time. The protein levels were determined byWestern blot assays. mRNA levels of Parkin were measured by real-time PCRand normalized with actin. Data are presented as mean ± SD (n = 3). (A andB) p53 induces Parkin expression at both protein (A) and mRNA (B) levels inH460-con cells under both nonstressed and stressed conditions. (C and D) p53activation by Nutlin-3a (5 μM) induces Parkin expression at both protein (C)and mRNA (D) levels in cells. (E) p53 induces Parkin expression in H460-concells treated with Etoposide (10 μM for 24 h) as detected by IF staining.

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Fig. 2. p53 binds to and transactivates the p53 responsive element in hu-man Parkin gene. (A) The putative REs in human Parkin gene predicted bythe p53MH algorithm. Numbers indicate the nucleotide position relative tothe ATG site. (Upper) Sequences of the p53 RE. N, any nucleotide; Pu, purine;Py, pyrimidine. (B) ChIP analysis of interactions between p53 and the p53 REin human Parkin gene in cells. H460-con and H460-p53siRNA cells weretreated with Etoposide (10 μM for 16 h) before ChIP assays. Input DNA, 1/20DNA of ChIP. (C) p53 activates the luciferase reporter vector containing theputative p53 RE in intron 1 of human Parkin gene. Reporter vectors weretransfected into p53 null H1299 cells along with a wild-type (WT p53) orR273H mutant (Mut p53) p53 expression plasmid. The relative luciferaseactivity in cells cotransfected with mutant p53 was designated as 1. Data arepresented as mean ± SD (n = 3).

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p53 deficiency leads to the Warburg effect in tumors, which ischaracterized by higher glucose uptake, a higher rate of glycol-ysis, and higher lactate production in tumor cells than normalcells (9, 10). We found that Parkin, as a downstream target ofp53, contributes to the role of p53 in regulating glucose metab-olism and the Warburg effect in cells. As shown in Fig. 4 A–C,ectopic expression of Parkin in H460-p53siRNA cells signifi-cantly decreased glucose uptake, the rate of glycolysis, and lac-tate production. Furthermore, knockdown of endogenous Parkinin H460-con cells significantly enhanced glucose uptake, the rateof glycolysis, and lactate production. Similar effects of Parkin onthe Warburg effect were also observed in Parkin−/− MEF com-pared with Parkin+/+ MEF cells (Fig. 4 A–C, Right). Theseresults demonstrate that Parkin deficiency results in the Warburgeffect, whereas restoration of Parkin expression reverses theWarburg effect in cells. Furthermore, Parkin mediates the role ofp53 in glucose metabolism. As shown in Fig. 4D, p53 knockdownsignificantly enhanced glucose uptake, the rate of glycolysis, andlactate production in H460 cells. Simultaneous knockdown ofp53 and Parkin results in higher glucose uptake, rate of glycol-ysis, and lactate production compared with individual knock-down of p53 or Parkin in cells, but the effects are less thanadditive effects. These results suggest that Parkin is one of theimportant mediators for p53’s role in glucose metabolism.Parkin deficiency leads to mitochondrial dysfunction in neu-

ronal cells, which contributes to the development of PD (16).Therefore, the role of Parkin in preventing the Warburg effectcould be mainly due to its function in maintaining mitochondrialrespiration. Consistent with the role of p53 in enhancing mito-chondrial respiration (Fig. 4E, first panel), ectopic expression ofParkin in H460-p53siRNA cells enhanced oxygen consumption(Fig. 4E, second panel). Furthermore, Parkin knockdown inH460-con and Parkin knockout in MEF cells (Parkin−/− MEF)decreased oxygen consumption (Fig. 4E, third and fourth pan-els), which indicates reduced mitochondrial respiration. Parkinknockout in mice was reported to result in the decreased ex-pression of several mitochondrial proteins, including pyruvatedehydrogenase E1α1 (PDHA1), in the mouse brain as detectedby 2D gel electrophoresis and mass spectrometry analysis (16).PDHA1 is a critical component of the pyruvate dehydrogenase(PDH) complex, which catalyzes the conversion of pyruvate intoacetyl-CoA and serves as a critical link between glycolysis andmitochondrial respiration. It is unknown whether Parkin canregulate the expression of PDHA1 in human cells, which may inturn affect the activity of the PDH complex and therefore the

