Protects Cancer Cells Against Hypoxia and Drug - Molecular Cancer ...

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 Peroxiredoxin-3 (Prdx3) is a mitochondrial member of the antioxidant family of thioredoxin peroxidases that uses mitochondrial thioredoxin-2 (Trx2) as a source of reducing equivalents to scavenge hydrogen peroxide (H2O2). Low levels of H2O2 produced by the mitochondria regulate physiological processes, including cell proliferation, while high levels of H2O2 are toxic to the cell and cause apoptosis. WEHI7.2 thymoma cells with stable overexpression of Prdx3 displayed decreased levels of cellular H2O2 and decreased cell proliferation without a change in basal levels of apoptosis. Prdx3-transfected cells showed a marked resistance to hypoxia-induced H2O2 formation and apoptosis. Prdx3 overexpression also protected the cells against apoptosis caused by H2O2, t-butylhydroperoxide, and the anticancer drug imexon, but not by dexamethasone. Thus, mitochondrial Prdx3 is an important cellular antioxidant that regulates physiological levels of H2O2, leading to decreased cell growth while protecting cells from the apoptosisinducing effects of high levels of H2O2.

Introduction The mitochondria are an important site for the production of cellular reactive oxygen species (ROS). The mitochondrial electron transport chains consume oxygen by oxidative phosphorylation to form cellular energy in the form of ATP. During this process, between 0.4% and 4% of the consumed oxygen is released in the mitochondria as ROS that include hydrogen peroxide (H2O2), superoxide (O2 ), singlet oxygen (O), and hydroxyl radical (HO ) (1). H2O2 is stable enough to diffuse out of the mitochondria and to have a cytoplasmic effect (2). Low levels of H2O2 are important to the cell because they regulate physiological processes such as receptor-mediated cell signaling pathways, normal cell proliferation, and transcriptional activation (3, 4). However, aberrant increases in

Received 1/21/03; revised 5/13/03; accepted 5/13/03. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Grant support: CA52995 and CA772049. Requests for reprints: Garth Powis, Arizona Cancer Center, University of Arizona, 1515 North Campbell Avenue, Tucson, AZ 85724-5024. Phone: (520) 626-6704; Fax: (520) 626-4848. E-mail: [email protected] Copyright D 2003 American Association for Cancer Research.

mitochondrial H2O2 can induce apoptosis by causing the release of proapoptotic factors from the mitochondria, such as cytochrome c and apoptosis-inducing factor, through the opening of a nonselective mitochondrial permeability transition pore (5 – 7). Increases in H2O2 can be caused by a variety of cell stresses including drug exposure and pathological conditions, particularly neurodegenerative diseases and diseases that cause hypoxic/ischemic episodes (8 – 10). Cells contain a variety of peroxidases including catalase, glutathione peroxidases, and peroxiredoxins (Prdxs) that control the constitutive levels of H2O2 in the cell and protect against ROS-induced damage by catalyzing the reduction of the H2O2 into water (11 – 13). There are six known mammalian Prdxs. Prdxs1 – 4 have two conserved cysteine residues, whereas Prdxs5 and 6 have only one of the cysteine residues involved in the peroxidase activity (14 – 16). In some species, Prdxs5 and 6 have additional cysteines in less conserved NH2-terminal regions of the protein that are required for peroxidase activity (17). Prdxs1 – 4 have high amino acid sequence homology with each other (60 – 80%) but lower homology to Prdxs5 and 6 (20%). Prdxs1 – 4 belong to the thioredoxin peroxidase subfamily and require the small redox protein thioredoxin (Trx) as an electron donor to remove H2O2, whereas Prdxs5 and 6 can use other cellular reductants, such as glutathione, as their electron donor (17, 18). Prdx1, Prdx2, and Prdx6 are found in the cytoplasm and nucleus (14, 19 – 21). Prdx3 contains a mitochondrial localization sequence, is found exclusively in the mitochondrion, and uses mitochondrial thioredoxin-2 (Trx2) as the electron donor for its peroxidase activity (22). Prdx4 has an NH2-terminal secretion signal sequence and is found in the endoplasmic reticulum and the extracellular space (23). Prdx5 is expressed as a long form associated with the mitochondria and a short form found with the peroxisomes (24), and a nuclear localization has also been reported (19). Prdx expression is increased in several human cancers. Prdx1 levels are increased relative to normal tissue 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). Prdx3 (MER5, SP-22, and AOP-1) was originally cloned out of murine erythroleukemia cells (29). Prdx3 expression is induced by oxidants in the cardiovascular system and is thought to play a role in the antioxidant defense system and homeostasis within the mitochondria (30, 31). Prdx3 is a c-myc target gene, and antisense experiments have shown that its expression is 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 Prdx3expressing 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, Prdx310, 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 Prdx3overexpressing cells (Fig. 2A). The mitochondrial membrane potential (DW) also showed a small significant decrease in the Prdx3-overexpressing cells (Fig. 2B). The expression of mitochondrial Trx2, thioredoxin reductase-2 (TrxR2), and other mitochondrial antioxidant proteins superoxide dismutase-2 (SOD-2) and catalase measured by Western blot analysis was not changed (Fig. 2C). Cell Proliferation Cell proliferation was significantly decreased in the Prdx3overexpressing 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 vector-transfected WEHI7.2/neo cells (P < 0.01 in both cases) (Fig. 3A). Cell cycle analysis revealed that there was no change in the fraction of cells in each phase of the cell cycle (Fig. 3B) and no change in basal rates of apoptosis (Fig. 3C). Protection Against Apoptosis Exposure of cells to hypoxia is known to increase cellular ROS and eventually to cause apoptosis (35). Prdx3-overexpressing WEHI7.2 cells were protected against hypoxiainduced apoptosis, with only 12 – 15% of the cells being apoptotic by 16 h in 1% O2 compared with 50% in the empty vector-transfected WEHI7.2/neo cells (P < 0.01 in both cases)

