Impaired mitochondrial function protects against free radical-mediated cell death

Free Radical Biology & Medicine, Vol. 33, No. 9, pp. 1209 –1220, 2002 Copyright © 2002 Elsevier Science Inc. Printed in the USA. All rights reserved 0891-5849/02/$–see front matter

PII S0891-5849(02)00984-X

Original Contribution IMPAIRED MITOCHONDRIAL FUNCTION PROTECTS AGAINST FREE RADICAL-MEDIATED CELL DEATH DARLENE DAVERMANN,* MARCIA MARTINEZ,* JUDITH MCKOY,* NIMA PATEL,* DIETRICH AVERBECK,† CAROL WOOD MOORE*

and

*Department of Microbiology and Immunology, City University of New York Medical School/Sophie Davis School of Biomedical Education and Graduate Programs in Biochemistry and Biology, New York, NY, USA; and †Institut Curie-Section de Recherche, Centre Universitaire d’Orsay, Orsay, France (Received 13 December 2001; Revised 17 April 2002; Accepted 6 June 2002)

Abstract—Free radical damage can have fatal consequences. Mitochondria carry out essential cellular functions and produce high levels of reactive oxygen species (ROS). Many agents also generate ROS. Using the yeast Saccharomyces cerevisiae as a eukaryotic model, the role of functional mitochondria in surviving free radical damage was investigated. Respiratory-deficient cells lacking mitochondrial DNA (␳0) were up to 100-fold more resistant than isogenic ␳⫹ cells to killing by ROS generated by the bleomycin-phleomycin family of oxidative agents. Up to approximately 90% of the survivors of high oxidative stress lost mitochondrial function and became “petites.” The selective advantage of respiratory deficiency was studied in several strains, including DNA repair-deficient rad52/rad52 and blm5/blm5 diploid strains. These mutant strains are hypersensitive to lethal effects of free radicals and accumulate more DNA damage than related wild-type strains. Losses in mitochondrial function were dose-dependent, and mutational alteration of the RAD52 or BLM5 gene did not affect the resistance of surviving cells lacking mitochondrial function. The results indicate that inactivation of mitochondrial function protects cells against lethal effects of oxygen free radicals. © 2002 Elsevier Science Inc. Keywords—Free radicals, Reactive oxygen species, ROS, Oxidative, Mitochondrial damage, Petite, Saccharomyces cerevisiae, Bleomycin, Phleomycin, RAD52, BLM5

INTRODUCTION

is associated with the inner mitochondrial membrane where oxidative phosphorylation occurs and large amounts of ROS are produced. In fact, mutations caused by damage to mtDNA are associated with a variety of human diseases including degenerative and aging disorders, cancers, diabetes mellitus, and Parkinson’s [6 –14]. Oxygen free radical damage to the mitochondrial genome of the yeast Saccharomyces cerevisiae can render mtDNA nonfunctional and the mitochondria functionally defective, resulting in respiratory deficiency. These mutant cells lack mitochondrial protein synthesis [15–17] and grow “petite” rather than “grande” (␳⫹). Cytoplasmic petite mutants can be devoid of mtDNA (␳0) or retain a small fraction of their mtDNA (␳⫺). Mitochondrial DNA comprises approximately 5% to 15% of the total DNA in ␳⫹ and ␳⫺ Saccharomyces cerevisiae [18, 19], but can become as high as 25% of total cellular DNA [20]. Distinguishing properties of ␳⫺ and ␳0 cells permit

Oxidative reactions produce free radicals that are deleterious to cells [1– 4]. Thus, to understand mechanisms that cells use to survive free radical damage as well as the cellular actions of agents that generate high levels of reactive oxygen species (ROS) is of considerable biological and medical importance. High levels of endogenous ROS are produced during mitochondrial oxidative phosphorylation. The major source of superoxide radicals (O2⫺) and H2O2 during cellular energy metabolism is the mitochondrial electron transport chain [2,5]. Thus, mitochondria are exposed to these high levels of ROS. Mitochondrial DNA (mtDNA) Address correspondence to: Dr. Carol Wood Moore, City University of New York Medical School/Sophie Davis School of Biomedical Education and Graduate Programs in Biochemistry and Biology, Department of Microbiology and Immunology, Convent Avenue at 138th Street, New York, NY 10031, USA; Tel: (212) 650-6926; Fax: (212) 650-7797; E-Mail: [email protected]. 1209

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D. DAVERMANN et al. Table 1. Saccharomyces cerevisiae Strains

