P53-Induced Up-Regulation of Mnsod and Gpx but Not Catalase Increases Oxidative Stress and Apoptosis

[CANCER RESEARCH 64, 2350–2356, April 1, 2004] p53-Induced Up-Regulation of MnSOD and GPx but not Catalase Increases Oxidative Stress and Apoptosis

S. Perwez Hussain, Paul Amstad, Peijun He, Ana Robles, Shawn Lupold, Ichiro Kaneko, Masato Ichimiya, Sagar Sengupta, Leah Mechanic, Shu Okamura, Lorne J. Hofseth, Matthew Moake, Makoto Nagashima, Kathleen S. Forrester, and Curtis C. Harris Laboratory of Human Carcinogenesis, National Cancer Institute, Center for Cancer Research, Bethesda, Maryland

ABSTRACT DNA strand breaks; however, a simultaneous increase in CAT or GPx corrected this hypersensitivity (5). p53-mediated apoptosis may involve the induction of redox-controlling There are two forms of intracellular SOD in mammalian cells: (a) genes, resulting in the production of reactive oxygen species. Microarray Cu,Zn-SOD, localized in the cytoplasm and nucleus; and (b) MnSOD, expression analysis of doxorubicin exposed, related human lymphoblasts, p53 wild-type (WT) Tk6, and p53 mutant WTK1 identified the p53- which is found in the mitochondrial matrix. MnSOD (SOD2) is dependent up-regulation of manganese superoxide dismutase (MnSOD) localized to chromosome 6q25, a region that is frequently deleted in and glutathione peroxidase 1 (GPx). Consensus p53 binding sequences certain types of human cancer (6, 7). A decreased level of MnSOD has were identified in human MnSOD and GPx promoter regions. A 3-fold been reported in tumor cells (8). Overexpression of MnSOD in murine increase in the MnSOD promoter activity was observed after the induc- and human cell lines leads to a decrease in the colony-forming ability tion of p53 in Li-Fraumeni syndrome (LFS) fibroblast, TR9-7, expressing and a reduction in the tumor formation in athymic nude mice (9–12). p53 under the control of a tetracycline-regulated promoter. An increased Furthermore, both human and mouse cell lines with MnSOD overex- protein expression of endogenous MnSOD and GPx also positively corre- pression have reduced growth rate, plating efficiency, and viability lated with the level of p53 induction in TR9-7 cells. However, catalase compared with the control cells (11, 13). In addition to a slower (CAT) protein expression remained unaltered after p53 induction. We growth rate, MnSOD-overexpressing cells show increased sensitivity also examined the expression of MnSOD, GPx, and CAT in a panel of normal or LFS fibroblasts, containing either WT or mutant p53. We to oxygen radical treatment, and this sensitivity is abrogated by found increased MnSOD enzymatic activity, MnSOD mRNA expression, pyruvate, a H2O2 scavenger (14). and MnSOD and GPx protein in LFS fibroblasts carrying a WT p53 allele GPx is a selenoprotein and occurs in five different isoforms. GPx1 when compared with homozygous mutant p53 isogenic cells. The CAT is the major isoform that metabolizes H2O2 to O2 and H2O. GPx protein level was unchanged in these cells. We observed both the release overexpression has been found to block MnSOD-induced tumor ؉ of cytochrome C and Ca2 from the mitochondria into the cytoplasm and growth inhibition (15). CAT is a tetrameric enzyme with a ferripro- an increased frequency of apoptotic cells after p53 induction in the TR9-7 toporphyrin group in each subunit. Although GPx is found mainly in cells that coincided with an increased expression of MnSOD and GPx, and the cytosol and mitochondrial matrix, CAT is largely present in the level of reactive oxygen species. The increase in apoptosis was reduced peroxisomes. The hypersensitivity of Cu-Zn SOD-expressing mouse by the antioxidant N-acetylcysteine. These results identify a novel mechanism of p53-dependent apoptosis in which p53-mediated up-regulation of epidermal cells to H2O2-induced DNA damage is corrected by CAT MnSOD and GPx, but not CAT, produces an imbalance in antioxidant expression in the double transfectant (5). A decrease in the CAT enzymes and oxidative stress. activity has been found in a variety of animal tumors (16). Fibroblasts from cancer-prone Xeroderma-pigmentosum cases possess a 5-fold

