(Thioredoxin Peroxidase-2) Protects Cancer Cells Against Hypoxia and Drug-Induced Hydrogen Peroxide-Dependent Apoptosis

682 Vol. 1, 682–689, July 2003 Molecular Cancer Research

Increased Expression of Mitochondrial Peroxiredoxin-3 (Thioredoxin Peroxidase-2) Protects Cancer Cells Against Hypoxia and Drug-Induced Hydrogen Peroxide-Dependent Apoptosis

Larisa Nonn, Margareta Berggren, and Garth Powis

Arizona Cancer Center, University of Arizona, Tucson, AZ

Abstract mitochondrial H2O2 can induce apoptosis by causing the Peroxiredoxin-3 (Prdx3) is a mitochondrial member of the release of proapoptotic factors from the mitochondria, such as antioxidant family of thioredoxin peroxidases that uses cytochrome c and apoptosis-inducing factor, through the mitochondrial thioredoxin-2 (Trx2) as a source of reduc- opening of a nonselective mitochondrial permeability transition

ing equivalents to scavenge hydrogen peroxide (H2O2). pore (5–7). Increases in H2O2 can be caused by a variety of cell Low levels of H2O2 produced by the mitochondria stresses including drug exposure and pathological conditions, regulate physiological processes, including cell prolif- particularly neurodegenerative diseases and diseases that cause

eration, while high levels of H2O2 are toxic to the cell and hypoxic/ischemic episodes (8–10). cause apoptosis. WEHI7.2 thymoma cells with stable Cells contain a variety of peroxidases including catalase, overexpression of Prdx3 displayed decreased levels of glutathione peroxidases, and peroxiredoxins (Prdxs) that control

cellular H2O2 and decreased cell proliferation without a the constitutive levels of H2O2 in the cell and protect against change in basal levels of apoptosis. Prdx3-transfected ROS-induced damage by catalyzing the reduction of the H2O2 cells showed a marked resistance to hypoxia-induced into water (11–13). There are six known mammalian Prdxs.

H2O2 formation and apoptosis. Prdx3 overexpression Prdxs1–4 have two conserved cysteine residues, whereas Prdxs5 also protected the cells against apoptosis caused by and 6 have only one of the cysteine residues involved in the

H2O2, t-butylhydroperoxide, and the anticancer drug peroxidase activity (14–16). In some species, Prdxs5 and 6 have imexon, but not by dexamethasone. Thus, mitochondrial additional cysteines in less conserved NH2-terminal regions Prdx3 is an important cellular antioxidant that regulates of the protein that are required for peroxidase activity (17).

physiological levels of H2O2, leading to decreased Prdxs1–4 have high amino acid sequence homology with each cell growth while protecting cells from the apoptosis- other (60–80%) but lower homology to Prdxs5 and 6 (20%).

inducing effects of high levels of H2O2. Prdxs1–4 belong to the thioredoxin peroxidase subfamily and require the small redox protein thioredoxin (Trx) as an electron Introduction donor to remove H2O2, whereas Prdxs5 and 6 can use other The mitochondria are an important site for the production of cellular reductants, such as glutathione, as their electron donor cellular reactive oxygen species (ROS). The mitochondrial (17, 18). Prdx1, Prdx2, and Prdx6 are found in the cytoplasm and electron transport chains consume oxygen by oxidative nucleus (14, 19–21). Prdx3 contains a mitochondrial local- phosphorylation to form cellular energy in the form of ATP. ization sequence, is found exclusively in the mitochondrion, and During this process, between 0.4% and 4% of the consumed uses mitochondrial thioredoxin-2 (Trx2) as the electron donor for oxygen is released in the mitochondria as ROS that include its peroxidase activity (22). Prdx4 has an NH2-terminal secretion ÀÁ signal sequence and is found in the endoplasmic reticulum and hydrogen peroxide (H2O2), superoxide (O2 ), singlet oxygen Á ÀÁ the extracellular space (23). Prdx5 is expressed as a long form (O ), and hydroxyl radical (HO ) (1). H2O2 is stable enough to diffuse out of the mitochondria and to have a cytoplasmic effect associated with the mitochondria and a short form found with the peroxisomes (24), and a nuclear localization has also been (2). Low levels of H2O2 are important to the cell because they regulate physiological processes such as receptor-mediated cell reported (19). Prdx expression is increased in several human signaling pathways, normal cell proliferation, and transcrip- cancers. Prdx1 levels are increased relative to normal tissue in tional activation (3, 4). However, aberrant increases in oral cancer (25), in follicular but not papillary thyroid cancer (26), in breast cancer (27), and in lung cancer (28). Prdxs2 and 3 are increased in breast cancer (27) while increased expression of all the Prdxs is found in mesothelioma (19). Received 1/21/03; revised 5/13/03; accepted 5/13/03. Prdx3 (MER5, SP-22, and AOP-1) was originally cloned out The costs of publication of this article were defrayed in part by the payment of of murine erythroleukemia cells (29). Prdx3 expression is page charges. This article must therefore be hereby marked advertisement in induced by oxidants in the cardiovascular system and is thought accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Grant support: CA52995 and CA772049. to play a role in the antioxidant defense system and homeostasis Requests for reprints: Garth Powis, Arizona Cancer Center, University of within the mitochondria (30, 31). Prdx3 is a c-myc target gene, Arizona, 1515 North Campbell Avenue, Tucson, AZ 85724-5024. Phone: (520) 626-6704; Fax: (520) 626-4848. E-mail: [email protected] and antisense experiments have shown that its expression is Copyright D 2003 American Association for Cancer Research. required for neoplastic transformation by c-myc (31).