levels of acetyl-CoA and glucose metabolism in cells. Results inFig. 5 A–D clearly show that Parkin knockdown in H460-concells decreased PDHA1 protein levels, the activity of the PDHcomplex, and levels of acetyl-CoA, whereas ectopic Parkin ex-pression in H460-p53siRNA cells increased PDHA1 proteinlevels, the activity of the PDH complex, and levels of acetyl-CoA.Consistently, PDHA1 levels were much higher in Parkin+/+

MEF than Parkin−/− MEF cells (Fig. 5A). Furthermore, PDHA1knockdown in H460-con cells significantly reduced mitochon-drial respiration (Fig. 5E), which in turn increased glucose up-take, the rate of glycolysis, and lactate production, leading to theWarburg effect (Fig. 5F). It is still unclear how Parkin regulatesPDHA1. Parkin does not regulate PDHA1 expression at themRNA level (Fig. S2A). Knockdown of Parkin or PDHA1 didnot decrease intracellular ATP levels (Fig. S2B), which suggeststhat enhanced glycolysis compensates the decreased mitochon-drial respiration for ATP generation in cells.

Parkin Contributes to the Role of p53 in Regulating AntioxidantDefense. The antioxidant function is a novel mechanism for p53in tumor suppression (8, 13). Parkin regulates antioxidant func-tion in neuronal cells (16). To investigate whether the inductionof Parkin by p53 also contributes to the role of p53 in antioxidantdefense, Parkin was overexpressed or knocked down in cells.Ectopic Parkin expression significantly reduced ROS levels inH460-p53siRNA cells (Fig. 6A) treated with or without H2O2.Parkin knockdown in H460-con (Fig. 6B) and Parkin knockout inMEF cells (Fig. 6C) significantly increased ROS levels. Fur-thermore, Parkin mediates the role of p53 in ROS regulation. Asshown in Fig. 6D, simultaneous knockdown of p53 and Parkinresults in higher intracellular ROS levels than individualknockdown of p53 or Parkin, but the effect is less than additive.These results suggest that Parkin is one of the important medi-ators for p53’s role in ROS regulation in cells.Reduced glutathione (GSH) is an important antioxidant mol-

ecule and a scavenger for ROS. GSH:GSSG (oxidized glutathi-one) balance reflects the redox state of cells. p53 has been reportedto up-regulate GSH levels and the GSH:GSSG ratio in cells (11,12). As shown in Fig. 6 E and F, H460-con displayed significantlyhigher GSH levels and GSH:GSSG ratio than H460-p53siRNAcells. Ectopic Parkin expression significantly increased GSH levelsand GSH:GSSG ratio in H460-p53siRNA cells. Furthermore,Parkin knockdown inH460-con and Parkin knockout inMEF cellssignificantly decreased GSH levels and the GSH:GSSG ratio (Fig.

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Fig. 3. p53 regulates theexpression of mouse Par-kin gene both in vitro andin vivo. (A) The putativep53 REs in mouse Parkingene predicted by thep53MH algorithm. Num-bers indicate the nucleo-tide position relative tothe initiation codon ATGsite. (B) p53 activates theluciferase reporter vec-tors containing putativep53 REs in mouse Parkingene. Reporter vectorswere transfected into p53null H1299 and p53−/−MEFcells along with a p53wild-type (WT p53) or R273H mutant (Mut p53) expression plasmid. Data are presented as mean ± SD (n = 3). (C and D) p53 induces the expression of Parkin inMEF cells at both protein (C) and mRNA (D) levels. p53+/+ and p53−/− MEFs were treated with H2O2 (200 μM), and Parkin mRNA levels were measured by real-time PCR and normalized to actin. (E and F) p53 induces mouse Parkin expression in vivo in response to IR. p53+/+ and p53−/− C57BCL/6 mice were irradiated(4 Gy). The levels of Parkin protein (E) and mRNA (F) in different tissues were measured by Western blot and real-time PCR assays, respectively, at 20 h after IR(n = 6). Represented is the induction of Parkin protein by IR in three mouse spleen tissues in a p53-dependent manner (E). The mRNA levels of Parkin andp21 were normalized to actin. The mRNA induction fold of Parkin and p21 was calculated as the mRNA levels in IR-treated mice compared with those incontrol mice without IR. Data are presented as mean ± SD (n = 6).