FIGURE 1. Stable transfection with Prdx3 shown by overexpression of Prdx3 mRNA, protein, and mitochondrial thioredoxin peroxidase activity in WEHI7.2 cells. A. Northern blot analysis on 2 Ag mRNA from WEHI7.2 cells stably transfected with empty vector (WEHI7.2/neo ) or Prdx3. A glyceraldehyde-3-phosphate dehydrogenase (GADPH ) probe was used as a loading control. Values are the levels of overexpression. B. Western blot analysis of Prdx3 protein in 20 Ag cell lysate and 10 Ag mitochondria from WEHI7.2/neo , Prdx3-10, and Prdx3-32 cells. Actin 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. Columns, means of three determinations; bars, SD. *P < 0.05, compared to WEHI7.2/neo cells.

(Fig. 4A). Annexin V positivity and uptake of propidium iodide was measured by fluorescence-activated cell sorting (FACS) analysis to distinguish live, apoptotic, and necrotic cells. Prdx3 overexpression completely prevented the hypoxia-induced increase in H2O2 seen in the WEHI7.2/neo cells (Fig. 4B). Transient overexpression of Prdx3 in HT-29 adherent solid tumor colon cancer cells showed a similar protection against hypoxia-induced apoptosis. After 36 h in 1% O2, HT-29 cells transfected with empty vector were 47.7 F 2.95% apoptotic, and Prdx3-transfected cells were 34.5 F 2.2% apoptotic (P V 0.01) (data not shown). Prdx3-overexpressing WEHI7.2 cells were protected against cell killing by H2O2, t-butylhydroperoxide, and imexon, a

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684 Prdx3 Protects Cells Against Apoptosis

droperoxide, but not to dexamethasone (Fig. 5). Fluorescent images of the Annexin V-positive cells showed intact membranes and apoptotic morphology after the drug treatments in empty vector-transfected WEHI7.2/neo and Prdx3overexpressing 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 Prdx3overexpressing 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 WEHI7.2 cells. A. CM-H2DCFDA fluorescence measurement of cellular H2O2 levels in the Prdx3-overexpressing cells compared with empty vector WEHI7.2/neo cells. Columns, means of three determinations; bars, SD. *P < 0.05, compared to WEHI7.2/neo cells. B. Tetramethylrhodamine methyl ester measurement of the DC in the Prdx3-overexpressing cells. Columns, means of three determinations; bars, SD. *P < 0.05, **P < 0.01, compared to WEHI7.2/neo cells. C. Levels of other mitochondrial antioxidant proteins in empty vector-transfected and Prdx3-transfected cells. Mitochondrial lysate (10 Ag) was probed by Western blotting for Prdx3, Trx2, TrxR2, Mn superoxide dismutase (MnSOD ), and catalase.

mitochondria damaging drug that increases mitochondrial H2O2 (36), but not by dexamethasone (Table 1). The increased cell survival was associated with a decreased apoptosis in the Prdx3-transfected cells after exposure to H2O2 and t-butylhy-

H2O2 levels are increased within in the cell in response to growth factors and act as an intracellular messenger (37). H2O2 inhibits protein tyrosine phosphatase function by the oxidation of key catalytic site cysteine residues, leading to activation of the kinase signaling (38). Blocking the accumulation of H2O2 by the addition of exogenous catalase, a cytoplasmic and mitochondrial peroxidase, prevents platelet-derived growth factor-induced mitogen-activated protein kinase activation (38). Treatment of cells with H2O2 mimics growth factor-induced signaling by inducing mitogen-activated protein kinase activation and stimulates cell growth (39). Probably because of the requirement for H2O2 in normal cell function, it is difficult to overexpress antioxidant proteins 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, representatives 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 2-fold increase in protein expression. Wonsey et al. (31) overexpressed Prdx3 in two different adherent cell lines and showed a similar low level of Prdx3 overexpression. In our