lower CAT activity and produces five times more H2O2 upon UV INTRODUCTION irradiation exposure when compared with cells from noncancer-prone An increase in the level of reactive oxygen species (ROS), termed trichothiodystrophy (17). Although both GPx and CAT decompose as oxidative stress, is controlled by multiple, interacting, low and high H2O2, their contributions vary depending on the amount and the site molecular weight components. Among them, superoxide dismutase of H2O2 production (18, 19). (SOD), glutathione peroxidase 1 (GPx), and catalase (CAT) play a ROS has been implicated in the induction of apoptosis (18, 20, 21). • central role (reviewed in Refs. 1, 2). The overall effect of the antiox- Mitochondria is the major source of superoxide anion (O2 ) produc- ϳ idant system depends on the intracellular balance between these tion in cells and 1–2% of oxygen reduced by mitochondria are • • antioxidant enzymes rather than a single component (3). In the anti- converted to O2 (22). The O2 is converted to H2O2 by MnSOD, • oxidant enzyme system, SOD catalyzes the dismutation of superoxide which is present in the mitochondria. Although O2 is primarily confined to the mitochondria, H2O2 is capable of passing through the radicals to H2O2, which is further metabolized to H2O and O2 by CAT and GPx (4). An imbalance in the coordinated expression/activity of mitochondrial membrane into the cytoplasm and the extracellular antioxidant enzymes can lead to the generation of oxidative stress (3, microenvironment (23). Excess of H2O2 that is not reduced by GPx 5). An overexpression of Cu, Zn-SOD in mouse epidermal cells inside the mitochondria can cross the mitochondrial membrane into resulted in the sensitization to oxidative chromosomal aberrations and the cytosol and is reduced by CAT and GPx to H2O and O2. Another • • free radical, nitric oxide (NO ) can enhance the generation of O2 and Received 8/12/02; revised 12/24/03; accepted 1/27/04. subsequent generation of H2O2 by inhibiting the cytochrome oxidase The costs of publication of this article were defrayed in part by the payment of page and thereby modulating ROS-mediated apoptosis (21). Increased ox- charges. This article must therefore be hereby marked advertisement in accordance with idative stress causes the release of cytochrome C from mitochondria 18 U.S.C. Section 1734 solely to indicate this fact. Note: S. P. Hussain and P. Amstad contributed equally to this work. P. Amstad is into the cytosol. Cytosolic cytochrome c can bind to Apaf1 and currently at the Scientific Review Program, National Institute of Allergy and Infectious activate caspase 9 in the apoptosome in response to diverse inducers Diseases, Bethesda, MD. S. Lupold is currently at the Johns Hopkins School of Medicine, of cell death (24–26). Cytochrome c is released from the intermem- Baltimore, MD. I. Kaneko is currently at the Trauma and Clinical Care Center, Tokyo University, Tokyo, Japan. M. Ichimiya is currently at the Department of Surgery, Kobe brane space of the mitochondria through openings in the outer mem- Teishin Hospital, Kobe, Japan. brane, which formed as a consequence of mitochondrial permeability Requests for reprints: Curtis C. Harris, Chief, Laboratory of Human Carcinogenesis, Building 37, Room 3068, National Cancer Institute, NIH, Bethesda, MD 20892-4255. transition, leading to the loss of the mitochondrial membrane potential Phone: (301) 496-2048; Fax: (301) 496-0497; E-mail: [email protected]. (reviewed in Refs. 27, 28). 2350