To further examine the antioxidant role of Prdx3, we have generated cell clones with stable overexpression of Prdx3. We have found that Prdx3 overexpression alters the mitochondrial membrane potential, reduces endogenous cellular H2O2 levels, and causes growth retardation. Prdx3-overexpressing cells are resistant to the increase in H2O2 and apoptosis caused by hypoxia. Increased Prdx3 also protects cells from apoptosis caused by H2O2, t-butylhydroperoxide, and imexon, a mitochondrial targeting anticancer drug, but not by dexamethasone.

Results Prdx3-Expressing Cells Prdx3 was stably transfected into WEHI7.2 mouse thymoma cells because of their well-characterized responsiveness to apoptotic stimuli (32–34). Measurement of Prdx3 gene expression in transfected WEHI7.2 cells by Northern blot showed many clones with overexpression of Prdx3 mRNA ranging from 5- to 8.7-fold (Fig. 1A). A medium Prdx3- expressing clone (Prdx3-10) and high Prdx3-expressing clone (Prdx3-32), as well as empty vector-transfected cells (WEHI7.2/neo), were used for subsequent experiments. Prdx3-10 and Prdx3-32 cells showed Prdx3 protein overexpression of 1.6- and 2.1-fold, respectively, by Western blot analysis of the total cell lysate (Fig. 1B), and cellular subfractionation confirmed that the expressed Prdx3 was confined to the mitochondria (data not shown). Western blotting of purified mitochondria from WEHI7.2/neo, Prdx3- 10, and Prdx3-32 cells showed a similar overexpression of Prdx3 as in the cell lysate Western blot (Fig. 1B). There was a significant increase in the peroxidase activity within the mitochondrial fraction of the Prdx3-transfected cells compared with the vector-alone cells (Fig. 1C). Cellular H2O2 levels showed a small but significant decrease in the Prdx3- overexpressing cells (Fig. 2A). The mitochondrial membrane FIGURE 1. Stable transfection with Prdx3 shown by overexpression potential (DW) also showed a small significant decrease in the of Prdx3 mRNA, protein, and mitochondrial thioredoxin peroxidase activity in WEHI7.2 cells. A. Northern blot analysis on 2 Ag mRNA from Prdx3-overexpressing cells (Fig. 2B). The expression of WEHI7.2 cells stably transfected with empty vector (WEHI7.2/neo)or mitochondrial Trx2, thioredoxin reductase-2 (TrxR2), and other Prdx3. A glyceraldehyde-3-phosphate dehydrogenase (GADPH) probe was used as a loading control. Values are the levels of overexpression. mitochondrial antioxidant proteins superoxide dismutase-2 B. Western blot analysis of Prdx3 protein in 20 Ag cell lysate and 10 Ag (SOD-2) and catalase measured by Western blot analysis was mitochondria from WEHI7.2/neo, Prdx3-10, and Prdx3-32 cells. Actin not changed (Fig. 2C). was used as a loading control in cell lysate and cytochrome c as loading control in mitochondria. C. Thioredoxin peroxidase activity of the mitochondria from WEHI7.2/neo, Prdx3-10, and Prdx3-32 cells. Cell Proliferation Columns, means of three determinations; bars, SD. *P < 0.05, compared to WEHI7.2/neo cells. Cell proliferation was significantly decreased in the Prdx3- overexpressing cells compared with the vector control cells, with doubling times for the Prdx3-10 and Prdx3-32 cells of 17.4 and 18.4 h, respectively, compared with 15 h for the empty (Fig. 4A). Annexin V positivity and uptake of propidium iodide vector-transfected WEHI7.2/neo cells (P < 0.01 in both cases) was measured by fluorescence-activated cell sorting (FACS) (Fig. 3A). Cell cycle analysis revealed that there was no change analysis to distinguish live, apoptotic, and necrotic cells. Prdx3 in the fraction of cells in each phase of the cell cycle (Fig. 3B) overexpression completely prevented the hypoxia-induced and no change in basal rates of apoptosis (Fig. 3C). increase in H2O2 seen in the WEHI7.2/neo cells (Fig. 4B). Transient overexpression of Prdx3 in HT-29 adherent solid Protection Against Apoptosis tumor colon cancer cells showed a similar protection against Exposure of cells to hypoxia is known to increase cellular hypoxia-induced apoptosis. After 36 h in 1% O2, HT-29 cells ROS and eventually to cause apoptosis (35). Prdx3-over- transfected with empty vector were 47.7 F 2.95% apoptotic, expressing WEHI7.2 cells were protected against hypoxia- and Prdx3-transfected cells were 34.5 F 2.2% apoptotic (P V induced apoptosis, with only 12–15% of the cells being 0.01) (data not shown). apoptotic by 16 h in 1% O2 compared with 50% in the empty Prdx3-overexpressing WEHI7.2 cells were protected against vector-transfected WEHI7.2/neo cells (P < 0.01 in both cases) cell killing by H2O2, t-butylhydroperoxide, and imexon, a

droperoxide, but not to dexamethasone (Fig. 5). Fluorescent images of the Annexin V-positive cells showed intact membranes and apoptotic morphology after the drug treat- ments in empty vector-transfected WEHI7.2/neo and Prdx3- overexpressing cells and less Annexin V-positive cells were observed in the Prdx3-overexpressing cells than in the WEHI7.2/neo cells (Fig. 5, A–L). Prdx3-overexpressing cells exposed to imexon for 24 h displayed decreased apoptosis compared with empty vector-transfected WEHI7.2/neo cells, as shown by FACS analysis of Annexin V/propidium iodide and fluorescent images of Annexin V-positive cells (Fig. 6A). Cellular H2O2 levels were measured and showed that Prdx3- overexpressing cells had attenuated H2O2 increases after imexon treatment compared with empty vector WEHI7.2 cells (Fig. 6B).