Strain A364A CM1069-40 CM-1293 CM-1477 CM-1489 CM-1492 STX433 STX434

Genotype at BLM5, RAD6, and RAD52 loci

Source or reference

BLM5, RAD6, RAD52 BLM5, RAD6, RAD52 BLM5/BLM5, RAD6/RAD6, RAD52/RAD52 BLM5/blm5-1, RAD6/RAD6, RAD52/RAD52 BLM5/BLM5, RAD6/RAD6, RAD52/RAD52 blm5-1/blm5-1, RAD6/RAD6, RAD52/RAD52 BLM5/BLM5, rad6-1/rad6-1, RAD52/RAD52 BLM5/BLM5, RAD6/RAD6, rad52-1/rad52-1

Yeast Genetic Stock Center Moore and Schmick, 1979 [109] Moore and Schmick, 1979 [109] This study This study This study Yeast Genetic Stock Center Dr. John Game, Yeast Genetic Stock Center

their detection and direct measurement among populations of ␳⫹ cells [21–23], enabling S. cerevisiae to be used as an in vivo eukaryotic cellular model for studying genomic instability in mitochondria and loss of mitochondrial function [24]. In the current study, ␳0 diploids were derived from their wild-type ␳⫹ parent to examine the influence of the loss of the mitochondrial genome on cellular survival after free radical damage. In addition, we determined the influence of free radical damage on mitochondrial function and killing, and examined the roles of two important genes and their encoded proteins that protect cells against free radical damage. Mutational alteration of the RAD52 or BLM5 gene confers exquisite hypersensitivity to killing by free radical damage, e.g., by oxidative DNA-damaging agents, and a deficiency in repairing nuclear DNA double-strand breaks in homozygous mutant diploid strains [25–35a]. Thus, these strains accumulate more chromosomal damage than strains bearing the corresponding normal genes. Also, homozygous blm5/blm5 mutant strains usually exhibit low spontaneous frequencies of petite mutants compared to genetically related homozygous normal BLM5/BLM5 or heterozygous BLM5/blm5 strains. The low molecular weight bleomycin group of metalloglycopeptides and anticancer antibiotics (Mr approximately 1550) cleaves DNA in an oxygen-dependent manner and causes the formation of superoxide (O2⫺) and hydroxyl (OH䡠) radicals [36 –39]. The site of oxygen activation on the metal-chelating bleomycin molecule is at the metal-binding domain, which is attached to a disaccharide group and binds redox-active transition metals [40 – 44]. Not only do the structurally complex bleomycins attack deoxyribose in DNAs by a free radical mechanism, the redox cycling of bleomycins themselves produces radicals [38,39]. As such, they were utilized in the current investigation along with structurally related phleomycin as useful model compounds for generating ROS in strains with varying resistance to killing. Gamma irradiation and the bleomycin-phleomycin chemical group cause some of the same types of free radical damage in DNA. Chromosomal DNA breaks after ␥ irradiation and bleomycin treatments increase approximately linearly with increasing doses of ␥ rays

[45] or bleomycin (e.g., bleomycin A2 or B2 [46]) in S. cerevisiae, and pathways are shared for the repair of nuclear DNA damage by ionizing radiation, bleomycins, and phleomycins [26 –28,34,35,45,47,48]. In comparison to some other genotoxic agents, such as ultraviolet light and psoralens, ␥ irradiation produces little damage to mtDNA [49 –52]. Thus, most mitochondrial genomes in surviving cells remain functional, and cells are respiratory proficient. In contrast, the results in this paper indicate that cells that have lost mitochondrial function are more resistant to the lethal effects of ROS generated by bleomycin and phleomycin than cells with functional mitochondria, and they constitute a high proportion of the survivors of high levels of ROS. MATERIALS AND METHODS

Yeast strains and culturing conditions The strains used in this study are listed in Table 1. Strains were routinely grown with aeration in nonsynthetic complete medium (YPAD [45,46] at 30°C). Fresh cells were inoculated into YPAD at 5 ⫻ 106 cells/ml and grown to the early stationary phase of growth (about 2 ⫻ 108 cells/ml). The growth phase was determined from growth curves for each strain. Treatments For bleomycin or phleomycin treatments, procedures were adapted from our published protocols [35,45,46]. Except where noted, cells were harvested by centrifugation at 4°C, washed three times, resuspended at 1 ⫻ 107 cells/ml of deionized water (pH 5), and incubated with or without drug for 20 or 30 min with aeration in sterile deionized water at 4°C. Bleomycins (Blenoxane, BristolMyers Squibb Co., Syracuse, NY, USA, and Evansville, IN, USA) were dissolved and diluted in deionized water (pH 5) just prior to use. Absorbance of bleomycin (Mr approximately 1500 –1600 [53,54]), whose extinction coefficient is 1.45 ⫻ 104, was monitored at 292 nm. The phleomycin D1 formulation was purchased from Invitrogen (Carlsbad, CA, USA) as a 1/20 dilution of phleomy-