p53-induced apoptosis can be mediated by ROS. One mechanism is continuous setting). Aliquots were removed from each sample to determine the p53 regulation of the expression of genes that are related to cellular protein concentration with the BCA reagent (Pierce, CA). Extracts were then redox status and involved in apoptosis (Ref. 20; reviewed in Ref. 29). centrifuged at 800 ϫ g for 5 min, and the supernatant was saved. An aliquot In the present study, we investigated the hypothesis that an oxidative of the cleared extract corresponding to 50 ␮g of protein was applied to a stress induced from an imbalance between H O -producing MnSOD nondenaturing 10% polyacrylamide gel to localize MnSOD activity as previ- 2 2 ously described (38) with the exception that no N,N,NЈ,NЈ-tetramethylethyl- and the peroxide-removing enzymes, GPx and CAT, plays a role in enediamine was used for staining. the p53-mediated apoptosis. ROS Measurement. Intracellular production of ROS was measured using a fluorescent dye, 2Ј,7Ј-dichlorofluorescin-diacetate (DCFH-DA). DCFH-DA MATERIALS AND METHODS is a nonpolar compound that is converted into a nonfluorescent polar-deriva- tive DCFH by cellular esterases, after it is incorporated into the cells (26). 4 Cell Lines and Cell Culture. The human lymphoblast cell line, TK6 with Approximately 10 TR9-7 cells were placed onto a glass grid inside a 35-mm wild-type (WT) p53, and the highly related pair, WTK1 with mutated (non- Petri dish. Cells were grown for 4 days in the presence or absence of 3 ␮g/ml functional) p53 (30), were grown in RPMI medium, supplemented with 15% tetracycline. For the assay, medium was replaced with Hank’s solution con- fetal bovine serum. The cell lines MDAH 041, MDAH 087, and MDAH 172 taining 5 ␮M DCFH-DA. After 5 min of incubation at room temperature, the were derived from fibroblasts of patients with Li-Fraumeni syndrome (LFS; fluorescent intensity of ϳ50 cells for each treatment condition was measured Ref. 31). These cell lines were kindly provided by Michael Tainsky (Case by using confocal laser scanning microscopy. The excitation wavelength at 513 Western Reserve University, Cleveland, OH). The cells were grown in an nm and emission wavelength at 530 nm were used. Relative fluorescence intensity was determined with the NIH Image Analysis program (39). atmosphere of 5% CO2 in DMEM, supplemented with 10% fetal bovine serum. LFS fibroblast, (TR9-7), engineered to produce p53 only in the absence of Adeno-CAT Infection and Immunofluorescence. Adenovirus vector tetracycline (32), was obtained from George Stark (Cleveland Clinic Founda- containing the human CAT cDNA (adeno-CAT) was kindly provided by Drs. tion, OH). The cells were cultured in DMEM plus 10% fetal bovine serum and Toren Finkel (NIH, Bethesda, MD) and Kaikobad Irani (Johns Hopkins School 5 maintained in the presence of 600 ␮g/ml G418, 50 ␮g/ml hygromycin, and 3 of Medicine, Baltimore, MD). TR9-7 cells (3 ϫ 10 cells/100-mm dish) were ␮g/ml tetracycline. Under these conditions, p53 expression is undetectable. plated in a 100-mm dish, containing a coverslip in the center, and grown 24 h Construction of pGL3-MnSOD and Reporter Gene Assay. The MnSOD in the presence of tetracycline. Cells were then infected with either adeno-CAT promoter region was amplified by PCR using DNA, isolated from normal or Adeno-␤Gal in 3 ml of tetracycline-free medium and incubated for 1 h. human fibroblasts, as a template. Using the long PCR kit (Perkin-Elmer, Cells were then allowed to grow for 24, 48, 96, and 120 h with the addition of Branchburg, NJ), the 2.3-kb MnSOD promoter region (Ϫ2215 to ϩ42) was 7 ml of tetracycline-free medium. At these time points, the cells on the amplified and cloned into the HindIII site of the basic luciferase reporter coverslips were used for immunocytochemistry, and the monolayer cells on the plasmid pGL3 (Promega, Madison, WI). The nucleic acid sequence of the plates were collected for protein isolation and Western blot analysis. Briefly, cloned MnSOD promoter region was confirmed by using “Long distance the cells on the coverslip were washed with PBS and fixed with acetone: sequencer” method as described by Hagiwara et al. (33). For transient trans- methanol (1:1) for 3 min. The coverslips were air dried for 10 min, and the fection, TR9-7 cells were grown for 4 days with and without tetracycline. Cells cells were blocked with 10% normal chicken serum for 20 min. The cells were were washed once in serum-free medium (Opti-MEM; Life Technologies, Inc., washed once with PBS and incubated overnight with human anti-CAT anti- Rockville, MD) and cotransfected for2hin3mlofOpti-MEM