Abstract
Recently, mitochondria have been suggested to act in tumor suppression. However, the underlying mechanisms by which mitochondria suppress tumorigenesis are far from being clear. In this study, we have investigated the link between mitochondrial dysfunction and the tumor suppressor protein p53 using a set of respiration-deficient (Res(-)) mammalian cell mutants with impaired assembly of the oxidative phosphorylation machinery. Our data suggest that normal mitochondrial function is required for γ-irradiation (γIR)-induced cell death, which is mainly a p53-dependent process. The Res(-) cells are protected against γIR-induced cell death due to impaired p53 expression/function. We find that the loss of complex I biogenesis in the absence of the MWFE subunit reduces the steady-state level of the p53 protein, although there is no effect on the p53 protein level in the absence of the ESSS subunit that is also essential for complex I assembly. The p53 protein level was also reduced to undetectable levels in Res(-) cells with severely impaired mitochondrial protein synthesis. This suggests that p53 protein expression is differentially regulated depending upon the type of electron transport chain/respiratory chain deficiency. Moreover, irrespective of the differences in the p53 protein expression profile, γIR-induced p53 activity is compromised in all Res(-) cells. Using two different conditional systems for complex I assembly, we also show that the effect of mitochondrial dysfunction on p53 expression/function is a reversible phenomenon. We believe that these findings will have major implications in the understanding of cancer development and therapy.
Highlights
Phorylation (OxPhos),2 which is carried out by four electron transport chain (ETC)/respiratory chain (RC) complexes (I–IV) and the ATP synthase
Reversible Regulation of p53 by Mitochondrial Dysfunction and tensin homolog) and activation of genes implicated in survival signaling (e.g. AKT1, HIF␣) by ETC/RC deficiencies can contribute to the process of tumorigenesis [24]
Impaired p53 signaling is the most common genetic alteration found in cancer and it plays a critical role in apoptosis [26]. p53 controls cell fate by transcription-dependent and transcription-independent mechanisms following stress signals such as DNA damage, oncogene activation, and hypoxia [27,28,29]. p53 has been implicated in regulating cellular metabolism [30]
Summary
The cells were transfected at ϳ50% confluency with the indicated plasmids using Lipofectamine 2000 (Invitrogen) following the manufacturer’s instructions. The oligomycin was excluded from the experiments toward determining the maximal respiratory capacity of cells This effect is observed irrespective of the substrate used such as glucose versus pyruvate. The rotenone was added at the indicated concentration 1 h prior to or after ␥IR (1–20 h), whereas the chloramphenicol (50 g/ml) was added 24 – 48 h prior to ␥IR, and relative cell death was monitored 24 h later as described above. Real Time PCR—RNA was isolated using TRIzol reagent (Invitrogen) according to the manufacturer’s description, and the PCR was performed as described previously [44, 50].
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