Discussion FIGURE 2. Mitochondrial redox status of Prdx3-overexpressing H2O2 levels are increased within in the cell in response to WEHI7.2 cells. A. CM-H2DCFDA fluorescence measurement of cellular growth factors and act as an intracellular messenger (37). H2O2 levels in the Prdx3-overexpressing cells compared with empty vector WEHI7.2/neo cells. Columns, means of three determinations; bars, SD. H2O2 inhibits protein tyrosine phosphatase function by the *P < 0.05, compared to WEHI7.2/neo cells. B. Tetramethylrhodamine oxidation of key catalytic site cysteine residues, leading to methyl ester measurement of the DC in the Prdx3-overexpressing cells. activation of the kinase signaling (38). Blocking the Columns, means of three determinations; bars, SD. *P < 0.05, **P < 0.01, compared to WEHI7.2/neo cells. C. Levels of other mitochondrial accumulation of H2O2 by the addition of exogenous catalase, antioxidant proteins in empty vector-transfected and Prdx3-transfected a cytoplasmic and mitochondrial peroxidase, prevents cells. Mitochondrial lysate (10 Ag) was probed by Western blotting for platelet-derived growth factor-induced mitogen-activated Prdx3, Trx2, TrxR2, Mn superoxide dismutase (MnSOD), and catalase. protein kinase activation (38). Treatment of cells with H2O2 mimics growth factor-induced signaling by inducing mitogen-activated protein kinase activation and stimulates mitochondria damaging drug that increases mitochondrial H2O2 cell growth (39). (36), but not by dexamethasone (Table 1). The increased cell Probably because of the requirement for H2O2 in normal survival was associated with a decreased apoptosis in the cell function, it is difficult to overexpress antioxidant proteins Prdx3-transfected cells after exposure to H2O2 and t-butylhy- at very high levels (34, 40). We observed a tight regulation of

FIGURE 3. Prdx3 overexpression retards cell proliferation without alteration in cell cycle or apoptosis. A. Growth curves of WEHI7.2/neo empty vector (x), Prdx3-10 (n), and Prdx3-32 (E) cells. Points, repre- sentatives of three separate experiments with three measurements at each time point; bars, SD. **P < 0.01, compared to empty vector-transfected WEHI7.2/neo cells. B. Percentage of cells in each phase of the cell cycle was measured by propidium iodide staining of fixed cells and analyzed by FACScan. Filled bars are WEHI7.2/neo empty vector-transfected cells, shaded bars are Prdx3-10 cells, and open bars are Prdx3-32 cells. C. Percentage of apoptotic cells measured by Annexin V/propidium iodide staining and FACScan of WEHI7.2/neo, Prdx3-10, and Prdx3-32 cells. Columns, means of three determinations; bars, SD.

the antioxidant protein Prdx3 expression with a maximum Table 1. IC50 Values Acquired From 48 h Cytotoxicity 2-fold increase in protein expression. Wonsey et al. (31) Curves overexpressed Prdx3 in two different adherent cell lines and IC Values (AM) showed a similar low level of Prdx3 overexpression. In our 50 Drug Vector Prdx3-10 Prdx3-32

H2O2 60.28 F 11.1 172.23 F 8.4** 230.75 F 32.3** t-butylhydroperoxide 23.2 F 0.7 30.92 F 0.2** 35.79 F 0.7** imexon 92.32 F 6.59 136.82 F 1.8** 135.47 F 6.6** dexamethasone 0.022 F 0.004 0.024 F 0.004 0.025 F 0.004

**P = 0.001.

study, Prdx3 overexpression was associated with decreased DW, decreased cellular H2O2, and decreased cell proliferation. Wonsey et al. (31) reported a similar decrease in cellular H2O2 but did not observe a change in cell growth or DW. The reason for this difference is not known, but may be due to cell line differences because Wonsey et al. used adherent cell lines and we used the WEHI7.2 suspension cell line. A possible explanation for our findings in the WEHI7.2 cells is that Prdx3 expression lowers cellular H2O2 which decreases kinase growth factor signaling leading to decreased cell proliferation, although a direct relationship with any kinases has not been established. Whereas low levels of H2O2 are essential for cell growth, elevated levels of H2O2 are toxic to the cell and can lead to apoptosis (41). We have shown that Prdx3 overexpression is able to scavenge the excess H2O2 and protect cells from H2O2-, t-butylhydroperoxide-, and imexon-induced apoptosis. Imexon is an anticancer agent that acts as mitochondrial toxin (36). However, Prdx3 does not prevent apoptosis induced by all agents, as demonstrated by the unaltered sensitivity of the Prdx3-overexpressing cells to dexamethasone. Dexamethasone is used for the treatment of hematological tumors and causes apoptosis through binding the glucocorticoid receptor (42). The precise mechanism of dexamethasone-induced apoptosis is not known, but it involves the mitochondrial release of cytochrome c (43). When H2O2 levels were increased by exposure of the cells to hypoxia, the Prdx3-overexpressing cells were protected against apoptosis. During hypoxia, the ROS are specifically generated in the mitochondria by the disruption of oxidative phosphorylation (44). This may explain why mitochondrial Prdx3 offers greater protection against apoptosis caused by hypoxia than to added H2O2 and various drugs where the effects may also be cytoplasmic.