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6 E and F). These results strongly suggest that as a target of p53,Parkin contributes greatly to the role of p53 in antioxidant defense.

Parkin Deficiency Sensitizes Mice to IR-Induced Tumorigenesis. Re-cent studies have suggested that Parkin is a potential tumorsuppressor (18–22). It has been well-established that IR inducestumorigenesis in mice. To study the impact of Parkin deficiencyupon tumorigenesis, 2-mo-old Parkin+/+ and Parkin−/− C57BL6/Jmalemice (28) were subjected to a single dose of 4-Gy IR.As shownin Fig. 7A, Parkin knockout did not enhance the rate of spontaneoustumors but sensitized mice to IR-induced tumorigenesis; Parkin−/−

mice displayed a shorter tumor latency induced by IR comparedwith wild-type mice (P < 0.01). The IR-induced tumor spectrum issimilar between Parkin−/− and wild-type mice; IR mainly inducedlymphomas in the spleen in both mice (Fig. 7B). Interestingly, ourresults have shown that IR specifically induced Parkin expression ina p53-dependent manner in mouse spleen (Fig. 3 E and F), whichsuggests that the induction of Parkin by p53 in response to IR maycontribute to the role of Parkin in preventing IR-induced lympho-mas in the spleen. These results provide further direct evidence tosupport the role of Parkin as a potential tumor suppressor.

DiscussionParkin has recently been suggested to be a potential tumorsuppressor (18–22). Here we demonstrate that Parkin deficiencysensitizes mice to IR-induced tumorigenesis, providing furtherdirect evidence to support a role of Parkin in tumor suppression.Diminished expression of Parkin has frequently been observed invarious tumors, but the mechanisms are not well-understood.Parkin mutations do not account for all of the decreased Parkinexpression in tumors (18–22). Our finding that p53 regulatesParkin expression not only provides a mechanism for the regu-lation of Parkin but also suggests that loss of p53, a commonevent in tumors, is an important mechanism contributing to thefrequently decreased expression of Parkin in tumors.Recently, Parkin was reported to transcriptionally repress p53

(29). Our results show that Parkin does not repress p53 expres-sion in H460 or HCT116 cells in which p53 induces Parkin ex-pression (Fig. S3 A–D). Interestingly, in human neuroblastomaSH-SH5Y cells, whereas Parkin represses p53 expression andtranscriptional activity (Fig. S3 A–D), which is consistent with theprevious report (29), p53 does not regulate Parkin expression(Fig. S4 A and B). So far, no negative feedback loop between p53and Parkin was observed in these three cell lines and several

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Fig. 4. Parkin regulates the Warburg effect in cells. (A)Parkin reduces glucose uptake in H460 and MEF cells. (B) Parkinreduces the rate of glycolysis in H460 and MEF cells. (C) Parkinreduces the lactate production of H460 and MEF cells. (D)Parkin contributes to the role of p53 in glucose metabolism.Simultaneous knockdown of p53 and Parkin in H460 cellsresults in higher glucose uptake, rate of glycolysis, and lactateproduction than individual knockdown of p53 or Parkin, butthe effects are less than the additive effects. (E) Parkinincreases oxygen consumption of H460 and MEF cells. Firstpanel: p53 deficiency reduces oxygen consumption in cells. ForA–C and E, H460-p53siRNA cells were transfected with Parkinexpression vector, and H460-con cells were transfected withsiRNA oligos against Parkin 24 h before assays. For D, H460 cellswere transfected with siRNA oligos against p53 and/or Parkin24 h before assays. Three different siRNA oligos were used, andsimilar results were obtained as the represented one. Data arepresented as mean ± SD (n = 3). #P < 0.05; *P < 0.01.

