Sustained inhibition of oxidative phosphorylation impairs cell proliferation and induces premature senescence in human fibroblasts

Experimental Gerontology 41 (2006) 674–682 www.elsevier.com/locate/expgero

Sustained inhibition of oxidative phosphorylation impairs cell proliferation and induces premature senescence in human fibroblasts Petra Sto¨ckl 1, Eveline Hu¨tter 1, Werner Zwerschke, Pidder Jansen-Du¨rr * Institute for Biomedical Aging Research, Molecular and Cellular Biology, Austrian Academy of Sciences, Rennweg 10, A-6020 Innsbruck, Austria Received 14 March 2006; received in revised form 10 April 2006; accepted 20 April 2006

Abstract The mitochondrial theory of aging predicts that functional alterations in mitochondria contribute to the aging process. Whereas this hypothesis implicates increased production of reactive oxygen species (ROS) as a driving force of the aging process, little is known about molecular mechanisms by which mitochondrial impairment might contribute to aging. Using cellular senescence as a model for human aging, we have recently reported partial uncoupling of the respiratory chain in senescent human fibroblasts. In the present communication, we address a potential cause-effect relationship between mitochondrial impairment and the appearance of a senescence-like phenotype in young cells. We found that treatment by antimycin A delays proliferation and induces premature senescence in a subset of the cells, associated with increased reactive oxygen species (ROS) production. Quenching of ROS by antioxidants did however not restore proliferation capacity nor prevent premature senescence. Premature senescence is also induced upon chronic exposure to oligomycin, irrespective of ROS production, and oligomycin treatment induced the up-regulation of the cdk inhibitors p16, p21 and p27, which are also up-regulated in replicative senescence. Thus, besides the well-established influence of ROS on proliferation and senescence, a reduction in the level of oxidative phosphorylation is causally related to reduced cell proliferation and the induction of premature senescence. q 2006 Elsevier Inc. All rights reserved. Keywords: Oxidative phosphorylation; Senescence; Mitochondria; Reactive oxygen species; Antimycin A; Oligomycin

1. Introduction Mitochondrial function is essential for post-mitotic tissues with a high demand of energy, such as neurons or muscle cells. However, it has not been formally established whether intact mitochondria are required for the proper function of cells with high proliferation potential, e.g. fibroblasts from normal skin. Moreover, the role of mitochondrial function for the proliferation of mammalian cells is unknown. Cell homeostasis and cell division are both dependent on chemical energy that is supplied in the form of ATP. In mammalian cells, ATP can be generated by the glycolytic breakdown of glucose and carbohydrates

Abbreviations: BrdU, bromo-desoxy uridine; ROS, reactive oxygen species; DHE, dihydroethidium; DHR, dihydrorhodamine; NAC, N-acetylcysteine; JC-1, 5,5 0 ,6,6 0 -tetrachloro-1,1 0 ,3,3 0 -tetraethylbenzimidazolylcarbocyanine iodide. * Corresponding author. Tel.: C43 512 583919 44; fax: C43 512 583919 8. E-mail address: [email protected] (P. Jansen-Du¨rr). 1 Petra Sto¨ckl and Eveline Hu¨tter contributed equally to this work. 0531-5565/$ - see front matter q 2006 Elsevier Inc. All rights reserved. doi:10.1016/j.exger.2006.04.009

and/or by oxidative phosphorylation. Previous studies on tumor cells have explored effects of metabolic inhibitors on cell proliferation and cell death. Thus, short-term treatment of Ehrlich ascites tumor cells with oligomycin resulted in complete growth arrest (Kroll et al., 1983), and the combined treatment of HeLa cells with oligomycin and 2-deoxyglucose, thereby blocking both mitochondrial and glycolytic ATP production, resulted in a rapid induction of apoptosis (Izyumov et al., 2004). It has been argued that many proliferating cells derive most of their energy from glycolysis and avoid mitochondrial ATP generation to minimize the damage associated with increased oxidative stress (Brand and Hermfisse, 1997), which is known as an important byproduct of oxidative phosphorylation (Cadenas and Davies, 2000). For example, in rat thymocytes a considerable increase of glycolytic enzymes occurs during transition from the resting to the proliferating state, which enables the cells to meet the enhanced energy demand by increased aerobic glycolysis (Brand, 1997). Similarly, it has been known since decades that tumor cells use glycolytic rather than mitochondrial energy production leading to a strong increase in the production of lactate, also known as the Warburg effect (for review, see Semenza et al., 2001). The