Protection against ROS

cin (Zeocin). EDTA was added at the end of each incubation period to a final concentration of 0.025 M to minimize further drug activity. Cells were pelleted, washed three times in 0.025 M EDTA, diluted, and plated on YPAD medium. If cells were treated in phosphate buffer, EDTA was not added since phosphate buffer ameliorates lethal effects of bleomycin [55]. Gamma irradiation was conducted according to our published procedures [35,45]. Cells were grown and washed as for drug treatments and suspended at 107 cells/ml. Irradiation was carried out on ice in a cobalt-60 irradiator (J. L. Shepard and Associates, Glendale, CA, USA) kindly made available by Dr. Christopher Lawrence (University of Rochester, Rochester, NY, USA). The dose rate was determined from the decay constant of 60 Co after periodic ferrous sulfate dosimetry. Unirradiated and irradiated cells were immediately pelleted by centrifugation at 4°C and converted to spheroplasts as described previously [35,45,46]. [2-14C]- and [6-3H]-prelabeled DNAs and sedimentation of DNAs These procedures were carried out as previously described [35,45,46]. Cells were grown approximately 14 h from starting inocula of fresh cells (5 ⫻ 106 cells/ml) in supplemented synthetic minimal medium [56,57]. The [2-14C] uracil (specific activity, 40 to 60 Ci/mmol; New England Nuclear Corp., Boston, MA, USA) was added to 5 ␮Ci/ml or [6-3H] uracil (specific activity, 20 to 30 Ci/mmol; New England Nuclear Corp.) was added to 7 ␮Ci/ml. The [2-14C]- and [6-3H]-prelabeled DNAs were sedimented together through precalibrated, isokinetic alkaline sucrose gradients [25,35,45,46,58 – 60]. RNA was hydrolyzed completely after velocity sedimentation. No differences were observed in replicated experiments between profiles of [2-14C]- or [6-3H]-prelabeled DNAs.

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remained white, while ␳⫹ colonies turned red or pink. Throughout the studies, no sectored colonies were observed. To confirm respiratory deficiency, representative white colonies were replated on YPAD and the same medium in which dextrose was replaced with the nonfermentable carbon source, glycerol. RESULTS

Spontaneous petite mutations The spontaneous frequencies of petite mutants were investigated in six diploid strains constructed from different genetic backgrounds. As shown in Table 2, spontaneous frequencies were low, ranging from less than 0.03% to approximately 0.6% for all but one of the strains. The spontaneous frequency was more than 2% in the DNA repair-deficient rad52-1/rad52-1 diploid. Derivation of ␳0 strains and their enhanced resistance to oxidative injury One ␳0 diploid strain was derived from the wild-type CM-1293 ␳⫹ diploid after treatment with ethidium bromide. The technique of velocity sedimentation of cellular DNAs through precalibrated, isokinetic alkaline sucrose gradients at low centrifugal speeds was then used to confirm that the petite strain was devoid of mtDNA. Figure 1 illustrates the sedimentation profiles of nuclear and mtDNAs from the ␳⫹ strain and only nuclear DNA from the ␳0 strain. In the gradients, the nuclear DNA sediments in a major peak (circa Mr [2–2.4] ⫻ 108) approximately 70 –75% from the top of the gradients, and mtDNA sediments in a minor peak in the top 25% under the sedimentation conditions routinely used (8000 rpm for 19.5 h at 20°C). Both peaks are present in DNAs from the ␳⫹ strain, but only the major peak of nuclear Table 2. Spontaneous Frequencies of Petite Mutants

Petite mutagenesis assays Respiratory-deficient cytoplasmic petite mutants were identified by the tetrazolium overlay technique [61,62]. In this assay, nuclear petites constitute less than 1 ⫻ 10⫺7 of the populations of respiratory-deficient cells. A 0.1% solution containing 2,3,5 triphenyl-tetrazolium chloride (TTC; Sigma Chemical Co., St. Louis, MO, USA) was used. Bacto-agar (1.5%) was dissolved in 0.067 M sodium phosphate buffer at pH 7.0, autoclaved, and cooled to 55– 60°C. The TTC in deionized water (1 g/100 ml) was then added. This mixture was gently poured on the agar plates containing colonies, and the plates were incubated for at least 1 h at 30°C. The red and white colonies were counted. The ␳⫺ and ␳0 colonies