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other cell lines we tested. These results strongly suggest that theregulation of Parkin by p53, and the regulation of p53 by Parkin,could be cell type- or tissue-specific.

Our results demonstrate that as a newly identified importantcomponent of the p53 signaling pathway, Parkin contributes tothe functions of p53 in both energy metabolism and antioxidantdefense. Parkin deficiency results in the Warburg effect, whereasectopic expression of Parkin reverses the Warburg effect in cells.These results indicate that decreased expression of Parkin, whichhas been frequently observed in tumors, should be an importantmechanism for the Warburg effect in tumors. The reduced mi-tochondrial respiration resulting from Parkin deficiency could bean important mechanism that contributes to enhanced glycolysisand the Warburg effect in tumor cells. The decreased expressionof mitochondrial proteins resulting from Parkin deficiency, suchas PDHA1, contributes to reduced mitochondrial respiration,which in turn promotes the Warburg effect. Recently, severaladditional mechanisms by which Parkin regulates mitochondrialfunction have been proposed, including regulating autophagy toclear damaged mitochondria (30), promoting mitochondrial fis-sion (31), and maintaining mitochondrial genome integrity (32),all of which may contribute to the role of Parkin in regulation ofthe Warburg effect. p53 has also been reported to play similarroles in some of these processes, such as autophagy and main-taining mitochondrial genome integrity (6). It is possible thatParkin also contributes to the role of p53 in the regulation ofthese processes. Thus, maintaining the homeostasis of energymetabolism and preventing the Warburg effect could be an im-portant mechanism contributing to the tumor-suppressive func-tion of Parkin. Furthermore, Parkin enhances GSH levels anddecreases ROS levels. Considering the important role of ROS intumorigenesis, the antioxidant function of Parkin should alsocontribute greatly to its role in tumor suppression. Thus, as adirect p53 target, Parkin contributes to the functions of p53 intumor suppression through the regulation of energy metabolism,especially the Warburg effect, and antioxidant defense.

Materials and MethodsCell Culture. p53-deficient H460-p53siRNA cells were established by stabletransduction of a p53 shRNA retroviral vector (pSuper-puro-si-p53) in p53 wild-type H460 cells. H460-con cells are H460 cells with stable transduction ofacontrol retroviralvector.p53wild-typeHCT116-conandp53-deficientHCT116-p53siRNA cells were generous gifts from M. Oren (Weizmann Institute of Sci-ence, Rehovot, Israel) (33). MEF Parkin+/+ and Parkin−/− cells were establishedfrom wild-type and Parkin−/− C57BL6/J mice (The Jackson Laboratory) (28).

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Fig. 5. The regulation of PDHA1 by Parkin contributes to the role of Parkin inregulating the Warburg effect in cells. (A) Parkin regulates the expression ofPDHA1. (B) PDHA1 knockdown by siRNA in H460-con cells. (C) The levels of Parkinand PDHA1 affect PDH complex activity in H460 cells. (D) Parkin and PDHA1regulate the intracellular levels of acetyl-coA. (E) PDHA1 knockdown reducesoxygen consumption inH460-con cells. (F) PDHA1knockdown results in enhancedglucose uptake, rate of glycolysis, and lactate production in H460-con cells. Threedifferent siRNAoligosagainst ParkinorPDHA1wereused forall assays, and similarresults were observed. Data are presented as mean ± SD (n = 3). *P < 0.01.