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reasons for this metabolic switch are not entirely understood, but this commonly observed event could well reflect a reduced requirement of proliferating cells for mitochondrial function. Reactive oxygen species (ROS) that are produced by the mitochondria and by other enzyme systems are known to play key roles in the regulation of many biological processes, such as stress-related signalling, immune function, vascular function, etc. In particular, the available evidence suggests that ROS are essential players in the regulation of cell proliferation and cell death (Behrend et al., 2003). Whereas low levels of ROS appear to play a major positive role in mitogenic signalling (Burdon et al., 1995), increased levels of ROS were shown to inhibit cell proliferation in vitro (Chen and Ames, 1994; von Zglinicki et al., 1995). Mitochondria and ROS are thought to play a key role in aging, based primarily on studies with lower eukaryotic model organisms. For example, increased replicative longevity in Saccharomyces cerevisiae, due to caloric restriction, has been linked to enhanced mitochondrial respiratory activity, which was found to decrease the rate of mitochondrial ROS production (Barros et al., 2004; reviewed by Heeren et al., 2004). In the nematode Caenorhabditis elegans, lifespan can be extended by targeting mitochondrial electron transport, either through genetic manipulations (siRNA mediated gene silencing and the introduction of specific mutations, respectively) or by the addition of drugs, e.g. antimycin A (Dillin et al., 2002; for review, see Anson and Hansford, 2004). These observations suggest that mitochondrial activity per se bears the inherent potential to restrict lifespan, at least in these model organisms (reviewed by Houthoofd et al., 2005). Data derived from studies with lower eukaryotic model organisms further suggest that reactive oxygen species, irrespective of their actual source, play a major role in aging processes in vivo, although no direct mechanistic links have been established so far (for recent review, see Sohal et al., 2002). For example, reducing the level of antioxidant enzymes, such as superoxide dismutase (SOD) in many species, including mice, leads to a consistent reduction of the lifespan, and premature aging (Kokoszka et al., 2001). Accordingly, extending the antioxidative capacity by either overexpression of SOD/catalase or by pharmacological intervention has been described to extend lifespan in both Drosophila melanogaster (Orr and Sohal, 1994) and C. elegans (Melov et al., 2000), respectively. However, more recent data questioned the ability of SOD/catalase overexpression to extend lifespan in normal strains of Drosophila (Orr et al., 2003); similarly, the ability of SOD/catalase mimetics to extend lifespan in C. elegans has been questioned by recent studies (Keaney et al., 2004). Thus, the precise role of ROS in aging may depend on the context and remains to be defined. While the role of reactive oxygen species in human aging is unknown, it was shown that the exposure of normal human cells to increased oxidative stress induces premature senescence in vitro (Chen and Ames, 1994; von Zglinicki et al., 1995), providing a potential mechanism by which ROS could contribute to certain aspects of human aging. At present, it is unknown whether changes in mitochondrial function

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contribute to the phenotype of cellular senescence (for recent review, see von Zglinicki and Passos, 2005), and mitochondriarelated pathways that might be involved have to be elucidated. Using cellular in vitro senescence of human diploid fibroblasts as model system for human aging, we have recently shown that partial uncoupling of the respiratory chain can be observed in senescent fibroblasts (Hutter et al., 2004), supporting the idea that mitochondrial impairment may contribute to cellular senescence. In support of a role for mitochondria in cellular aging in vivo, others have reported a significant decline in the efficiency of oxidative phosphorylation in human fibroblasts derived from the skin of elderly donors (Greco et al., 2003). In the present communication, we have addressed the question if manipulations of mitochondrial function in young cells might influence the replicative potential and induce premature senescence in these cells. 2. Material and methods 2.1. Cell culture Normal human diploid fibroblasts (HDF) were isolated from human foreskin (Durst et al., 1987) and cultured in Dulbecco’s modified Eagle’s medium (Sigma, Vienna, Austria), supplemented with penicillin/streptomycin solution and 10% fetal calf serum (Gibco Life Technologies, Vienna, Austria). The cells were subcultured in an atmosphere of 5% CO2 at 37 8C. Population doublings (PDL) were estimated using the following equation: nZ(log10FKlog10I)/0,301 (with nZ population doublings, FZnumber of cells at the end of one passage, IZnumber of cells that were seeded at the beginning of one passage). Oligomycin (Sigma, Vienna, Austria) and antimycin A (Sigma, Vienna, Austria) were dissolved in ethanol and stored as aliquots at K20 8C. The antioxidant N-acetyl-cysteine (NAC; Sigma, Vienna, Austria) was dissolved in Aqua bidest, adjusted to pH 7.2, and sterilized by filtration. For the different treatments, cells were plated in cell culture dishes 10 cm in diameter, usually in triplicates. The substances were given in fresh medium every day including 1 mM antimycin A or 8 mM oligomycin in the respective experimental groups. Since these chemicals are dissolved in ethanol, cells were treated with the corresponding amount of ethanol as vehicle control. NAC was used at a final concentration of 4 mM. 2.2. SA-ß-galactosidase staining The senescent status of cells was verified by in situ staining for senescence-associated b-galactosidase (SA-b-gal), as described (Dimri et al., 1995). 2.3. Measurement of reactive oxygen species For in situ evaluation of reactive oxygen species by oxidantsensitive dyes, fibroblasts were grown overnight on 60 mm diameter cell culture dishes. For staining, cells were washed twice with PBS and incubated either with dihydrorhodamine