containing 2 body (Cortex Biochem), diluted 1:50 in normal chicken serum, at 4°C. The ␮g of pGL3-MnSOD plasmid, containing the MnSOD promoter linked to the cells were again washed three times with PBS and incubated with FITC- luciferase reporter gene, 2 ␮gof␤-galactosidase-expression plasmid pCMV- conjugated donkey antirabbit secondary antibody (diluted 1:50 in normal ␤-gal as transfection control, and 40 ␮l of Lipofectamine (Life Technologies, chicken serum) for1hatroom temperature. The cells were finally washed Inc.). After transfection, the medium was replaced with full growth medium thrice with PBS and mounted with Vectashield containing 4Ј,6-diamidino-2- with and without tetracycline. After 48 h of incubation, cells were lysed, and phenylindole for nuclear staining. cellular extracts were prepared for luciferase and ␤-galactosidase assay. Pro- cDNA Microarray Hybridization and Analysis. Cells were lysed with moter activity is expressed as luciferase activity in relative light units/mU Trizol reagent (Life Technologies, Inc.), and total RNA was extracted accord- ␤-galactosidase. Each experiment was repeated three or more times. ing to the manufacturer’s instructions at 0, 6, and 24 h after treatment with 0.5 MnSOD and GPx mRNA Expression. For the analysis of MnSOD and mg/ml doxorubicin. Fluorescent-labeled cDNA probes were generated using Gpx mRNA expression, 2 ϫ 105 TR9-7 cells were plated in a 10-cm culture 40 ␮g of total RNA by a single round of reverse transcription in the presence dish in growth medium containing 3 ␮g/ml tetracycline. Forty-eight h after of aminoallyl-dUTP (Sigma), followed by a coupling reaction to Cy3 or Cy5 plating, the medium containing tetracycline was replaced by growth medium monofunctional NHS-ester (Amersham Pharmacia, Piscataway, NJ). Complex containing no tetracycline. In the control plates, the medium was changed with probes (usually containing untreated Cy3-labeled cDNA and doxorubicin- complete growth medium containing tetracycline. One, 2, 4, and 5 days after treated Cy5-labeled cDNA) were denatured and hybridized to glass slides the withdrawal of tetracycline from the growth medium, the cells were lysed, featuring 9180 cDNA elements: 7102 named genes; 1179 expressed sequence and total RNA was prepared according to the procedure of Chomczynski et al. tag clones; and 122 Incyte clones (NCI Microarray Facility, Advanced Tech- (34). Total RNA (10 ␮g) was used for Northern blot analysis using MnSOD, nology Center, Gaithersburg, MD) and incubated overnight at 42°C. Slides GPx, and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) riboprobes were washed successively in 1ϫ SSC/0.1% SDS, 1ϫ SSC, and 0.2ϫ SSC for (3, 35, 36). 2 min each, then rinsed in 0.5ϫ SSC and spin-dried. The two fluorescent MnSOD, GPx, CAT, and p53 Protein Expression. TR9-7 cells were intensities were measured simultaneously using a GenePix 4000A scanner, and cultured in the absence of tetracycline for 2, 4, and 5 days. In a control dish, the acquired image was processed with GenePix Pro 3.0 software (Axon cells were cultured in the presence of 3 ␮g/ml tetracycline. Other LFS Instruments, Union City, CA). The basic raw data and derived ratio measure- fibroblasts were grown to 75–80% confluency. Protein lysate was prepared in ments were then uploaded to the NCI MicroArray Database system, which radioimmunoprecipitation assay buffer and subjected to Western blot analysis, provides the bioinformatics and analysis tools necessary for the interpretation as described earlier (37), using one of the following: anti-MnSOD monoclonal of gene expression data. Spot size and intensity filters were applied to all antibody (Chemi-Con International, Temecula, CA), anti-GPx monoclonal measurements in each array, duplicate arrays were averaged, and the averaged antibody (MBL Corporation, Boston, MA), anti-CAT [gift from Alain Baret data were analyzed, based on an arbitrary threshold of 2.0-fold up- or down- (Nante, France) and Cortex Biochem (San Leandro, CA)], or anti-p53 poly- regulation. clonal antibody CM-1 (Signet Pathology System, Dedham, MA). MnSOD Enzymatic Activity Measurement. MnSOD activity was deter- RESULTS mined by activity gel assay (38). Cells were collected in cold PBS and sedimented at 1600 ϫ g for 5 min. The cell pellet was then resuspended in 200 Induction of MnSOD and GPx after Doxorubicin Treatment in ␮lof50mM potassium phosphate buffer with a pH of 7.8 and followed by TK6 Cells with WT p53. We examined the induction of antioxidant sonication for four 15-s bursts on a Branson sonicator B15 (position 2, enzymes MnSOD, GPx, and CAT in TK6 and WTK1 cells, after 2351