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Fig. 6. Parkin reduces ROS levels and increases GSH levels in cells. (A and B) Parkin reduces ROS levels in cells. H460-p53siRNA cells were transfected with Parkinexpression vectors (A), and H460-con cells were transfected with siRNA oligos against Parkin (B) for 24 h. The cells were then treated with H2O2 (200 μM) for 6 hbefore assays. (C) Parkin knockout increases ROS levels in MEF cells. (D) Parkin contributes to the role of p53 in ROS regulation. Simultaneous knockdown of p53and Parkin in H460 cells results in higher ROS levels than individual knockdownof p53 or Parkin, but the effect is less than additive. (E and F) Parkin regulates GSHlevels (E) and the GSH:GSSG ratio (F) in H460 and MEF cells. The levels of GSH and GSSG were measured in cells at 24 h after transfection. Three different siRNAoligos against Parkin were used for all assays, and similar results were observed. Data are presented as mean ± SD (n = 3). #P < 0.05; *P < 0.01.

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ChIP and Luciferase Activity Assays. ChIP assays were performed in H460-conand H460-p53siRNA cells treated with Etoposide (10 μM for 16 h) to activatep53 as described (11, 25). The pGL2 firefly luciferase reporter containingputative p53 REs in human and mouse Parkin genes was constructed, andluciferase activity assays were performed as described (11, 25).

Real-Time PCR, Western Blot Analysis, and IF Staining. TaqMan real-time PCRwas performed as described (11, 25). Western blot analysis and IF stainingwere performed as previously described (11).

Measurement of Glucose Uptake, Glycolysis Rate, Lactate Production, andOxygen Consumption. Glucose uptake was measured by determining theuptake of 2-[3H]deoxyglucose (American Radiolabeled Chemicals) by cells aspreviously described (34). Glycolysis rate was measured by monitoring theconversion of 5-[3H]glucose to 3H2O as described (5, 9). Lactate levels in theculture media of cells were determined by using a Lactate Assay Kit (Bio-vision). Oxygen consumption in cells was measured by using the BD OxygenBiosensor System (BD Biosciences) as described (11).

Measurements of Activity of PDH and Levels of Acetyl-CoA, ATP, ROS, and GSH.The activity of PDH was measured by using a PDH Enzyme Activity Assay Kit(MitoSciences). The intracellular levels of acetyl-CoA were measured by usingan Acetyl-CoA Assay Kit (Biovision). The intracellular levels of ATP weremeasured by using an ATP Bioluminescence Assay Kit (Roche) as described(11). ROS levels were measured by flow cytometry as described (9, 11). Thelevels of GSH and GSSG were measured by using a glutathione detection kit(Biovision) as described (9, 11).

γ-Irradiation and IR-Induced Tumorigenesis of Mice. Two-month-old p53+/+

and p53−/− male C57BL6/J mice were treated with IR (4 Gy). Mice were killedat different times after IR (n = 6 for each time point), and different tissueswere collected to determine Parkin expression. For tumorigenesis assays, 2-mo-old wild-type and Parkin−/− C57BL6/J mice (28) (The Jackson Laboratory)were subjected to 4-Gy IR. Mice were examined three times/wk until mori-bund. The statistical differences in tumor latency were analyzed by Kaplan–Meier analysis.

See SI Materials and Methods for details.

ACKNOWLEDGMENTS. This work was supported by grants from the NationalInstitutes of Health (1R01CA143204-01), New Jersey Commission on CancerResearch (NJCCR), and Foundation of the University of Medicine andDentistry of New Jersey (to Z.F.), and by a grant from the National Institutesof Health (1P30CA147892-01) (to A.J.L. and W.H.). C.Z. is supported by apostdoctoral fellowship from the NJCCR.

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Fig. 7. Loss of Parkin sensitizes mice to IR-induced tumorigenesis. Two-month-old wild-type and Parkin−/− C57BL6/J male mice were subjected toa single dose of 4-Gy IR and monitored for survival. (A) Kaplan–Meier curvedemonstrates that Parkin−/− mice had a significantly shorter tumor latencyinduced by IR compared with wild-type mice (P < 0.01). (B) The similar IR-induced tumor spectrum between Parkin−/− and wild-type mice.

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