Strain number CM-1293 CM-1293 CM-1477 CM-1477 CM-1477 CM-1477 CM-1489 CM-1489 CM-1492 CM-1492 STX433 STX434

Experiment 1 2 1 2 3 4 1 2 1 2 1 1

Total number Number of Frequency of of colonies petite colonies petite colonies 3761 4180 3103 3338 1661 2866 1803 2298 2648 2701 491 194

0 26 9 8 3 10 8 15 1 1 3 5

⬍ 0.00027 0.0062 0.0029 0.0024 0.0018 0.0035 0.0045 0.0065 0.00038 0.00037 0.006 0.025

For each experiment, the strain was grown to the early stationary phase of growth and washed three times. Cells were then diluted and plated on YPAD medium and incubated 3 d at 30°C.

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Fig. 1. Sedimentation profiles of mitochondrial and nuclear DNAs from early stationary phase cells. Œ ⫽ ␳⫹ cells (strain CM-1293); ⽧ ⫽ ␳0 cells (strain CM-1293-4). The ␳0 cells have lost the DNA fraction corresponding to the mtDNA.

DNA is present in the ␳0 strain. A spontaneous ␳0 diploid strain was also isolated for these studies from the ␳⫹ CM-1293 diploid and found to be devoid of mitochondrial DNA. The grande and petite strains were studied under a variety of treatment doses and conditions. Unexpectedly, ␳0 cells were found to exhibit substantially higher resistance than ␳⫹ cells to killing by the oxidative DNAdamaging agent. This is illustrated in Fig. 2. While the plating efficiencies of both types of cells were identical if the cells were not exposed to the drug, the survival of ␳0 cells was up to 100-fold higher than the survival of ␳⫹ cells after exposure to bleomycin. Breaks in mtDNA after treatments with oxidative DNA-damaging agents Efforts were made to simultaneously monitor breaks introduced into mitochondrial and nuclear DNAs of ␳⫹ cells by sedimenting the DNAs in precalibrated, isokinetic alkaline sucrose gradients. The single-strand breaks (SSBs) assayed in this system include closely and more distantly opposed SSBs. Alkali-labile lesions left in DNA by the release of free bases are also converted to DNA breaks, and thus are included in the SSBs assayed in this system. Figure 3 (panel A) illustrates typical profiles of nuclear and mitochondrial DNAs from ␳⫹ strains. Profiles of DNAs from one haploid and one diploid strain are

shown. Using the strain for which the mitochondrial peak was more discrete, the expectation was that low numbers of breaks introduced into the mitochondrial fraction could be detected and quantitated as they were previously for nuclear fractions [35,45]. A small change in the sedimentation of DNAs in the region of the profile containing the mtDNA would correspond to a substantial change in molecular weight. Thus, the DNAs were analyzed from cells exposed to low concentrations of bleomycin (Fig. 3, panel B) and structurally related phleomycin (Fig. 3, panel C). For comparison, DNAs from cells exposed to an equitoxic dose of ␥ irradiation (Fig. 3, panel D) were also studied. Both mitochondrial and nuclear DNAs from treated cells (panels B–D) appeared to sediment in lower molecular weight fractions than the corresponding DNAs from untreated cells (panel A), indicating damage to both DNA fractions in the cells. The lowest molecular weight fractions contained more DNA after bleomycin or phleomycin treatment than after ␥ irradiation, suggesting fewer breaks may have been introduced into the mtDNA after the drug treatments than after irradiation. Moreover, the peaks of mtDNA fractions remained more distinct in profiles after drug treatments than after irradiation. These findings were confirmed by adjusting the sedimentation conditions to retain only mtDNAs in the middle of the gradients (profiles not shown). In spite of using a variety of sedimentation conditions, however, the actual molecular weights of the mtDNA fractions from treated cells could not be accurately determined. Dose dependency of losses of mitochondrial function The knowledge that ␳0 cells were more resistant than isogenic ␳⫹ cells to lethal effects of bleomycin together with the detection of bleomycin and phleomycin damage to mtDNA led to the hypothesis that the loss of mitochondrial function may be a favored way of surviving free radical damage caused by the oxidative treatments. To test this hypothesis, the frequencies with which mitochondria lost function were measured over a wide range of drug doses and in a variety of strain backgrounds. Frequencies of petites were consistent from experiment to experiment for a particular strain, but not comparable for all of the strains. Frequencies in a particular strain appeared to depend on the resistance of the strain to killing by the oxidative agents. This is illustrated in Fig. 4 by a comparison of two extremes. In Fig. 4, the typical response of the strains we tested is illustrated in panel A. This strain displays normal resistance to killing by bleomycin. Typically, petite frequencies were dose dependent and increased sharply (Fig. 4, panel A) as survival decreased (Fig. 4, panel C) from 0 ␮g/ml to 1.0 ␮g/ml. Frequencies of petite mutants usually reached a