FIGURE 4. Prdx3-overexpressing cells are protected against hypoxia- induced apoptosis and H2O2 formation. A. Apoptosis measured by Annexin V/propidium iodide staining and FACScan. Typical dotplots of data are shown for WEHI7.2/neo, Prdx3-10, and Prdx3-32 cells grown in 1% O2 for 16 h as well as a histogram showing the mean of three determinations (columns) and SD (bars). **P V 0.01, compared to hypoxia-exposed WEHI7.2/neo cells. B. CM-H2DCFDA fluorescence measurement of cellular H2O2 levels in the Prdx3-overexpressing cells compared with empty vector WEHI7.2/neo cells grown in air (filled bars) and after 16 h at 1% O2 (open bars). Columns, means of three determinations; bars, SD. **P V 0.01, compared to hypoxia-exposed WEHI7.2/neo cells.

FIGURE 5. Overexpression of Prdx3 protects against apoptosis induced by H2O2 and t-butylhydroperoxide but not dexamethasone. Apoptosis was measured by Annexin V/propidium iodide staining after 24 h exposure of WEHI7.2/neo, Prdx3-10, and Prdx3-32 cells to 300 AM H2O2,50AM t-butylhydroperoxide, and 20 AM dexamethasone. Typical dotplots of data are shown for WEHI7.2/neo, Prdx3-10, and Prdx3-32 cells and right panel shows a histogram showing the mean of three determinations (columns) and SD (bars). Columns, means of three determinations; bars, SD. **P < 0.05, compared to WEHI7.2/neo cells. Microscopic fluorescent images of Annexin V-Fluos (488 nm) were used to show apoptotic cells in the WEHI7.2/neo and Prdx3-32 cells; DNA fluorescence (350 nm) was used to visualize total cell number. WEHI7.2/neo after 24 h exposure to 300 AM H2O2 (A and B), 50 AM t-butylhydroperoxide (E and F), and 20 AM dexamethasone (I and J). Prdx3-32 after 24 h exposure to 300 AM H2O2 (C and D), 50 AM t-butylhydroperoxide (G and H), and 20 AM dexamethasone (K and L).

Human solid tumors frequently show regions of hypoxia, as hypoxia and caused by some drugs, Prdx3 protects cells against the growing tumor outstrips its blood supply (45). Prdx3 is apoptosis. Elevated levels of Prdx3 could offer tumor cells a overexpressed in some human cancers (19, 27) where it may survival advantage in areas of hypoxia as well as protection protect the growing tumor against hypoxia-induced apoptosis against drug-induced apoptosis. due to increased H2O2 production. The results of the study suggest that mitochondrial Prdx3 is Materials and Methods an important regulator of H2O2 in the cell. At low physiological Chemicals and Reagents levels of H2O2 formation, Prdx3 inhibits the growth-stimulating All chemicals and reagents were obtained from Sigma- effects of H2O2. At higher levels of H2O2 formation, as seen in Aldrich (St. Louis, MO), unless otherwise stated. Imexon was

provided by Dr. Robert Dorr (University of Arizona). Primary GATCGAATTCCTACTGATTTAACCTTCTGAAAGT-3V). antibodies used were rabbit polyclonal antibody against human The PCR product and pCMV-script vector (Stratagene, La Jolla, Trx2, TrxR2, and Prdx3 (46), bovine catalase (Rockland, CA) were digested with EcoRI/NotI and ligated overnight at Gilbertsvill, PA), and monoclonal antibody against human Mn 14jC. Ligation and orientation were verified by sequence superoxide dismutase (Upstate Biotechnology, Inc., Lake analysis. Empty vector or vector containing Prdx3 were Placid, NY). transfected into WEHI7.2 cells by electroporation. Individual clones were selected with 1.2 mg/ml G418 in soft agar. Cell Culture WEHI7.2 mouse thymoma cells were obtained from RNA Purification and Northern Blot Analysis American Type Culture Collection (Rockville, MD). Cells Total RNA was extracted from 108 cells using Trizol j were grown at 37 Cin5%CO2 in DMEM (MediaTech Reagent (Invitrogen, Carlsbad, CA). Polyadenylated RNA was CellGro, Herndon, VA) with 10% fetal bovine serum. For purified from total RNA using Oligotex beads (Qiagen, hypoxia studies, 15 mM HEPES (pH 7.4) was added to the Valencia, CA). Polyadenylated RNA (2 Ag) was separated by DMEM. Hypoxic conditions were set at 1% O2 using a Reming electrophoresis on a 1.2% agarose/4% formaldehyde gel and Bioinstruments chamber and oxygen regulator (Reming RNA was transferred to nitrocellulose membranes (Osmonics, Bioinstruments, Redfield, NY). Westborough, MA) for Northern blot analysis. cDNA probes were radiolabeled with 32P-a-dCTP (NEN, Boston, MA) with Prdx3 Cloning and Transfection Random Prime Labeling Kit (Invitrogen). Blots were hybri- Prdx3 was cloned out of human cDNA by PCR (primers: 5V- dized with radiolabeled probes overnight at 42jC in UltraHyb GATCGCGGCCGCATGGCGCTGCTGTAGGAC-3V and 5V- Buffer (Ambion, Austin, TX). Nonspecific probe was removed by a series of washes. Northern blots were visualized by autoradiography and a Molecular Dynamics PhosphorImager (Amersham Biosciences, Piscataway, NJ) and quantified using Image Quant software.

