Effects of KU-55933 on HCT116 p53+/+ and HCT116 p532/ 2 Cells
We examined the effects of KU-55933 on mitochondrial function as assessed by reduction of resazurin in isogenic p53 wild type and p53 loss of function HCT116 cells, and observed that even in the absence of p53, the kinase inhibitor reduced mitochondrial function, demonstrating that inhibition of ATMdependent p53 activation with subsequent p53-mediated dependent SCO2 activation cannot account for the effect of KU-55933 on mitochondrial function (Figure 5). The ability of KU-55933 to inhibit oxidative phosphorylation in p53-null cells also argues against a mediating role of TIGAR, a p53-dependent mitochondrial regulator [29].Effects of KU-55933 and Metformin on TCA Metabolites
In order to better understand the consequences of KU-55933 and metformin on cellular energy metabolism, we measured intracellular levels of the metabolites indicated in Figure 6A and Table S1. Interestingly, both compounds increased intracellular lactate and glucose, consistent with data in Figure 2 concerning glucose absorption and lactate excretion. Both KU-55933 and metformin significantly reduced the concentrations of the TCA cycle intermediates fumarate, malate, citrate, and alpha ketoglutarate. The compounds differed with their effects on succinate level, which was increased more than 5-fold by KU-55933, but reduced by metformin. NAD+ levels were significantly reduced only by metformin. The underlying mechanisms require further study, but these data suggest that in the case of metformin, effects on respiratory complex I are important, and that the compound reduces generation of NAD+ by complex I. Thus, metformin may not only reduce oxidative phosphorylation, but also inhibit the TCA cycle via its effect on redox status, given that the TCA enzymes isocitrate dehydrogenase and alpha ketoglutarate dehydrogenase require NAD+.
Inhibition of ATM by KU-55933 Decreases SCO2 Levels in MCF-7 Cells
As ATM activates p53 [26] and p53 upregulates oxidative phosphorylation by increasing SCO2 [27], we considered the possibility that ATM inhibition may act to decrease p53 activation and therefore decrease SCO2 levels, which would be expected to decrease oxidative phosphorylation, as observed. This potential mechanism was appealing in view of a recent report [28] showing that in muscle, ATM inhibition reduces cytochrome c oxidase activity (by an unspecified mechanism), an action that is the expected consequence of SCO2 reduction, and which would result in the reduced mitochondrial function. As shown in Figure 3, KU55933 had a major time-dependent effect in reducing SCO2 levelFigure 1. Growth inhibition by the ATM inhibitor KU-55933 and metformin. (A) MCF-7 (LKB+/+) and (B) HeLa (LKB2/2) cancer cells in exponential stages of growth were seeded into 96-well plates with 10% FBS and after 24 hrs exposed to increasing concentrations of KU-55933 (ATM inhibitor) in media containing 1% FBS for 72 hrs. Cell growth was estimated by Alamar Blue dye reduction (resazurin (3 mM)). Data are presented as mean 6 S.E.M. from 3 independent experiments done in triplicate. (C) MCF-7 HepG2, HeLa and MCF-10A cells were growth inhibited by KU-55933 and metformin. Cells were seeded into 96-well plates in the presence of 1% FBS and after 24 hrs treated with KU-55933 (10 mM) or metformin (5 mM). Data are presented as mean 6 S.E.M. from 4 independent experiments done in triplicate. * indicates a result significantly different from that obtained in the absence of KU-55933 or metformin as determined by 2-way ANOVA (P,0.0001). (G) MCF-7 cells were transfected with 50 nM ATM-siRNA or with control siRNA. Twenty-four hours after transfection, cells were treated with KU-55933 (10 mM) or metformin (5 mM) and incubated for 48 hrs in RPMI containing 1% FBS. Cell growth in each well was measured by counting cells using Trypan blue. Results using cell number or Alamar blue as endpoints yielded the same conclusions. Columns, mean of 3 independent experiments carried out in triplicate (n = 9); bars, S.E.M. (H) After transfecting MCF-7 cells with 50 nM ATM-siRNA or with control siRNA, cells were lysed and prepared for immunoblot analyses using antibodies against ATM. ?actin is shown as a loading control.
none to ubiquinol. The former reaction is part of the TCA cycle, while the latter forms part of the respiratory chain of oxidative phosphorylation. It is conceivable that KU-55933 may directly or indirectly cause complex II dysfunction in a manner that reduces oxidative phosphorylation as well as conversion of succinate to fumarate, leading to accumulation of succinate and inhibition of the Krebs cycle.Subcellular Localization of ATM in MCF-7 Cells
Our observations raise the possibility of a direct role for ATM in the mitochondria. While traditionally considered a nuclear protein, there is prior evidence [30] for cytoplasmic localization of ATM, but ATM has not previously been localized to mitochondria. We prepared a subcellular fraction highly enriched for mitochondria, and detected immunoreactivity to a mitochondrial marker VDAC and to ATM, but neither to the cytoplasmic marker tubulin nor the nuclear marker Ki67 (Figure 7).
Discussion
ATM-related proteins are ancient in evolutionary terms [31], and our findings add to recent evidence suggesting that these kinases have important functions in addition to those initially described that are related to DNA repair [2]. A prior study of fibroblasts obtained from a patient with the ataxia telangiectasia syndrome [16] provided early evidence that ATM deficiency is associated with abnormalities in mitochondrial function that could not be accounted for by DNA repair deficits. Our studies extend this work by showing that pharmacologic inhibition of ATM with KU-55933 results in reduced mitochondrial membrane potential, reduced coupled respiration, and reduced ATP levels, while increasing glucose uptake and lactic acid production. These actions are similar to those of metformin, a compound known to partially inhibit respiratory complex I. We speculate that the increased glucose uptake and lactic acid production are a consequence of increased glycolysis that partially compensates for the decrease in mitochondrial ATP production in the setting of loss of function of ATM, suggesting that neoplasms involving loss of function of ATM will exhibit a “Warburg” metabolic phenotype. Although inhibition of ATM by KU-55933 decreased expression of SCO2 (a protein required for cytochrome c oxidase assembly) in a p53-dependent fashion, the compound retained antiproliferative activity in p53-null cells, indicating that the actions of p53 are dispensable for the effects of KU-55933 on metabolism and proliferation. ATM has other substrates than p53 [32,33], including Sp1 [34], that may alter nuclear gene expression patterns in ways that influence metabolism. Alternatively, the evidence that ATM is present in mitochondria raises the possibility that it may play a more proximal role in regulating oxidative phosphorylation. The significant increase in succinate concentration associated with exposure to the ATM inhibitor allows speculation that this drug may have a direct or indirect effect that compromises the activity of respiratory complex II (succinate:ubiquinone oxidoreductase) (Figure 6?). Little is known about potential regulation of the activity of this complex by phosphorylation [35], but we did not detect a consensus sequence for the kinase activity of ATM against any of the subunits, arguing against a direct effect of ATM on this complex, despite the evidence for a requirement of ATM for optimum mitochondrial function.