Table 1. IC50 Values Acquired From 48 h Cytotoxicity Curves IC50 Values (AM) Drug

Vector

Prdx3-10

H2O2 t -butylhydroperoxide imexon dexamethasone

60.28 23.2 92.32 0.022

172.23 30.92 136.82 0.024

**

F 11.1 F 0.7 F 6.59 F 0.004

F F F F

Prdx3-32 8.4** 0.2** 1.8** 0.004

230.75 35.79 135.47 0.025

F F F F

32.3** 0.7** 6.6** 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 hypoxiainduced 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.

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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, 50 AM 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 the growing tumor outstrips its blood supply (45). Prdx3 is overexpressed in some human cancers (19, 27) where it may protect the growing tumor against hypoxia-induced apoptosis due to increased H2O2 production. The results of the study suggest that mitochondrial Prdx3 is an important regulator of H2O2 in the cell. At low physiological levels of H2O2 formation, Prdx3 inhibits the growth-stimulating effects of H2O2. At higher levels of H2O2 formation, as seen in

hypoxia and caused by some drugs, Prdx3 protects cells against apoptosis. Elevated levels of Prdx3 could offer tumor cells a survival advantage in areas of hypoxia as well as protection against drug-induced apoptosis.

Materials and Methods Chemicals and Reagents All chemicals and reagents were obtained from SigmaAldrich (St. Louis, MO), unless otherwise stated. Imexon was


provided by Dr. Robert Dorr (University of Arizona). Primary antibodies used were rabbit polyclonal antibody against human Trx2, TrxR2, and Prdx3 (46), bovine catalase (Rockland, Gilbertsvill, PA), and monoclonal antibody against human Mn superoxide dismutase (Upstate Biotechnology, Inc., Lake Placid, NY). Cell Culture WEHI7.2 mouse thymoma cells were obtained from American Type Culture Collection (Rockville, MD). Cells were grown at 37jC in 5% CO2 in DMEM (MediaTech CellGro, Herndon, VA) with 10% fetal bovine serum. For hypoxia studies, 15 mM HEPES (pH 7.4) was added to the DMEM. Hypoxic conditions were set at 1% O2 using a Reming Bioinstruments chamber and oxygen regulator (Reming Bioinstruments, Redfield, NY). Prdx3 Cloning and Transfection Prdx3 was cloned out of human cDNA by PCR (primers: 5VGATCGCGGCCGCATGGCGCTGCTGTAGGAC-3V and 5V-

GATCGAATTCCTACTGATTTAACCTTCTGAAAGT-3V). The PCR product and pCMV-script vector (Stratagene, La Jolla, CA) were digested with EcoRI/NotI and ligated overnight at 14jC. Ligation and orientation were verified by sequence analysis. Empty vector or vector containing Prdx3 were transfected into WEHI7.2 cells by electroporation. Individual clones were selected with 1.2 mg/ml G418 in soft agar. RNA Purification and Northern Blot Analysis Total RNA was extracted from 108 cells using Trizol Reagent (Invitrogen, Carlsbad, CA). Polyadenylated RNA was purified from total RNA using Oligotex beads (Qiagen, Valencia, CA). Polyadenylated RNA (2 Ag) was separated by electrophoresis on a 1.2% agarose/4% formaldehyde gel and RNA was transferred to nitrocellulose membranes (Osmonics, Westborough, MA) for Northern blot analysis. cDNA probes were radiolabeled with 32P-a-dCTP (NEN, Boston, MA) with Random Prime Labeling Kit (Invitrogen). Blots were hybridized with radiolabeled probes overnight at 42jC in UltraHyb 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 icecold lysis buffer (0.5% NP40, 0.5% Na-deoxycholate, 50 mM NaCl, 1 mM EDTA, 1 mM NaVO3, 50 mM HEPES, pH 7.5, 1 mM phenylmethylsulfonyl fluoride), and incubated on ice for 20 min, with vortexing every 5 min before being cleared by centrifugation at 10,000 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.

FIGURE 6. Overexpression of Prdx3 protects against apoptosis and increased cellular H2O2 induced by imexon. A. FACS analysis of Annexin 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, means of three determinations; bars, SD. *P < 0.05, compared to empty vector 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. B. Effect of exposure to 150 AM imexon on cellular H2O2 levels measured by CM-H2DCFDA fluorescence. Filled bars are without imexon and empty bars are with imexon. Columns, means of three determinations; bars, SD. *P < 0.05, compared to WEHI7.2/neo cells.