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123 (DHR123, Sigma, Vienna, Austria, 0.6 mg/ml in complete DMEM for 30 min), or dihydroethidium (DHE, Molecular Probes, Leiden, Holland, 20 mM in serum free DMEM for 30 min). All incubations were carried out at 37 8C in the incubator. After two final washing steps with PBS, cells were immediately analyzed by confocal laser scanning microscopy. For FACS analysis of DHE fluorescence, cells were trypsinized after the in situ staining, resuspended in DMEM, centrifuged, washed with PBS, centrifuged again and analysed in PBS. 2.4. Evaluation of mitochondrial membrane potential For JC-1 staining, cells were grown overnight on 60 mm dishes, washed with PBS, and incubated in MiR05, to which 20 mg/ml digitonin, 10 mM succinate (Merck, Darmstadt, Germany), 0.5 mM rotenone and 0.5 mg/ml JC-1 (Molecular Probes, Leiden, NL) were added. After 15 min incubation, cells were washed with MiR05 supplemented with 10 mM succinate and analyzed by confocal laser scanning microscopy. MiR05 consisted of 110 mM sucrose, 60 mM K-lactobionate (Fluka Chemie, Buchs, Switzerland), 0.5 mM EGTA, 1 g/l BSA essentially fat free, 3 mM MgCl2, 20 mM taurine (Merck, Darmstadt, Germany), 10 mM KH2PO4 (Merck, Darmstadt, Germany), 20 mM HEPES adjusted to pH 7.1 with KOH at 37 8C (Renner et al., 2003). A standardized mitochondrial resting state was obtained by electron supply from the complex II substrate succinate in the presence of the complex I inhibitor rotenone. Under these conditions, a maximum mitochondrial membrane potential is maintained by state 2 respiration. After confocal laser scanning microscopy, the same cells were uncoupled by 20 mM FCCP according to the experimental setup, and another image was obtained to define the reference point of a low mitochondrial membrane potential after uncoupling with 20 mM FCCP. 2.5. Proliferation and apoptosis assays Proliferation was assessed by measurement of bromodesoxy uridine (BrdU) incorporation into DNA (anti-BrdU immunofluorescence staining kit, Roche, Mannheim, Germany), and counting BrdU-positive nuclei. Cells were seeded and treated on glass cover slips. DAPI (Sigma, Vienna, Austria) was incubated along with the secondary antibody as nuclear counterstaining. BrdU-index was calculated as percentage of BrdU-positive stained cells per total cell number. Apoptosis was analyzed by in situ detection of DNA strand breaks (TUNEL enzymatic labeling assay, Roche, Mannheim, Germany), and FACS analysis of the cell cycle profile. Briefly, cells were trypsinized and DNA was stained with propidium iodide (Sigma, Vienna, Austria) in PBS/TritonX-100 for 30 min. Sub-G1-DNA content was evaluated by plotting propidium iodide fluorescence against cell number by FACS, as described (Mannhardt et al., 2000). 2.6. Preparation of cell extracts and Western blotting To prepare whole cell extracts, cells were washed twice in