Protection against ROS

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Fig. 2. Comparisons of responses of isogenic ␳⫹ and ␳0 diploid strains to killing by bleomycin. The ␳⫹ (CM-1293) and ␳0 (CM-1293-4 and CM-1293-81) strains were grown to stationary phase and washed three times prior to treatments. (A–C) CM-1293 ␳⫹ and CM-1293-4 ␳0. Three independent experiments. Cells were incubated at 20°C for 20 min with 0, 50, 75, and 100 ␮g/ml bleomycin in 0.05 M phosphate buffer. (D) CM-1293 ␳⫹ and CM-1293-81 ␳0. Cells were incubated at 30°C for 30 min with 0, 75, 100, and 150 ␮g/ml bleomycin in deionized water. After treatments, cells were washed again, diluted, and plated. For determining plating efficiencies, cells were handled exactly as for treatments except that no drug was added during the incubation period.

plateau when they reached between 50% and 90% of the surviving population, and this plateau was accompanied by a plateau in the fraction of the population that survived. For contrast, a strain that is unusually highly resistant to killing by the very high doses of 10 to 50 ␮g/ml bleomycin is shown in Fig. 4, panels B and D. Petites were not found above spontaneous levels in this strain. Role of the RAD52 and BLM5 gene products The finding that the frequencies of cells that lost mitochondrial function were high among the survivors of oxidative treatments led us to examine additional wildtype strains and to investigate the role of the RAD52 and BLM5 genes. Mutations in these genes cause cells to become hypersusceptible to lethal effects of oxidative DNA damaging agents and to accumulate higher levels of DNA damage than wild-type strains [27,28,35,35a]. Thus, we determined if mutational alteration of the genes influenced the resistance of cells with lost mitochondrial

function. In fact, petite mutants were consistently produced at far higher frequencies in the rad52/rad52 and blm5/blm5 mutant strains after low treatment doses than in the three strains with normal resistance. This initially suggested that the mutations influenced damage to the mitochondrial genome. However, since drug treatments caused higher killing of the hypersensitive mutant strains than the other strains, it was deemed better to compare frequencies of lost mitochondrial function among strains with differing susceptibilities to killing as a function of the fractions of the cell populations that survived the oxidative treatments rather than as a function of treatment doses. By doing this, the effects of equitoxic treatments on mitochondrial function could be compared among the different strains. The results for wild-type and mutant strains are plotted in Fig. 5. What is apparent is that between 100% and 10% survival, mitochondrial function was retained in nearly all of the survivors. Yet, remarkably, frequencies of petite mutants rose sharply in all strains when survival

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Fig. 3. Sedimentation profiles of mitochondrial and nuclear DNAs from untreated and treated ␳⫹ cells. (A) Early stationary phase, no treatment. CM-1293 diploid strain (Œ) and CM1069-40 haploid (E). For comparisons, the CM-1293 profile from A (Œ) is repeated in B–D. (B) ƒ early stationary phase CM-1293 cells treated with 0.2 ␮g/ml bleomycin for 30 min with aeration. (C) ƒ logarithmic phase CM-1293 cells treated with 0.2 ␮g/ml phleomycin for 30 min with aeration. (D) ƒ early stationary phase CM-1293 cells treated with 100 Gy.

dropped below ten percent. Petites reached nearly 90% among strains with wild-type resistance and 75% among the mutant strains. All data points appeared to fall along a similar steep sigmoidal curve when survival was between 10% and 1% (Fig. 5), indicating genotype was not a contributing factor to the resistance of petite strains.

function when phleomycin D1 was used to produce oxidative stress. Despite the fact that the commercial (diluted) phleomycin D1 formulation used in these studies was less cytotoxic than the more concentrated mixture of phleomycins and the clinical formulation of the bleomycin congeners, petite mutants were more resistant than ␳⫹ cells.