Downloaded from cancerres.aacrjournals.org on September 29, 2021. © 2004 American Association for Cancer Research. p53-INDUCED UP-REGULATION OF MnSOD AND GPx treatment with doxorubicin, using microarray hybridization technique. activity, TR9-7 cells were cotransfected with pGL3-MnSOD-Luc and We have earlier reported that treatment of TK6 cells (functional p53) pCMV-␤gal, as described in “Materials and Methods.” Cells were with 0.5 ␮g/ml doxorubicin induces apoptosis in 39 Ϯ 2% incubated in the presence or absence of tetracycline and used for the (mean Ϯ SD) cells by 24 h; however, WTK1 cells (mutant p53) were measurement of luciferase activity. Removal of tetracycline from the completely resistant to the same treatment (40). Both MnSOD and growth medium (induction of p53) increased the MnSOD promoter GPx were found among the gene cluster showing increased induction activity by 3-fold when compared with the controls in the presence of in TK6 cells compared with WTK1 cells after 24 h exposure to tetracycline in the growth medium (repression of p53; P ϭ 0.002, Fig. doxorubicin (Supplemental Table 1). CAT mRNA expression was not 1D). A p21 reporter construct was used as a positive control, which found to be altered in this assay. resulted in a 5-fold increase in the promoter activity in the presence of MnSOD, GPx, and CAT Expression in Isogenic Pairs of LFS p53 (P ϭ 0.0001, Fig. 1E). These results indicate that p53 transacti- Fibroblasts. To determine the effect of p53 on the up-regulation of vates the MnSOD promoter. Similar results for GPx have been re- MnSOD, we measured MnSOD expression and activity in LFS cell ported previously (42). lines, carrying different p53 genotypes, as well as TR9-7 cells in To further investigate the association between WT p53 and which p53 expression is regulated by tetracycline. MnSOD activity, H2O2-generating MnSOD, we used TR9-7 cells, which express p53 mRNA and protein expression, and GPx and CAT protein expression in the absence of tetracycline in the culture medium. Fig. 2A were compared in three pairs of LFS cell lines, expressing either WT compares MnSOD mRNA expression of both 1- and 4-Kb mRNA p53, mutated p53, or were null for p53 (Fig. 1, A and B). The 041ϩ/Ϫ species, generated by alternate splicing and polyadenylation in cell line expresses only WT p53 because there is a frameshift mutation TR9-7 cells grown either in the presence or absence of tetracycline. of one p53 allele at codon 184, whereas in 041Ϫ/Ϫ cells, the normal The induction of p53 increased MnSOD mRNA expression in a p53 allele is lost. The 041ϩ/Ϫ cells showed more MnSOD mRNA time-dependent manner, reaching a maximum after 5 days. As and enzymatic activity, and MnSOD and GPx protein expression shown in Fig. 2C, MnSOD protein expression was positively when compared with the p53-null 041Ϫ/Ϫ cells. Similar results were correlated with the change in MnSOD mRNA expression. Further- obtained with 087 cells. The 087ϩ/Ϫ cells contain a codon 248Trp more, increased MnSOD expression was consistent with the in- heterozygous mutation, whereas 087Ϫ/Ϫ cells lack the remaining WT allele. In 172ϩ/Ϫ cells that contain a codon 175His heterozygous crease in p53 induction. We also performed immunofluorescent mutation, MnSOD mRNA expression is increased when compared staining for MnSOD in TR9-7 cells. Cells that were induced to with 172Ϫ/Ϫ cells that lack the remaining WT allele; however, no express p53 by the removal of tetracycline for 4 days showed an change in MnSOD enzymatic activity nor MnSOD and GPx protein intense staining pattern for MnSOD when compared with cells expression was observed. These results indicate that MnSOD and GPx without p53 expression (Supplemental Fig. 1). expression in LFS cells correlates with the p53 genotype. However, We next determined if p53 expression affects the expression of no change in CAT expression was observed in these isogenic LFS hydrogen peroxide-removing enzymes, GPx and CAT, which act cells. downstream to MnSOD activity. The balance between hydrogen Regulation of MnSOD, GPx, and CAT Expression by p53. The peroxide-producing MnSOD and peroxide-removing GPx and CAT promoters of human MnSOD and GPx, but not CAT, contain se- can influence the availability of ROS in the cellular environment. We quences that correspond to the consensus p53 binding element, as determined GPx mRNA and GPx and CAT protein expression in described earlier [Ref. 41; Fig. 1C). These p53 consensus binding sites TR9-7 cells in the presence (p53 suppressed) or absence of tetracy- are present Ϫ2009 to Ϫ2032 bp upstream of the transcription start site cline (p53 induced; Fig. 2, B and C). We observed an increase in GPx in MnSOD (GTGCTTGTTC-4 bp-GGGCATGTCC) and Ϫ694 to mRNA and protein expression after the induction of p53 in a time- Ϫ720 bp in GPx (AGGTTTGTTG-8 bp-GGACTAGCTT) promoters. dependent manner. However, we did not find any change in CAT To determine the effect of p53 expression on the MnSOD promoter expression up to 5 days after p53 induction.