ice-cold PBS and scraped from the culture dish with a rubber policeman. Cells were lysed for 30 min on ice in a buffer containing 50 mM Tris–HCl, pH 7.5, 300 mM NaCl, 1% NP40, 0.1% SDS, 0.5% Na-deoxycholate, 0.2 mM phenylmethylsulfonyl-fluoride, 1 mM NaF, 10 mg/ml aprotinin, 10 mg/ml leupeptin and 10 mM b-glycerophosphate. The lysates were centrifuged at 20,000g for 15 min at 4 8C, and the supernatants separated on SDS-polyacrylamide gels. Protein concentrations were determined with the DC Protein Assay Kit (Biorad, Vienna, Austria). The amounts of loaded extract corresponded to equal protein concentrations. After electrophoresis, the proteins were transferred to a PVDF membrane by wet electroblotting in a buffer containing 25 mM Tris–HCl, 190 mM glycine, 0.5% SDS and 10% methanol. Transfer was controlled by staining the membranes with Ponceau S. Membranes were blocked by incubation in 5% non-fat dried milk in TBS-T (Tris buffered salineC0.1% Tween 20) for 1 h at room temperature. Incubation with the primary antibody was performed for either 60 min at room temperature or overnight at 4 8C. Membranes were washed twice with TBS-T and incubated with the secondary antibody for 30 min. After four washes with TBS-T and one wash with TBS, immunoreactive proteins were detected using an enhanced chemiluminescence system (Amersham Life Science, Braunschweig, Germany). The following antibodies were used for Western blot analysis: mouse monoclonal anti-p21 and mouse monoclonal anti-p16 (Becton-Dickinson, Vienna, Austria), rabbit polyclonal antip27 (Santa Cruz, Heidelberg, Germany), monoclonal mouse anti-b-Actin (clone AC-15, Sigma, Vienna, Austria); all secondary antibodies were obtained from DAKO (Glostrup, Denmark). To monitor the performance of the antibodies to cdk inhibitors p16, p21 and p27, U-2OS osteosarcoma cells were transfected with the appropriate pX-derived expression vectors using Effectene (Quiagen, Hilden, Germany), as described previously (Zerfaß-Thome et al., 1997), and cellular lysates were prepared for Western blot. 3. Results 3.1. Inhibition of mitochondrial activity reduces the rate of cell proliferation in human diploid fibroblasts To analyze a potential contribution of mitochondrial activity to cellular proliferative capacity, human diploid fibroblasts, widely used for studies of in vitro cellular senescence, were chronically exposed to specific inhibitors of mitochondrial function, targeting either the electron transport chain or mitochondrial ATP synthase. We reasoned that pharmacological inhibition of mitochondrial function gives more reliable consistent mitochondrial impairment, as opposed to RNAi-mediated knockdown of mitochondrial subunits. Such procedures have been successfully applied in studies with C. elegans (Dillin et al., 2002); however, protocols for longterm knockdown of genes in primary human cells are currently not available. To assess the role of mitochondrial electron transport chain

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Fig. 1. Inhibition of mitochondrial function inhibits cell proliferation without induction of cell death. (A) Changes in mitochondrial membrane potential monitored by JC-1. Cells were either grown in the absence of any inhibitors, or chronically exposed to oligomycin and antimycin A for 14 days, as indicated. For assessment of mitochondrial membrane potential, cells were permeabilized with 20 mg/ml digitonin, to allow uniform mitochondrial staining (Hutter et al., 2004), and JC-1 was added to a final concentration of 0.5 mg/ml. Where indicated, FCCP was added to the cells at a final concentration of 20 mM, to define the reference point for minimal mitochondrial membrane potential (Hutter et al., 2004). (B) Effects on cell proliferation. Cells were either grown in the absence of any inhibitors, or chronically exposed to oligomycin and antimycin A, as indicated. Cells were passaged until they reached about 80% confluency; proliferation rate was determined by counting the cells at every passaging. Population doublings (PDL) were determined, as described previously (Hutter et al., 2004). While in standard respirometric regimes that can be performed within 3 h, oligomycin is typically used at a concentration of 1 mM (Hutter et al., 2004), a higher concentration (8 mM) of oligomycin was required to establish long-term effects on the cellular phenotype. The reason for this apparent discrepancy is not clear at present but may reflect limited metabolic stability of oligomycin in the long-term exposure experiments described here. (C) Assessment of S-phase entry by BrdU incorporation analysis. Cells were either grown in the absence of any inhibitors, or chronically exposed to oligomycin and antimycin A, as indicated. Cells were stained for DNA synthesis by the BrdU incorporation assay. A graphical representation of the results (nZ3) is shown. (D) Detection of apoptosis by PI-FACS. Cells were either grown in the absence of any inhibitors, or chronically exposed to oligomycin and antimycin A, as indicated. Cells were analyzed for the appearance of apoptotic nuclei by PI-FACS. Please note the low level of apoptotic cell death throughout the experiment.