Protection against phleomycin D1: a structural analog of bleomycin

DISCUSSION

Finally, we determined whether the high losses of mitochondrial function were uniquely found in bleomycin-treated populations or if they also were found among survivors of treatments with the phleomycin D1 structural analog. Phleomycin causes considerably more killing and DNA damage than bleomycin among yeast strains [27,46,63,64]. Table 3 summarizes the frequencies of survivors and the fractions of the survivors that lost mitochondrial

Cells lacking their entire mitochondrial genome were remarkably more resistant than cells with functional mitochondria to killing by ROS and oxidative stress. Mitochondrial function was inactivated in up to approximately 90% of the survivors after short exposures to bleomycin and structurally related phleomycin, while frequencies of respiratory deficiency were low in the absence of treatment. The fact that no sectored colonies were found among the petite colonies indicates that mi-

Protection against ROS

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Fig. 4. An example of the dose dependency of respiratory deficiency after short drug exposures in strains with normal resistance to killing by bleomycin, and an example of the lack of petites in highly resistant strains. Frequencies of petites are illustrated as a function of bleomycin dose for two related diploid strains with differing resistance to killing by bleomycin. Strains were grown to stationary phase and treated for 30 min as described in Fig. 2. (A) and (C) CM-1477 (normal resistance); (B) and (D) CM-1489 (a highly resistant strain). Open and closed symbols are the data from independent experiments. For each data point, the frequencies of petite mutants in untreated cell populations were subtracted from the frequencies of petite mutants among the survivors in the treated populations.

tochondrial damage was induced in the mother cells and these cells were unable to transmit functional mitochondria to their daughter cells. This finding is similar to that observed with drugs such as ethidium bromide interacting directly with mtDNA [65]. Thus, the oxidative bleomycin-phleomycin family, like ethidium bromide, acted differently from euflavine [66] or monofunctional furocoumarins [67] that do not transform mother cells but do efficiently transform daughter cells or buds into petites [67]. Since the ␳⫹ cells were killed preferentially, inactivation of mitochondrial function appears to favor survival in the presence of high quantities of ROS. In turn, bleomycin or phleomycin would not become activated in the absence of adequate amounts of oxygen and without cytochromes. The results indicate that the mitochondria certainly are sensitive targets of ROS produced by the action of the bleomycin-phleomycin family of drugs. We conclude that resistance to killing by ROS is enhanced by inactivating mitochondrial function. Free radicals and ␳⫹ vs. ␳⫺ and ␳0 cells A reason that ␳0 and ␳⫺ cells were more resistant than ␳ cells is undoubtedly due to the complicated free ⫹

Fig. 5. Comparisons of petite induction among five diploid strains as a function of survival. Experiments were carried out as for Figs. 2D, 3, and 4, and total survivors as well as the numbers of petites were calculated as for Figs. 2 and 4. Results of sixteen independent experiments are plotted. The results for the five different strains are represented by the five symbols. The strains, all examined at the early stationary phase of growth, exhibit a wide range of resistance. For each data point, the frequencies of petite mutants in untreated cell populations were subtracted from the frequencies of petite mutants among the survivors in the treated populations. ⽧ ⫽ CM-1293; Œ ⫽ CM-1477; ● ⫽ CM-1489; ■ ⫽ CM-1492; octagon ⫽ STX434.

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Table 3. Loss of Mitochondrial Function Among Phleomycin D1–treated Cells Dose (␮g/ml) 0 50 100 200

Total numbers of cells

Surviving fractions

Numbers of petite mutants

Frequencies

3761 30 68 43

(100%) 0.77% 0.37% 0.11%

0 12 46 31

⬍ 0.03% 40% 67.7% 72.1%

Cells (strain CM-1293) were grown to the early stationary phase before 30 min treatments. Experiments were carried out as for Fig. 3–5, except that phleomycin D1 (Zeocin) was used instead of bleomycin.

radical chemistry involving oxygen free radicals induced by mitochondrial metabolism and by the mechanisms of action of the bleomycin and phleomycin structural analogs. Mitochondria contain large amounts of oxygen, and free radicals accumulate. The mechanism of action of the analogs with DNA is oxygen dependent and causes the formation of O2⫺ and OH䡠 radicals [37–39,68]. Moreover, a dysfunctional electron transport system could generate additional ROS [69]. Thus, we propose that the synergy of free radicals from both sources confronts the cells with an “oxidative load,” and that reducing or eliminating the high levels of ROS in mitochondria would reduce oxidative stress. Accordingly, cells that lose mitochondrial function would have a selective survival advantage. The data presented in the current report support this view and clearly indicate that functional mitochondria are not required for cellular protection against the lethal effects of the bleomycin-phleomycin antibiotics. The precalibrated, isokinetic alkaline sucrose gradients in Fig. 3 were probably not optimized for quantitating damage to mtDNA. Even if they were, losses in mitochondrial function could result from mutational alterations, which would not change the molecular weight of mtDNA. MtDNA vs. nuclear DNA: lack of nucleosomes in mtDNA The fact that yeast mtDNA is not “protected” by the types of histones or other proteins associated with nuclear DNAs could make mtDNA more vulnerable than nuclear DNAs to free radical damage. It is well known that bleomycin preferentially cleaves between nucleosomes in nuclear DNAs [64,70 –73] where the DNA is “unprotected.” Previously, mtDNA damage was found to be more extensive than nuclear DNA damage in human cells following oxidative stress [74]. Ionizing radiation vs. bleomycin and phleomycin Initially, our working hypothesis was that frequencies of petite mutants after bleomycin and phleomycin treat-