Western Blot Analysis Cells were washed in ice-cold PBS, then resuspended in ice- cold lysis buffer (0.5% NP40, 0.5% Na-deoxycholate, 50 mM NaCl, 1 mM EDTA, 1 mM NaVO3,50mM HEPES, pH 7.5, 1mM phenylmethylsulfonyl fluoride), and incubated on ice for 20 min, with vortexing every 5 min before being cleared by centrifugation at 10,000 Â g for 15 min and 4jC. Protein was quantified using Bio-Rad Protein Reagent (Bio-Rad, Hercules, CA). Cleared lysate (20 Ag) was loaded onto a 10% NuPAGE gel (Invitrogen). The proteins were separated by electrophoresis and then transferred to a polyvinylidene fluoride membrane (NEN). Western blotting was performed in Tris-buffered saline/ 0.1% Tween 20 using the appropriate primary antibody followed by horseradish peroxidase-conjugated anti-rabbit or anti-mouse secondary antibody. The results were visualized by Western lightening chemiluminescence (NEN) followed by autoradiography.

Mitochondrial Thioredoxin Peroxidase Activity Mitochondria Isolation. Cells were harvested and washed once with ice-cold PBS (pH 7.4), resuspended, and incubated in a hypotonic buffer (10 mM Tris, 10 mM NaCl, 3 mM MgCl2, 1mM EDTA, 1 mM EGTA) for 30 min on ice. Cells were lysed by 15 strokes with a tight-fitting dounce homogenizer, FIGURE 6. Overexpression of Prdx3 protects against apoptosis and then nuclei and unbroken cells were pelleted by centrifugation increased cellular H2O2 induced by imexon. A. FACS analysis of Annexin for 15 min at 600 Â g and 4jC. The supernatant was V positivity and exclusion of propidium iodide after 24 h exposure of WEHI7.2/neo, Prdx3-10, and Prdx3-32 cells to 150 AM imexon. Columns, transferred to a fresh tube and mitochondria pelleted by means of three determinations; bars, SD. *P < 0.05, compared to empty centrifugation for 20 min at 12,000 Â g and 4jC. Mitochondria vector WEHI7.2/neo cells. Microscopic fluorescent images of Annexin were resuspended in lysis buffer [100 mM Tris-HCl (pH 7.4)/ V-Fluos (488 nm) were used to show apoptotic cells in the WEHI7.2/neo and Prdx3-32 cells; DNA fluorescence (350 nm) was used to visualize total 0.1% Triton X-100] and broken by sonification for 15 s, and cell number. B. Effect of exposure to 150 AM imexon on cellular H2O2 membranes were removed by centrifugation at 12,000 Â g for 15 levels measured by CM-H2DCFDA fluorescence. Filled bars are without j imexon and empty bars are with imexon. Columns, means of three min at 4 C. Mitochondrial protein was quantified using Bio-Rad determinations; bars, SD. *P < 0.05, compared to WEHI7.2/neo cells. Protein Reagent.