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(ETC) for cell proliferation, antimycin A, which inhibits the ETC at complex III, was added daily at a concentration of 1 mM, which is sufficient to reduce oxygen consumption in human diploid fibroblasts to a background value of nonmitochondrial respiration, as determined by respirometry (data not shown; see Hutter et al., 2004). Chronic exposure to antimycin A led to a drastic reduction in mitochondrial membrane potential throughout the time of exposure, as shown by staining with the potentiometric dye JC-1 (Mathur et al., 2000; Hutter et al., 2004) (Fig. 1(A)). To determine if inhibition of the mitochondrial ETC would interfere with the rate of cell proliferation, cell number was determined at regular intervals. As is shown in Fig. 1(B), addition of antimycin A led to a significant delay in cell proliferation, and antimycin A-treated cells reached approximately six population doublings in 30 days, whereas control cells reached eleven population doublings in the same time interval. To assess the role of mitochondrial ATP synthesis for cell proliferation, cells were treated with oligomycin, which specifically inhibits mitochondrial ATP synthase (Slater, 1967) and leads to an increased mitochondrial membrane potential (Slater, 1967). Fresh medium containing oligomycin was added daily to proliferating cells; in a pilot study, the effect of increasing oligomycin concentrations on overall cell proliferation rate was determined (data not shown; see also below, Fig. 1(B)). Upon chronic exposure to oligomycin at a concentration of 8 mM, significant growth retardation was observed, and oligomycin exposure led to a drastic increase in mitochondrial membrane potential, as shown by JC-1 staining (Fig. 1(A)). Chronic exposure to oligomycin lead to a significant delay in cell proliferation, and oligomycin-treated cells reached approximately nine population doublings in 30 days (Fig. 1(B)). Whereas inhibition of oxidative phosphorylation was shown to kill certain tumor cells (see Section 1), we report here for the first time phenotypical consequences of a sustained inhibition of oxidative phosphorylation in normal human cells, achieved through chronic exposure to an elevated concentration of oligomycin. To further study growth retardation induced by these mitochondrial inhibitors, the amount of cells in S-phase was determined by BrdU (bromo-desoxy uridine) incorporation analysis. Whereas in untreated cultures, more than 80% of the cells entered S-phase in a period of 24 h, the fraction of S-phase cells was significantly reduced (to roughly 60% in either case) by both inhibitors (Fig. 1(C)). This observation suggests that the reduction in the number of population doublings is primarily due to a reduced rate of S-phase entry. Cell death by apoptosis or necrosis was not observed under these conditions, as was shown by both FACS analysis of propidium iodide stained cells (Fig. 1(D)) and staining for apoptotic cells in situ by the TUNEL assay (data not shown). Our data suggest that sustained inhibition of mitochondrial ATP synthase, either directly by chronic exposure to oligomycin or indirectly through antimycin A-induced ETC inhibition, slows down proliferation of human diploid fibroblasts without inducing complete growth arrest or cell death.

3.2. The role of reactive oxygen species (ROS) in growth arrest due to mitochondrial impairment Manipulations of the mitochondrial respiratory chain by complex III inhibitors, such as antimycin A, bear the potential to induce reactive oxygen species (ROS) (Staniek and Nohl, 2000; Turrens, 2003), which are known to slow down cell proliferation in many cell types including human diploid fibroblasts (Chen and Ames, 1994; von Zglinicki et al., 1995). To address this point, ROS levels were determined in antimycin A-treated cells by in situ staining of living cells with redox-sensitive dyes. Senescent human fibroblasts, which are known to stain positive with such dyes (Hutter et al., 2004), were included as a positive control. Addition of antimycin A to young fibroblast cultures induced sustained oxidation of dihydroethidium (DHE), as revealed by increased red fluorescence (Fig. 2(A)), suggesting that antimycin A induced ROS production in human fibroblasts, as was shown by others before (Wang et al., 2003). This finding raises the possibility that increased ROS levels are responsible for growth arrest induced by antimycin A. The high mitochondrial membrane potential caused by inhibition of ATP synthase is known to increase ROS generation (Slater, 1967). Interestingly, oligomycin treatment of human T cell clones was shown to reduce ROS levels (Slater, 1967; for recent review, see Turrens, 2003). In our hands, chronic exposure to oligomycin indeed induced the oxidation of DHE (Fig. 2(A)), although the effect was less pronounced than with antimycin A. Increased oxidative stress was also documented by strongly increased DCFDA staining (data not shown). Cells were also stained by dihydrorhodamine 123 (DHR123), another redox-sensitive dye, which has been widely used to monitor the production of reactive oxygen species in living cells, including senescent human fibroblasts (Goldstein and Korczack, 1981; Martinez et al., 1987). However, others have shown that DHR123 does not detect generation of reactive oxygen species directly; rather, the oxidation of DHR123 reflects a more general change in the cellular redox state (Walrand et al., 2003). In keeping with this observation, it was found that inhibition of mitochondria by antimycin A or oligomycin, while inducing DHE staining, failed to produce any significant staining with DHR123 (Fig. 2(B)). This result suggests that DHR123 staining is not a direct measurement of ROS generation, and the positive DHR123 staining observed in senescent cells (Fig. 2(B)) rather reflects so far unknown metabolic events, which are not visible in young cells after exposure to either antimycin A or oligomycin. To address the possibility that the antiproliferative effects of chronic exposure to antimycin A might be due to increased ROS production, N-acetyl-cysteine (NAC), an antioxidant widely used to scavenge ROS (Aruoma et al., 1989), was added. In a pilot study, using DHE staining followed by flow cytometry (data not shown, see also below), the effective concentration of NAC was determined that is required to efficiently quench ROS induced through chronic exposure to antimycin A. We found that the addition of 4 mM NAC is