ments would be low since the oxidative agents are socalled “radiomimetic” in yeast [26,27] and ␥ irradiation is a poor inducer of petite mutants [52,75]. Bleomycin was previously classified as a weakly effective petite mutagen [76] in S. cerevisiae growing in the presence of the drug— experimental conditions quite different from those used in the current studies where cells were not grown with the drug. In another previous study, deletion of a single gene involved in cytochrome oxidase biogenesis in S. cerevisiae, OXA1 [77], led to a 4-fold increase in sensitivity to bleomycin [48]. Using the experimental conditions in the current study, the drug family could be used to enrich for cells with impaired mitochondrial function. Reasons for the major differences between ionizing radiation and the bleomycin-phleomycin family are undoubtedly multiple. Firstly, ␥ irradiation, unlike bleomycin, does not preferentially cleave DNA between nucleosomes; thus, nuclear DNA and mtDNA are likely similarly susceptible to damage by ␥ irradiation. Secondly, the nature of the DNA damage caused by the two agents is different. For example, more double-strand breaks (DSBs) are produced in S. cerevisiae chromosomes after bleomycin treatments than after equitoxic ionizing radiation [35]. In mammalian cells, bleomycin causes SSBs and DSBs at a ratio of 5:1 while ␥-rays induced only one DSB for every 20 SSBs [38]. Similarly, bleomycin was more potent than x-rays in inducing whole-chromosome loss in lymphocytes [78]. Thirdly, only small amounts of oxygen would be expected in the nucleus, where both ␥ irradiation and bleomycin efficiently cleave DNA. This is not the case in the mitochondria where ␥ irradiation is a poor inducer of petite mutants [49 –52]. Maintenance of mtDNA: efficient repair pathways for oxidative damage to mtDNA At the outset of this study, we expected that mutational alteration of the RAD52 or BLM5 genes would cause increased mtDNA damage in comparison to wildtype cells since the mutations render cells hypersensitive to DNA damage and killing by ionizing radiation, bleomycin, and phleomycin. The finding that comparable frequencies of petite mutants were found in bleomycintreated RAD52/RAD52 and rad52/rad52 strains is quite different from earlier results obtained after 254 nm UV irradiation with less well-defined recombination-deficient strains [79]. In those studies, a significantly lower frequency of petites per unit dose and per survivor was obtained in the mutant strains than in the corresponding wild-type strain. The repair of bulky adducts by nucleotide excision repair (NER), but not base excision repair (BER), is

Protection against ROS

deficient in mitochondria [80 – 83]. The lack of the NER mechanism for lesions induced in mtDNA parallels what has been observed in bifunctional furocoumarins plus UVA-induced damage in treated cells [84]. In the latter case, the damage induced in nuclear DNA appeared to be processed by nuclear excision repair and recombination, allowing cells to survive, whereas in mtDNA lesions accumulated and led to respiratory deficiency. The maintenance of mtDNA requires that it be replicated and packaged into nucleoids, then segregated from each other and partitioned into the buds and daughter cells. Normal cellular respiration requires that cells resulting from mitotic division receive a sufficient number of functional mtDNA molecules. S. cerevisiae contains an average of 50 to 100 copies of the 80 kb mitochondrial genome, depending on growth conditions and ploidy [85]. Replication of the mtDNA occurs throughout the cell cycle in association with extensive recombination [86 – 88]. The RAD52 [52] and REV2 and REV3 [89] genes were among the DNA repair genes influencing the induction of petites, although petite induction after ␥ irradiation in rad52/rad52 diploids resulted from the indirect effect of chromosome losses [52]. Indeed, several lines of evidence point to the existence of efficient repair pathways in mitochondria dealing with oxidative damage. Certainly the Mgm101p in yeast has an essential role in the repair of oxidatively damaged mtDNA and is likely to be important for the maintenance of mtDNA [90]. Furthermore, DNA base excision repair N-glycosylases are involved in the repair of oxidative damage, high-fidelity replication, and recombination of mtDNA [91]. These enzymes include the Ntgp [92], the Mip1p, a pol-␥ DNA polymerase with polymerase and 3⬘ to 5⬘ exonuclease activity [93], the mismatch repair protein Msh1p [94,95], the singlestrand-binding protein Pif1p [96], the helicase Abf2p [97], and the endonuclease Mgt1p encoded by the MHR1 gene [98]. Functional mitochondria appear to require the active MHR1 to keep the spontaneous oxidative damage in mtDNA tolerable [99]. Recently, it has been shown that the Ogg1p (Ntg1p) plays an important role in the protection of the mitochondrial genome against spontaneous and induced oxidative damage [100]. The Ogg1p is involved in the repair of oxidative damage such as 8-hydroxyguanine, 2,6-diamino-4-hydroxy-5(N-methylforamamido) pyrimidine, 2,6-diamino-4-hydroxy-5-formamidopyridine (Fapy), and 7,8-hydroxy-8-oxoadenine placed opposite a cytosine or a 5-methyl cytosine. In addition, the Ogg2p is involved in the repair of a large variety of oxidative lesions including 8-hydroxyguanine, thymine glycol, dihydrothymidine, dihydroxyuracil, and acts as a suppressor of oxidative mutagenesis in mitochondria [100]. Finally, recovery from spontaneous and induced