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, 1 mM EDTA, 1 mM EGTA) for 30 min on ice. Cells were lysed by 15 strokes with a tight-fitting dounce homogenizer, then nuclei and unbroken cells were pelleted by centrifugation for 15 min at 600 g and 4jC. The supernatant was transferred to a fresh tube and mitochondria pelleted by centrifugation for 20 min at 12,000 g and 4jC. Mitochondria were resuspended in lysis buffer [100 mM Tris-HCl (pH 7.4)/ 0.1% Triton X-100] and broken by sonification for 15 s, and membranes were removed by centrifugation at 12,000 g for 15 min at 4jC. Mitochondrial protein was quantified using Bio-Rad Protein Reagent.

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Peroxidase Assay. Mitochondrial proteins (100 Ag) and lysis buffer containing 0.25 mM NADPH, recombinant human DTrx2 (Trx2 without the mitochondrial localization sequence), purified human placental thioredoxin reductase-1 (TrxR1) (47), and cleared mitochondrial lysate (100 Ag) were added and equilibrated at room temperature. The reaction was initiated with 1 mM H2O2 and oxidation of NADPH was measured at 339 nm for 3 min on Hitachi U-3310 spectrophotometer. Cytotoxicity Cells (5 103) were seeded in 24-well plates in 1 ml DMEM/10% fetal bovine serum/1.2 mg/ml G418. Drugs were added at increasing concentrations and cells were allowed to grow for 48 h before trypan blue was added. Toxicity was measured by cell counting of live cells that did not take up trypan blue. Apoptosis Cells (12.5 105) were treated with drug for described times 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 CaCl2) containing Annexin V-Fluos (Boehringer Mannheim, Roche Applied Science, Indianapolis, IN) and 50 mg/ml propidium iodide (Molecular Probes, Eugene, OR). Cells were analyzed on a FACScan (Becton Dickinson, Franklin Lakes, NJ) using 488 nm excitation by an argon laser and a 515-nm bandpass filter for fluorescein detection and a filter >560 nm for propidium iodide detection. Compensation settings for FACS were FL1- 1.6% FL2, FL236.5% FL1, FL2- 0.0% FL3, and FL3- 22.6% FL2. When the data are analyzed by dotplot with X axis = Annexin V and Y = propidium iodide fluorescence, live cells are found in lower left quadrant, necrotic cells in upper left quadrant, and apoptotic cells in right quadrants. For microscopic images of Annexin V-Fluos, cells were attached to slides using Cell Tak (BD Biosciences, San Jose, CA) for 30 min in Annexin V-Fluos containing calcium buffer. Cells were fixed in 4% formaldehyde/PBS then mounted using ProLong Mounting Media (Molecular Probes). Annexin was visualized at 488 nm and DNA autofluorescence was visualized at 350 nm. Cell Cycle Analysis Cells (12.5 105) were pelleted and washed with PBS. Icecold ethanol was added, and the cells were pelleted and rehydrated in PBS before being resuspended in 100 mM TrisHCl (pH 7.4), 150 mM NaCl, 1 mM CaCl2, 0.5 mM MgCl2, and 0.1% NP40 containing 3 mM propidium iodide. Cell cycle was analyzed on a FACScan. H2O2 Assay Cellular H2O2 was measured by a modification of the method of Eposti (48). Cells (104 – 105) were washed three times in PBS and incubated in DMEM without phenol red (MediaTech CellGro) containing 5 AM 5-(and 6-)chloromethyl2V,7V-dichlorodihydrofluorescein diacetate, acetyl ester (CMH2DCFDA) (Molecular Probes) for 30 min at 37jC. Excess CM-H2DCFDA was removed from the cells by a wash in PBS.

Dichlorohydrofluorescein levels in cells were measured on a Gemini XS microplate spectroflourometer (Molecular Devices, Sunnyvale, CA) using 488 nm excitation and a 520 nm emission for detection. H2O2 levels were quantitated by comparison with a standard curve generated using dichlorohydrofluorescein. Mitochondrial Membrane Potential Cells (105) were incubated in phenol red-free DMEM containing 20 nM tetramethylrhodamine methyl ester (Molecular Probes) for 30 min at 37jC. Cells were analyzed and fluorescence was measured on a FACScan using 488 nm excitation and a 515-nm bandpass filter for detection of tetramethylrhodamine methyl ester.

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 Seaver from the shared sources at the Arizona Cancer Center. The statistics were performed at the Arizona Cancer Center Biometry Shared Service by Dr. Haiyan Cui.

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