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A-induced DHE fluorescence and its quenching by NAC was also detected by FACS analysis of DHE-stained cells (Fig. 3(A)). Neutralization of ROS by NAC failed to abolish the proliferation blockage by antimycin A (Fig. 3(B)), suggesting that, besides the increased ROS production, additional effects of complex III inhibitors may limit sustained cell proliferation. 3.3. Induction of premature senescence by mitochondrial inhibitors As it was shown before that mitochondrial function is compromised in senescent human fibroblasts (Hutter et al., 2004), we determined whether the modulation of mitochondrial

Fig. 2. Production of reactive oxygen species (ROS) in cells treated by mitochondrial inhibitors. (A) DHE staining. Cells were either grown in the absence of any inhibitors, or chronically exposed to oligomycin and antimycin A for 14 days, as indicated. ROS production was analyzed by DHE staining followed by fluorescence microscopy. Senescent HDF are included as controls. (B) DHR 123 staining. Cells were either grown in the absence of any inhibitors, or chronically exposed to oligomycin and antimycin A for 14 days, as indicated. Cells were analyzed by DHR123 staining followed by fluorescence microscopy. Senescent HDF are included as controls.

sufficient to reduce to background the increased ROS levels caused by chronic exposure to 1 mM antimycin A. This was shown by microscopical inspection, where bright nuclear staining was induced by antimycin A exposure (see also Fig. 2(A)) and quenched by NAC (data not shown). Antimycin

Fig. 3. Elimination of ROS by NAC does not restore cell proliferation. (A) DHE staining determined by FACS analysis. Cells were either grown in the absence of any inhibitors, or chronically exposed to oligomycin and antimycin A for 14 days, as indicated. ROS production was analyzed by DHE staining followed by flow cytometric analysis. Where indicated, 4 mM NAC has been added to quench ROS. (B) Growth curves. Cells were either grown in the absence of any inhibitors, or chronically exposed to oligomycin and antimycin A for 14 days, as indicated. The rate of cell proliferation rate was determined by counting the cells at every passaging. Where indicated, 4 mM NAC has been added to quench ROS. PDL, population doublings.

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P. Sto¨ckl et al. / Experimental Gerontology 41 (2006) 674–682 Table 1 Induction of premature senescence by mitochondrial inhibitors Treatment (12 days)

n

SA-b-Gal pos (%) mean G SD

Control NAC 4 mM Oligomycin 8 mM Oligomycin 8 mMCNAC Antimycin A 1 mM Antimycin A 1 mMCNAC

6 6 3 3 3 3

1.9G1.5 8.1G4.3 ** 9.0G4.0 ** 31.6G10.3 *** 22.7G8.5 *** 24.2G11.5 **

Human diploid fibroblasts in early passage were either grown in the absence of any inhibitors, or chronically exposed to oligomycin, antimycin A, and NAC for 12 days, as indicated. Cells were stained for SA-b-galactosidase (Dimri et al., 1995) and counted in the microscope. Statistical analysis was performed by one-way ANOVA with a subsequent Fisher PLSD post-hoc test, where, p!0.05 was defined as significant. *p!0.05, **p!0.01, ***p!0.001.