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mitochondrial damage appears to rely on a single DNA ligase, Cdc9p [101]. These results strongly suggest that in the yeast S. cerevisiae, as in mammalian cells, the BER pathway is operating on oxidative damage in mtDNA. Chemotherapy The current study indicates that mitochondrial damage could be an important activity of the bleomycins in chemotherapy. Since bleomycins are useful both as a single therapeutic agent in treating several human cancers and widely used in combination chemotherapy and radiotherapy, the current studies help us to better understand how the drug affects mitochondrial function and could lead to improved efficacy of the drug in cancer treatment. For example, cancer cells that become hypoxic may reduce their ability to activate bleomycin. Also, the lungs are a site of excessive levels of oxygen and their mitochondria are exposed to it. Thus, in addition to pulmonary fibrosis caused by bleomycin in susceptible patients [102,103], one could expect more mitochondrial damage in the lungs than in mitochondria of some other organs after exposures to agents that generate ROS and free radical damage. We further suggest that wherever bleomycin may actually compromise mitochondrial function, bleomycin-induced damage could be monitored on isolated mtDNA [9,104 –107]. This could be particularly useful since the genotoxicity of the bleomycin-phleomycin family is influenced by a number of factors [108]. MtDNA as a monitor of free radical damage In conclusion, we propose that mtDNA could be used to sensitively monitor the accumulation and modulation of damage by ROS contributed by multiple sources since such damage plays a critical role in aging, cell death, and pathogenesis. It was previously suggested that mtDNA damage could be a useful biomarker for diseases associated with ROS [74], and the results in the current report strongly support this recommendation. Acknowledgements — This work was supported by The National Science Foundation, National Institutes of Health (including the Research Centers in Minority Institutions AIDS Infrastructure and Minority Biomedical Research Programs), Aaron Diamond Foundation, NATO Collaborative Research Grants Program, and City University of New York Medical School/Sophie Davis School of Biomedical Education. D. A. and M. D. are indebted to the Institut Curie, Paris, Electricite´ de France, Paris, the CNRS and the Centre d’Energie Atomique (CEA), Fontenay aux Roses, France, for financial support. We thank Bristol Laboratories of Bristol-Myers Squibb Co., Pharmaceutical Research and Development Division (Syracuse, NY, USA and Evansville, IN, USA) for providing Blenoxane for these experiments. We are also grateful for the very helpful assistance provided by Christopher Lawrence, Ph.D., Richard Reynolds, Ph.D., Susan Man-

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cuso, Simone Averbeck, Michele Dardhalon, Ph.D., Ajay Pramanik, Ph.D., Sangeetha Nimmagadda, Avanella James, Olimpia Gheorghiu, Ronald Edwards, and Gertrude Fisher. This paper is dedicated to the memory of Susan Mancuso.

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EDTA—the chelating agent ethylenediaminetetraacetic acid ␥— gamma H2O2— hydrogen peroxide mtDNA—mitochondrial DNA O2⫺—superoxide radical OH䡠— hydroxyl radical ␳⫹— grande; contains nuclear and mitochondrial DNAs and functions in oxidative phosphorylation ␳⫺— cytoplasmic petite mutant that can not function in oxidative phosphorylation due to mutated mitochondrial genome ␳0— cytoplasmic petite mutant that has lost its mitochondrial genome ROS—reactive oxygen species S. cerevisiae—Saccharomyces cerevisiae SSB—single-strand DNA break TTC—2,3,5 triphenyl-tetrazolium chloride YPAD—nonsynthetic complete medium containing yeast extract, peptone, adenine, and dextrose