Fig. 1. Manganese superoxide dismutase (Mn- SOD) mRNA, protein expression, enzymatic activity, and glutathione peroxidase 1 (GPx) and catalase protein expression in Li-Fraumeni syndrome fibroblasts containing either wild-type, mutant, or nonfunctional p53 (A and B). Human MnSOD and GPx promoters contain sequences that correspond to the consensus p53-binding sequence 5Ј-RRRC- WWGYYY-3Ј (r ϭ purine, Y ϭ pyrimidine, and W ϭ A/T). These consensus p53 binding sites are present Ϫ2009 to Ϫ2032 bp upstream of the transcription start site in MnSOD (GTGCTTGTTC-4 bp-GGGCATGTCC) and Ϫ694 to Ϫ720 bp in GPx (AGGTTTGTTG-8 bp-GGACTAGCTT) promoters (C). MnSOD (D) and p21 (E) promoter-driven reporter gene expression. TR9-7 cells grown with and without tetracycline were cotransfected with either pMnSOD-Luc or p21-Luc and the control plasmid pCMV-␤-galactosidase. After 48 h of post- transfection, cell extracts were prepared for the measurement of luciferase and ␤-galactosidase activity.

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increased DCF fluorescence in cells expressing p53 when compared with cells without p53 expression. To confirm that the increase in DCF fluorescence in p53-expressing cells was due to the increased production of ROS, we measured DCF fluorescence in the presence of an antioxidant, NAC. NAC treatment reduced the p53-induced increase in DCF fluorescence (P ϭ 0.004). These data are consistent with the hypothesis that increased ROS production, after p53 expression in TR9–7 cells, can mediate p53-induced apoptosis. CAT Overexpression Inhibited p53-Mediated Apoptosis in TR9-7 Cells. Infection with adeno-CAT increased the expression of CAT in TR9-7 cells at different time points (Fig. 5A). Nuclei stained with 4Ј,6-diamidino-2-phenylindole were analyzed for apoptosis at 24, 48, 96, and 120 h after infection. CAT overexpression significantly inhibited apoptosis at 96 and 120 h (P Ͻ 0.01; Fig. 5, B and C). Cytochrome c Is Released from Mitochondria into the Cytosol after Induction of p53. We measured cytochrome c expression both in mitochondria and cytosol, after p53 induction in TR9-7 cells (Supplemental Fig. 2). The complete release of cytochrome c from the mitochondria 3 days after removal of tetracycline corresponded with an increase of cytochrome c in the cytosol. Ca2؉ Mobilization Occurs Late in p53-Induced Apoptosis. To examine the question whether Ca2ϩ release from the mitochondria was the cause or the consequence of p53-induced oxidative stress and Fig. 2. Manganese superoxide dismutase (MnSOD) and glutathione peroxidase 1 apoptosis, we measured intracellular calcium concentration Ca2ϩ as a (GPx) mRNA (A and B) and MnSOD, GPx, catalase, and p53 protein (C) expression in TR9-7 cells. Numbers below the bands indicate densitometry values as a ratio relative to function of time after p53 induction. As shown in Supplemental Fig. ϩ control values. 3, Ca2 began to increase by day 3 after the removal of tetracycline and induction of p53 and continued to rise past the last measurement at day 4. This result, combined with our observation that ROS pro- Increased p53 Expression Induces ROS Production and Apop- duction is high 2 days after p53 induction, indicates that changes in tosis. We examined the effect of p53 expression on apoptosis in mitochondrial calcium homeostasis occur as a consequence of ROS TR9-7 cells. Cells grown in either the presence or absence of tetra- generation at a later stage in apoptosis rather than participating in its cycline were immunostained for p53 with polyclonal anti-p53 anti- initiation. body CM-1 and followed by 4Ј,6-diamidino-2-phenylindole staining to score for apoptotic nuclei (Fig. 3A). Cells with condensed chroma- DISCUSSION tin and fragmented nuclei were considered apoptotic. At least 100 cells, treated with or without tetracycline for 4 days, were examined Several agents that induce p53-dependent apoptosis also generate for apoptosis. Cells that were grown in the absence of tetracycline oxygen radical species, which is consistent with the hypothesis that ROS showed positive p53 immunostaining and apoptotic nuclei when com- might play a critical role in p53-mediated apoptosis (20, 43). A model for pared with the control cells grown in the presence of tetracycline. The p53-dependent apoptosis was proposed (20), consisting of (a) the tran- addition of 5 mM N-acetyl cystein (NAC), an antioxidant, in the scriptional induction of redox-related genes, p53-induced genes; (b) the culture medium of cells expressing p53, decreased the number of formation of ROS; and (c) oxidative degradation of mitochondrial com- p53-positive apoptotic cells. Fig. 3, B and C, compares the frequency ponents. The electron transport chain in the mitochondria is the major of cells expressing p53 with the frequency of cells undergoing apo- source of intracellular ROS production, which makes it likely that alter- ptosis. Sixty-eight percent of cells grown in the absence of tetracy- ations in mitochondrial function are responsible for the increase in ROS cline expressed p53 (Fig. 3B), and only 33% of all of the cells were production during p53-induced apoptosis. In support of a role of mito- apoptotic (P ϭ 0.001, Student t test; Fig. 3C). Presence of NAC lead chondrial ROS generation in p53-induced apoptosis, it was shown that to a significant decrease in apoptosis (P ϭ 0.002). Cells grown in the bongkrekic acid, an inhibitor of mitochondrial permeability transition, presence of tetracycline showed only a small level of background p53 abolished p53-induced apoptosis and that cells undergoing p53-mediated staining (3%) as well as apoptosis (2%). These results indicate that the apoptosis showed increased mitochondrial lipid peroxidation (20). Fur- expression of p53 correlates with apoptosis and that ϳ45% of the thermore, p53-dependent apoptosis has been shown to be associated with p53-expressing cells undergo apoptosis. the loss of mitochondrial membrane potential and increased ROS pro- To determine whether p53-induced apoptosis is associated with an duction (43). increased production of ROS, we measured intracellular dichloro- One mechanism for the increased production of ROS is the imbal- fluorescin (DCF)-fluorescence in TR9-7 cells, grown either in the ance in the antioxidant enzyme system. Both MnSOD and GPx presence (p53 repressed) or absence of tetracycline (p53 expressed). showed a 2-fold higher induction in the TK6 (functional p53) cell line DCFH-DA diffuses into cells where it is deacetylated to DCFH, which after 24 h of doxorubicin treatment when compared with WTK1 fluoresces upon reaction with ROS. TR9-7 cells grown in the absence (mutant p53). However, CAT induction was not observed in the of tetracycline (p53 expressed) for 4 days showed strong DCF fluo- expression analyses. The p53-dependent increase in GPx expression rescence (P ϭ 0.0003, Fig. 4, A and B) when compared with cells in in our study is consistent with the earlier finding (42). Earlier studies the presence of tetracycline, where only background fluorescence is have shown that genes transactivated by p53 contain the consensus detected. Using the NIH Image analysis software (39), relative DCF p53 binding element (41). We identified potential p53 consensus fluorescence was compared in ϳ60 cells, cultured in the absence or binding sites in human MnSOD and GPx promoters. When we trans- presence of tetracycline. The results in Fig. 4B show quantitatively fected the MnSOD-luciferase promoter construct into TR9-7 cells, we 2353