Fig. 4. Induction of premature senescence by mitochondrial inhibitors. (A) Cells were either grown in the absence of any inhibitors, or chronically exposed to oligomycin and antimycin A for 14 days, as indicated. Cells were stained for SA-b-galactosidase activity and evaluated by microscopic inspection. Senescent HDF are shown as control. (B) and (C) Western blot analysis of cdk inhibitors. Cells were either grown in the absence of any inhibitors, or chronically exposed to oligomycin, antimycin A, and NAC for 14 days, as indicated. Cellular extracts were prepared, separated by SDS-PAGE, and probed by Western blot for the abundance of the cdk inhibitors p16, p21 and p27.In panel B, two parallel samples are shown for each treatment; b-actin served as loading control. For each antigen, specificity of the antibodies is documented by including extracts from U-2OS cells that had been transfected with expression vectors for p16, p21 and p27, respectively. Please note that the p16 protein encoded by the expression vector has a lower apparent molecular

activity would induce premature senescence in young human fibroblasts. To this end, fibroblasts were chronically exposed to mitochondrial inhibitors as described above, and stained for senescence-associated beta galactosidase (SA-b-gal; Fig. 4(A)). This experiment revealed that chronic exposure to antimycin A treatment resulted in 22.7G8.5% SA-b-gal positive cells, whereas chronic exposure to oligomycin lead to the appearance of 9.0G4.0% SA-b-gal positive cells within 14 days (Table 1), and the appearance of SA-b-gal positive cells was not prevented by addition of NAC (Table 1). Unexpectedly, chronic exposure to NAC induced SA-b-gal activity in 8.1G4.3% of the cells, and NAC further increased the number of SA-b-gal positive cells when combined with oligomycin (Table 1), suggesting that both effects are additive. Replicative senescence of human diploid fibroblasts is mediated by the simultaneous increase in the abundance of the cdk inhibitors p16 (INK4A), p21 (CIP1) and p27 (KIP1) (Alcorta et al., 1996; Hara et al., 1996; Wagner et al., 2001). Since, our data indicate that chronic exposure to drugs that target mitochondrial function leads to both cell cycle arrest and a phenotype of premature senescence, it was of interest to explore potential regulatory pathways that are involved. As a first step towards this goal, we analyzed effects of these compounds on the expression of the cdk inhibitors mentioned above. Chronic exposure to antimycin A had no discernable effect on the levels of either p16, p21 or p27 (Fig. 4(B) and (C)); hence, the pathway by which antimycin A induces cell cycle arrest (and SA-b-gal staining) remains to be established. Chronic exposure to oligomycin triggered a significant increase of p16, p21 and p27 protein levels (Fig. 4(B) and (C)). These findings suggest that cell cycle arrest and premature senescence induced by oligomycin are mediated by simultaneous up-regulation of these cdk inhibitors, which are also relevant for replicative senescence (Wagner et al., 2001). We also addressed potential cell cycle targets of antioxidant treatment using 4 mM NAC. While NAC had little if any effect on the level of p21, it triggered a robust increase of both p16 and p27 3 weight relative to p16 obtained from fibroblasts. The latter comigrated with p16 obtained from HeLa cells that were used as control (panel C). The reason for the different migration behaviour of transfected p16 is unclear at present.

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protein levels (Fig. 4(B) and (C)). However, the molecular basis for this effect remains to be clarified. 4. Discussion We describe in the present communication that prolonged exposure to the mitochondrial inhibitors antimycin A and oligomycin leads to growth inhibition and premature senescence in human diploid fibroblasts. The reduced degree of growth inhibition obtained with oligomycin compared to antimycin A might be due to partial inhibition of ETC in that case or to the different mechanisms of inhibition. This remains to be clarified. Hence, these cells appear to be partially independent of mitochondrial ATP production, consistent with our previous observation that diploid human fibroblasts are characterized by a high rate of aerobic glycolysis (Zwerschke et al., 2003). On the other hand, the reduced rate of proliferation, achieved upon oligomycin treatment, might be due to a partial ATP deficit. In support of this, reduced ATP levels were reported previously to contribute to growth arrest in senescent human diploid fibroblasts (Wang et al., 2003; Zwerschke et al., 2003), and semi-quantitative determination of ATP levels by commercially available fluorescence measurement kits suggests that there might indeed be a reduction of ATP levels in cells exposed to mitochondrial inhibitors in vitro (Hu¨tter et al., unpublished results); however, more work will be required to firmly establish that point. Neutralization of ROS by NAC failed to abolish the proliferation blockage by antimycin A (Fig. 3(B)), suggesting that, besides the increased ROS production, additional effects of complex III inhibitors may limit sustained cell proliferation. For example, the oxidation of mitochondrial substrates is impaired by antimycin A, which blocks electron transport through the respiratory chain, and the accumulation of reduced substrates may feed back to other metabolic pathways. Thus, glycolytic energy production, the only alternative source for ATP in mammalian cells, depends on the recycling of NADH to NAD, which occurs mainly in the mitochondria. However, it can not be formally excluded that antimycin A—induced delay of cell proliferation involves particular ROS species which cannot be quenched by NAC. Chronic exposure to NAC even further suppressed the rate of cell proliferation (Fig. 3(B)) and decreased the fraction of cells in S-phase. Together, these observations imply that growth arrest induced by antimycin A is not primarily caused by increased ROS production. Whereas the weak oligomycin-induced rise of DHE staining was reduced to background levels by NAC (Fig. 3(A)), the rate of cell proliferation was not increased upon the addition of NAC to oligomycin treated cells (Fig. 3(B)). Hence, increased ROS production does not play a major role in oligomycin-induced growth arrest. The findings reported here indicate that growth arrest and the induction of a senescence-like phenotype by mitochondrial inhibitors may not be directly caused by increased ROS but rather reflects a consequence of mitochondrial impairment, such as a reduced mitochondrial ATP production. Such conclusion would be consistent with the finding that senescent

