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

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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.

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 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 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 contribute to the phenotype of cellular senescence (for recent review, see von Zglinicki and Passos, 2005), and mitochondria-related 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.

Section snippets

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 °C. Population doublings (PDL) were estimated using the following equation: n=(log10F−log10I)/0,301 (with n=population doublings, F=number of cells

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

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

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.

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    Petra Stöckl and Eveline Hütter contributed equally to this work.

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