Peroxidase Assay. Mitochondrial proteins (100 Ag) and lysis Dichlorohydrofluorescein levels in cells were measured on a buffer containing 0.25 mM NADPH, recombinant human DTrx2 Gemini XS microplate spectroflourometer (Molecular Devices, (Trx2 without the mitochondrial localization sequence), puri- Sunnyvale, CA) using 488 nm excitation and a 520 nm fied human placental thioredoxin reductase-1 (TrxR1) (47), and emission for detection. H2O2 levels were quantitated by cleared mitochondrial lysate (100 Ag) were added and comparison with a standard curve generated using dichlorohy- equilibrated at room temperature. The reaction was initiated drofluorescein. with 1 mM H2O2 and oxidation of NADPH was measured at 339 nm for 3 min on Hitachi U-3310 spectrophotometer. Mitochondrial Membrane Potential Cells (105) were incubated in phenol red-free DMEM Cytotoxicity containing 20 nM tetramethylrhodamine methyl ester (Molec- Cells (5 Â 103) were seeded in 24-well plates in 1 ml ular Probes) for 30 min at 37jC. Cells were analyzed and DMEM/10% fetal bovine serum/1.2 mg/ml G418. Drugs were fluorescence was measured on a FACScan using 488 nm added at increasing concentrations and cells were allowed to excitation and a 515-nm bandpass filter for detection of grow for 48 h before trypan blue was added. Toxicity was tetramethylrhodamine methyl ester. measured by cell counting of live cells that did not take up trypan blue. Acknowledgments We thank Amy Coon for technical support and Dr. Emmanuelle Meuillet-May for her critical review of this manuscript. FACS data were collected by Norma Apoptosis Seaver from the shared sources at the Arizona Cancer Center. The statistics Cells (12.5 Â 105) were treated with drug for described times were performed at the Arizona Cancer Center Biometry Shared Service by Dr. Haiyan Cui. and conditions. The cells were pelleted and washed with PBS and resuspended in calcium buffer (10 mM HEPES/NaOH, pH 7.4, 140 mM NaCl, 5 mM CaCl ) containing Annexin V-Fluos References 2 1. Boveris, A. Determination of the production of superoxide radicals and (Boehringer Mannheim, Roche Applied Science, Indianapolis, hydrogen peroxide in mitochondria. Methods Enzymol., 105: 429 – 435, 1984. IN) and 50 mg/ml propidium iodide (Molecular Probes, 2. Melov, A. Mitochondrial oxidative stress: physiologic consequences and Eugene, OR). Cells were analyzed on a FACScan (Becton potential for a role in aging. Ann. NY Acad. Sci., 908: 219 – 225, 2000. Dickinson, Franklin Lakes, NJ) using 488 nm excitation by an 3. Simon, H. U., Yehia-Haj, A., and Levi-Schaffer, F. Role of reactive oxygen argon laser and a 515-nm bandpass filter for fluorescein species (ROS) in apoptosis function. Apoptosis, 5: 415 – 418, 2000. 4. Huang, R. P., Wu, J. X., Fan, Y., and Adamson, E. D. UV activates growth detection and a filter >560 nm for propidium iodide detection. factor receptors via reactive oxygen species. J. Cell Biol., 133: 211 – 220, 1996. Compensation settings for FACS were FL1- 1.6% FL2, FL2- 5. Zoratti, M. and Szabo, I. The mitochondrial permeability transition. Biochim. 36.5% FL1, FL2- 0.0% FL3, and FL3- 22.6% FL2. When the Biophys. Acta, 1241: 139 – 176, 1995. data are analyzed by dotplot with X axis=Annexin V and 6. Lui, X., Kim, C. N., Yang, J., Jemmerson, R., and Wang, X. Induction of Y =propidium iodide fluorescence, live cells are found in lower apoptotic program in cell-free extracts: requirement for dATP and cytochrome c. Cell, 86: 147 – 157, 1996. left quadrant, necrotic cells in upper left quadrant, and apoptotic 7. Susin, S. A., Zamzami, N., Castedo, M., Hiersch, T., Marchetti, P., Macho, A., cells in right quadrants. Daugas, E., Geuskens, M., and Kroemer, G. Bcl-2 inhibits the mitochondrial For microscopic images of Annexin V-Fluos, cells were release of an apoptogenic protease. J. Exp. Med., 184: 1331 – 1341, 1996. attached to slides using Cell Tak (BD Biosciences, San Jose, 8. Nicholls, D. G. Mitochondrial function and dysfunction in the cell: its CA) for 30 min in Annexin V-Fluos containing calcium buffer. relevance to aging and aging-related disease. Int. J. Biochem. Cell Biol., 34: 1372 – 1381, 2002. Cells were fixed in 4% formaldehyde/PBS then mounted using 9. Di Lisa, F. Mitochondrial contribution in the progression of cardiac ischemic ProLong Mounting Media (Molecular Probes). Annexin was injury. IUBMB Life, 52: 255 – 261, 2001. visualized at 488 nm and DNA autofluorescence was visualized 10. Dhalla, N. S., Temsah, R. M., and Netticadan, T. Role of oxidative stress in at 350 nm. cardiovascular disease. J. Hypertens., 18: 655 – 673, 2000. 11. Radi, R., Turrens, J. F., Change, L. Y., Bush, K. M., Crapo, J. D., and Freeman, B. A. Detection of catalase in rat heart mitochondria. J. Biol. Chem., Cell Cycle Analysis 266: 22028 – 22034, 2001. 5 Cells (12.5 Â 10 ) were pelleted and washed with PBS. Ice- 12. Knopp, E. A., Arndt, T. L., Eng, K. L., Caldwell, M., Leboef, R. C., Deeb, cold ethanol was added, and the cells were pelleted and S. S., and O’Brien, K. D. Murine phospholipids hydroperoxide glutathione peroxidase: cDNA sequence, tissue expression and mapping. Mamm. Genome, rehydrated in PBS before being resuspended in 100 mM Tris- 10: 601 – 605, 1999. HCl (pH 7.4), 150 mM NaCl, 1 mM CaCl2, 0.5 mM MgCl2, and 13. Chae, H. Z., Kim, H. J., Kang, S. W., and Rhee, S. G. Characterization of 0.1% NP40 containing 3 mM propidium iodide. Cell cycle was three isoforms of mammalian peroxiredoxin that reduce peroxides in the presence analyzed on a FACScan. of thioredoxin. Diabetes Res. Clin. Pract., 45: 101 – 112, 1999. 14. Kang, S. W., Chae, H. Z., Seo, M. S., Kim, K., Baines, I. C., and Rhee, S. G. Mammalian peroxidoxin isoforms can reduce hydrogen peroxide generated in H2O2 Assay response to growth factors and tumor necrosis factor-a. J. Biol. Chem., 273: 6297 – 6302, 1998. Cellular H2O2 was measured by a modification of the method of Eposti (48). Cells (104 –105) were washed three 15. Lim, Y. S., Cha, M. K., Yun, C. H., Kim, H. K., Kim, K., and Kim, I. H. Purification and characterization of thiol-specific antioxidant protein from human times in PBS and incubated in DMEM without phenol red red blood cell: a new type of antioxidant protein. Biochem. Biophys. Res. (MediaTech CellGro) containing 5 AM 5-(and 6-)chloromethyl- Commun., 199: 199 – 206, 1994. 2V,7V-dichlorodihydrofluorescein diacetate, acetyl ester (CM- 16. Kim, K., Kim, I. H., Lee, K. L., Rhee, S. G., and Stadtman, E. R. The isolation and purification of a specific ‘‘protector’’ protein which inhibits H2DCFDA) (Molecular Probes) for 30 min at 37jC. Excess inactivation by a thiol/Fe(III)O2 mixed-function oxidation system. J. Biol. Chem., CM-H2DCFDA was removed from the cells by a wash in PBS. 263: 4704 – 4711, 1988.