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human fibroblasts display reduced ATP levels and concomitantly up-regulated levels of AMP (Wang et al., 2003; Zwerschke et al., 2003). Moreover, the artificial up-regulation of intracellular AMP can induce proliferation arrest in tumor cells (Hugo et al., 1992) and premature senescence in diploid human fibroblasts (Zwerschke et al., 2003). Together, the data suggest that exposure to oligomycin causes changes in gene expression that are similar to those observed in spontaneous replicative senescence and that a functionally distinct pathway is triggered by antimycin A. More work will be required to elucidate molecular mechanisms underlying the observed effects of oligomycin on the abundance of growth-restricting inhibitors of cyclin-dependent kinases. Together, our results are compatible with a model where, besides the wellestablished role of high ROS for blocking proliferation and inducing senescence, a reduction in the level of mitochondrial ATP production is causally related to reduced cell proliferation and the induction of senescence in human diploid fibroblasts. Acknowledgements We thank Hans-Peter Viertler for excellent technical assistance and Erich Gnaiger for helpful discussions. This work was supported by the Austrian Science Funds (NRN S93), the European Union (MIMAGE project) and the Austrian Ministry of Science and Traffic. References Alcorta, D.A., Xiong, Y., Phelps, D., Hannon, G., Beach, D., Barrett, J.C., 1996. Involvement of the cyclin-dependent kinase inhibitor p16 (INK4a) in replicative senescence of normal human fibroblasts. Proc. Natl Acad. Sci. USA 93, 13742–13747. Anson, R.M., Hansford, R.G., 2004. Mitochondrial influence on aging rate in Caenorhabditis elegans. Aging Cell 3, 29–34. Aruoma, O.I., Halliwell, B., Hoey, B.M., Butler, J., 1989. The antioxidant action of N-acetylcysteine: its reaction with hydrogen peroxide, hydroxyl radical, superoxide, and hypochlorous acid. Free Radic. Biol. Med. 6, 593–597. Barros, M.H., Bandy, B., Tahara, E.B., Kowaltowski, A.J., 2004. Higher respiratory activity decreases mitochondrial reactive oxygen release and increases life span in Saccharomyces cerevisiae. J. Biol. Chem. 279, 49883–49888. Behrend, L., Henderson, G., Zwacka, R.M., 2003. Reactive oxygen species in oncogenic transformation. Biochem. Soc. Trans. 31, 1441–1444. Brand, K., 1997. Aerobic glycolysis by proliferating cells: protection against oxidative stress at the expense of energy yield. J. Bioenerg. Biomembr. 29, 355–364. Brand, K.A., Hermfisse, U., 1997. Aerobic glycolysis by proliferating cells: a protective strategy against reactive oxygen species. FASEB J. 11, 388–395. Burdon, R.H., Alliangana, D., Gill, V., 1995. Hydrogen peroxide and the proliferation of BHK-21 cells. Free Radic. Res. 23, 471–486. Cadenas, E., Davies, K.J., 2000. Mitochondrial free radical generation, oxidative stress, and aging. Free Radic. Biol. Med. 29, 222–230. Chen, Q., Ames, B.N., 1994. Senescence-like growth arrest induced by hydrogen peroxide in human diploid fibroblast F65 cells. Proc. Natl Acad. Sci. USA 91, 4130–4134. Dillin, A., Hsu, A.L., Arantes-Oliveira, N., Lehrer-Graiwer, J., Hsin, H., Fraser, A.G., Kamath, R.S., Ahringer, J., Kenyon, C., 2002. Rates of behavior and aging specified by mitochondrial function during development. Science 298, 2398–2401.

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