Fig. 3. p53 and 4Ј,6-diamidino-2-phenylindole (DAPI) staining of TR9-7 cells (A). Cells that were grown in the absence of tetracycline showed positive p53 immunostaining and apoptotic nuclei when compared with the control cells grown in the presence of tetracycline. The addition of N-acetyl cysteine (NAC), an antioxidant, in the culture medium of cells expressing p53, decreased the number of p53-positive apoptotic cells. Quantita- tion is presented as the percentage of p53-expressing cells (B) and the percentage of apoptotic cells (C) for 600 cells counted.

found a 3-fold increase in the MnSOD promoter activity after p53 induced up-regulation of MnSOD and GPx, without any alteration in expression, in the absence of tetracycline in the growth medium, when CAT, can produce oxidative stress in the cells. When we induced p53 in compared with the control cells that do not express detectable p53. TR9-7 cells, ϳ33% of the cells expressing p53 underwent apoptosis. p53 expression and the activation of a downstream target was also However, the number of apoptotic cells were reduced substantially in the confirmed by transfecting a p21/waf1-reporter construct, containing presence of an antioxidant, NAC, which supports the functional role of consensus p53 binding element. p53-induced up-regulation of luci- ROS in p53-mediated apoptosis in the present study. NAC has been • ferase activity, under the control of the GPx promoter, has been found to react with H2O2 but not O2 (47). CAT treatment, which reported previously (42). Our result, showing the up-regulation of degrades H2O2 to water and molecular oxygen, inhibits p53-mediated human MnSOD promoter activity by p53 in human lymphoblastoid apoptosis in human smooth muscle cells (45). It can be argued that CAT cells and fibroblasts, contrasts a recent study that describes down- plays a significant role in reducing p53-mediated oxidative stress, thus regulation of rat MnSOD promoter activity by p53 in human MCF-7 inhibiting apoptosis. In most cells, mitochondria lack CAT, and the H2O2 cancer cells (44). Although, the rat MnSOD promoter shows 36% that is generated inside mitochondria can be degraded by GPx. However, similarity with the human MnSOD promoter, it contains only one-half the activity of GPx depends on the level of glutathione and NADPH and of the consensus p53 binding sequence. Furthermore, overexpression may not be sufficient for complete removal of H2O2 under oxidative of p53 in rat smooth muscle cells did not generate ROS, whereas stress. Excess H2O2 can cross the mitochondrial membrane and can be human smooth muscle cells showed strong DCF fluorescence gener- degraded by CAT. Engineered HepG2 cells overexpressing CAT in the ated from the oxidative stress (45). Also, transient transfection of mitochondria are protected from oxidative injury by H2O2 (48). CAT- HeLa cells with WT p53 showed a reduction in the MnSOD level overexpressing thymocytes also are resistance to dexamethasone-induced (46). These results indicate that p53-mediated production of ROS may apoptosis (49). Furthermore, ceramide-induced inhibition of CAT and be cell type and species dependent. increased oxidative stress causes apoptosis in myeloid cells treated with We found that TR9-7 cells, when grown in the absence of tetracycline vesnarinone, an inotropic agent used for treating heart failure (50). CAT (p53 expressed), showed strong DCF fluorescence, a marker of increased overexpression also inhibited Vp16 or mitomycin C-induced apoptosis in oxidative stress when compared with cells in the presence of tetracycline HepG2 cells by decreasing phosphorylation and accelerating the degra- (p53 suppressed). These data support the earlier findings that an imbal- dation of p53 (51). In the present study, we tested the protective role of ance in the levels of antioxidant enzymes can lead to the production of CAT in oxidant-induced apoptosis by its overexpression in TR9-7 cells. oxidative stress (3, 5) and are consistent with our hypothesis that p53- Consistent with the hypothesis, the overexpression of CAT significantly 2354