17. Fisher, A. B., Dodia, C., Manevich, Y., Chen, J. W., and Feinstein, S. I. 33. Freemerman, A. J. and Powis, G. A redox-inactive thioredoxin reduces Phospholipid hydroperoxides are substrates for non-selenium glutathione growth and enhances apoptosis in WEHI7.2 cells. Biochem. Biophys. Res. peroxidase. J. Biol. Chem., 274: 21326 – 21334, 1999. Commun., 274: 136 – 141, 2000. 18. Singh, A. K. and Shichi, H. A novel glutathione peroxidase in bovine eye. 34. Briehl, M., Tome, M. E., Baker, A. F., Powis, G., and Payne, C. M. Catalase- Sequence analysis, mRNA level and translation. J. Biol. Chem., 273: 26171 – overexpressing thymocytes are resistant to steroid-induced apoptosis and exhibit 26178, 1998. increased net tumor growth. Am. Assoc. Cancer Res., 41: 828, 2000. 19. Kinnula, V. L., Lehtonen, S., Sormunen, R., Kaarteenaho-Wiik, R., Kang, 35. Chandel, N. S., McClintock, D. S., Feliciano, C. E., Wood, T. M., Melendez, S. W., Rhee, S. G., and Soini, Y. Overexpression of peroxiredoxins I, II, III, V, J. A., Rodriguez, A. M., and Schumacker, P. T. Reactive oxygen species generated and VI in malignant mesothelioma. J. Pathol., 196: 316 – 323, 2002. at mitochondrial complex III stabilize hypoxia-inducible factor-1a during 20. Mizusawa, H., Ishii, T., and Bannai, S. Peroxiredoxin I (macrophage 23 kDa hypoxia: a mechanism of O2 sensing. J. Biol. Chem., 275: 25130 – 25138, 2000. stress protein) is highly and widely expressed in the rat nervous system. Neurosci. 36. Dvorakova, K., Waltmire, C. N., Payne, C. M., Tome, M. E., Briehl, M. M., Lett., 283: 57 – 60, 2000. and Dorr, R. T. Induction of mitochondrial changes in myeloma cells by imexon. 21. Oberley, T. D., Verwiebe, E., Zhong, W., Kang, S. W., and Rhee, S. G. Blood, 11: 3544 – 3551, 2001. Localization of the thioredoxin system in normal rat kidney. Free Radic. Biol. 37. Rhee, S. G., Bae, Y. S., Lee, S. R., and Kwon, J. Hydrogen peroxide: a key Med., 30: 412 – 424, 2001. messenger that modulates protein phosphorylation through cysteine oxidation. 22. Watabe, S., Hiroi, T., Yamamoto, Y., Fujioka, Y., Hasegawa, H., Yago, N., Signal Transduct. Knowl. Environ., 53: 1 – 6, 2000. and Takahashi, S. Y. SP-22 is a thioredoxin-dependent peroxide reductase in 38. Sundaresan, M., Yu, Z-X., Ferrans, V. J., Irani, K., and Finkel, T. mitochondria. Eur. J. Biochem., 249: 52 – 60, 1997. Requirement for generation of H2O2 for platelet-derived growth factor signal 23. Okado-Matsumoto, A., Matsumoto, A., Fujii, J., and Taniguchi, N. transduction. Science, 270: 296 – 299, 1995. Peroxiredoxin IV is a secretable protein with heparin-binding properties under 39. Irani, K., Xia, Y., Zweier, J. L., Sollott, S. J., Der, C. J., Fearon, E. R., reduced conditions. J. Biochem., 127: 493 – 501, 2000. Sundaresan, M., Finkel, T., and Goldschmidt-Clermont, P. J. Mitogenic signaling 24. Seo, M. S., Kang, S. W., Kim, K., Baines, I. C., Lee, T. H., and Rhee, S. G. mediated by oxidants in Ras-transformed fibroblasts. Science, 275: 1649 – 1652, Identification of a new type of mammalian peroxiredoxin that forms an 1997. intramolecular disulfide as a reaction intermediate. J. Biochem., 275: 20346 – 40. Gallegos, A., Gasdaska, J. R., Taylor, C. W., Paine-Murrieta, G. D., 20354, 2000. Goodman, D., Gasdaska, P. Y., Berggren, M., Briehl, M. M., and Powis, G. 25. Yanagawa, T., Iwasa, S., Ishii, T., Tabuchi, K., Iwasa, S., Bannai, S., Omura, Transfection with human thioredoxin increases cell proliferation and a dominant K., Suzuki, H., and Yoshida, H. Peroxiredoxin I expression in oral cancer: a negative mutant thioredoxin reverses the transformed phenotype of breast cancer potential new tumor marker. Cancer Lett., 156: 27 – 35, 2000. cells. Cancer Res., 56: 5765 – 5770, 1996. 26. Yanagawa, T., Iwasa, S., Ishii, T., Tabuchi, K., Yusa, H., Onizawa, K., 41. Takeyama, N., Miki, S., Hirakawa, A., and Tanaka, T. Role of mitochondrial Omura, K., Suzuki, H., and Yoshida, H. Peroxiredoxin I expression in human permeability transition and cytochrome c release in hydrogen peroxide-induced thyroid tumors. Cancer Lett., 145: 127 – 132, 1999. apoptosis. Exp. Cell Res., 274: 16 – 24, 2002. 27. Noh, D. Y., Ahn, S. J., Lee, R. A., Kim, S. W., Park, I. A., and Chae, H. Z. 42. Cidlowski, J. A. and Scwartzman, R. A. Glucocorticoid-induced apoptosis of Overexpression of peroxiredoxin in human breast cancer. Anticancer Res., 21: lymphoid cells. Int. Arch. Allergy Appl. Immunol., 105(4): 347 – 354, 1994. 2085 – 2090, 2001. 43. Renner, K., Kofler, R., and Gnaiger, E. Mitochondrial function in 28. Chang, J. W., Jeon, H. B., Lee, J. H., Yoo, J. S., Chun, J. S., Kim, J. H., and glucocorticoid triggered T-ALL cells with transgenic bcl-2 expression. Mol. Yoo, Y. J. Augmented expression of peroxiredoxin I in lung cancer. Biochem. Biol. Rep. 29(1 – 2): 97 – 101, 2002. Biophys. Res. Commun., 289: 507 – 512, 2001. 44. Chandel, N. S., Maltepe, E., Goldwasser, E., Mathieu, C. E., Simon, M. C., 29. Yamamoto, Y., Mantsui, Y., Natori, S., and Obinata, M. Cloning of a and Schumacker, P. T. Mitochondrial reactive oxygen species trigger hypoxia- housekeeping-type gene (MER5) preferentially expressed in murine erythroleu- induced transcription. Proc. Natl. Acad. Sci. USA, 95: 15 – 10, 1998. kemia cells. Oncogene, 80: 337 – 343, 1989. 45. Richard, D. E., Berra, E., and Pouyssegur, J. Angiogenesis: How a tumor 30. Araki, M., Nanri, H., Ejima, K., Murasato, Y., Fujiwara, T., Nakashima, Y., adapts to hypoxia. Biochem. Biophys. Res. Commun., 266: 718 – 722, 1999. and Ikeda, M. Antioxidant function of the mitochondrial protein SP-22 in the 46. Nonn, L., Williams, R.R., Erickson, R. P., and Powis, G. Absence of cardiovascular system. J. Biol. Chem., 274: 2271 – 2278, 1999. mitochondrial thioredoxin-2 causes apoptosis, exencephaly and embryonic 31. Wonsey, D. R., Zeller, K. I., and Dang, C. V. The c-myc target gene PRDX3 lethality in homozygous mice. Mol. Cell. Biol., 23: 916 – 922, 2003. is required for mitochondrial homeostasis and neoplastic transformation. Proc. 47. Oblong, J. E., Gasdaska, P. Y., Sherrill, K., and Powis, G. Purification of Natl. Acad. Sci. USA, 99: 6649 – 6654, 2002. human thioredoxin reductase: properties and characterization by absorption and 32. Briehl, M. M., Cotgreave, I. A., and Powis, G. Downregulation of the circular dichroism spectroscopy. Biochemistry, 32: 7271 – 7277, 1993. antioxidant defense during glucocorticoid-mediated apoptosis. Cell Death Differ., 48. Eposti, M. Measuring mitochondrial reactive oxygen species. Methods 2: 41 – 46, 1995. Enzymol., 26: 335 – 340, 2002.