(60). Overexpression of c-myc or E2F-induced ROS accumulation and enhanced serum-deprived apoptosis in NIH 3T3 and Saos2 cells (61). In the same study, E2F1 was found to suppress ROS-induced MnSOD expression by inhibiting nuclear factor-␬B activity (61). However, it is not clear from this study how an increase in MnSOD expression relieves the cells from total ROS accumulation without any alteration in the peroxide removing enzymes. Furthermore, overexpression of c-myc, followed by the generation of ROS, DNA damage, and p53 accumulation in normal human fibroblast did not affect apoptosis. None of the studies, thus far, have addressed the imbalance of antioxidant enzymes that determines the generation of oxidative stress associated with p53-mediated apoptosis. As discussed earlier, the study of a single component of the antioxidant system does not yield a clear understanding of the overall effect. Although many antioxidant components are involved in the regulation of cellular redox status, in the present study, we have addressed this issue by investigating the regulation of MnSOD, GPx, and CAT expression by p53 and showed that their imbalance leads to oxidative stress and apoptosis. However, the degree of contribution and interdependence of several different redox-related genes in p53-induced apoptosis has yet to be determined. The imbalance in the antioxidant cascade described here, the previous studies demonstrating p53 regulation of genes encoding proteins that cause mitochondrial depolarization, and the release of cytochrome c or an essential component of the apoptosome, i.e., Apaf1, are all consistent with oxidative stress as the

Fig. 4. The production of ROS was determined by dichlorofluorescin (DCF) fluores- major mechanism of p53-dependent apoptosis. cence. DCF fluorescence was measured with a confocal microscope equipped with a laser at an excitation wavelength of 488 nm and emission wavelength of 525 nm (A). DCF fluorescence was quantified with the “NIH Image Analysis” software. B shows the mean of fluorescence of 50–100 cells. reduced p53-mediated apoptosis. These and other observations propose a protective role of CAT in oxidant-mediated apoptosis. Based on our results, showing an increase in MnSOD and GPx levels, but not in the CAT level, after p53 expression leading to the production of ROS and apoptosis, it can be hypothesized that an increase in GPx alone (without

CAT) does not suffice for the degradation of H2O2 produced by MnSOD. Furthermore, apoptosis induced by H2O2 and oxidized low-density li- poprotein in macrophages is associated with increased expression of p53 and MnSOD (52). Apoptosis, induced by a variety of stimuli, coincides with the release of cytochrome c from mitochondria into the cytoplasm where it activates a cascade of proteolytic enzymes crucial for the execution of apoptosis (20, 45). Our results are consistent with the previous studies showing that p53 induction leads to the release of cytochrome c from mitochondria into the cytosol (53). Cytochrome c release is a necessary intermediate in the p53 activation of downstream apoptotic targets as shown in a recent study, demonstrating that caspase-9 and its cofactor, Apaf-1, are essential downstream components of p53 in myc-induced apoptosis (54). Cytochrome c has been shown to be essential for the Apaf-1 binding to procaspase-9 promoting its dimer- ization and activation by autocatalysis (55, 56). Furthermore, p53 transcriptionally transactivates Apaf-1 (40, 57), which is a major downstream component in the p53-mediated apoptotic pathway (58). Although, p53-mediated apoptosis involves the generation of ROS, the role of MnSOD may appear to be contradictory in the literature. One scenario is that the decrease in the MnSOD level leads to • oxidative stress from the accumulation of superoxide anion (O2 ), which can mediate the p53-dependent apoptotic pathway (44). Other studies have suggested that a decrease or inhibition of MnSOD Fig. 5. Expression of CAT at different time points in TR9-7 cells following infection expression leads to the reduction in apoptosis (52), and its induction with Adeno-CAT expression vector or Adeno-␤GAL control in the absence of tetracycline (A). Immunocytochemical staining of TR9-7 cells infected with either Adeno-CAT or is associated with apoptosis (59). All-trans-retinoic acid induces Mn- Adeno-〉Gal control (B). CAT overexpressing cells show significant inhibition of apo- SOD expression and apoptosis in acute myeloblastic leukemia cells ptosis after 96 and 120 h. 2355

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S. Perwez Hussain, Paul Amstad, Peijun He, et al.

Cancer Res 2004;64:2350-2356.

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