Downloaded from mcr.aacrjournals.org on October 2, 2021. © 2003 American Association for Cancer Research. Increased Expression of Mitochondrial Peroxiredoxin-3 (Thioredoxin Peroxidase-2) Protects Cancer Cells Against Hypoxia and Drug-Induced Hydrogen Peroxide-Dependent Apoptosis 11CA52995 and CA772049.

Larisa Nonn, Margareta Berggren and Garth Powis

Mol Cancer Res 2003;1:682-689.

Updated version Access the most recent version of this article at: http://mcr.aacrjournals.org/content/1/9/682

Cited articles This article cites 46 articles, 14 of which you can access for free at: http://mcr.aacrjournals.org/content/1/9/682.full#ref-list-1

Citing articles This article has been cited by 28 HighWire-hosted articles. Access the articles at: http://mcr.aacrjournals.org/content/1/9/682.full#related-urls

E-mail alerts Sign up to receive free email-alerts related to this article or journal.

Reprints and To order reprints of this article or to subscribe to the journal, contact the AACR Publications Subscriptions Department at [email protected].

Permissions To request permission to re-use all or part of this article, use this link http://mcr.aacrjournals.org/content/1/9/682. Click on "Request Permissions" which will take you to the Copyright Clearance Center's (CCC) Rightslink site.