Cellular senescence, an irreversible cell cycle arrest induced by a diversity of stimuli, has been considered as an innate tumor suppressing mechanism with implications and applications in cancer therapy. The senescence phenomenon was first associated with aging as Hayflick and colleagues observed that normal human fibroblasts lost their proliferative capacity after a finite number of cell divisions in cell culture. Oncogene activation acting to potentiate or overstimulate normal growth signaling is a hallmark of cancer. However, in “normal” cells oncogene activation does not lead to transformation, but instead provokes cellular senescence. One characteristic feature of the oncogene induced senescence phenotypes involves formation of a unique type of chromatin, known as senescence associated heterochromatin foci. Histone modifications have been shown to be part of this chromatin formation. With new mass spectrometric technologies developed to analyze histone modifications, specifically acetylation and methylation, global histone modification changes in senescent cells were studied extensively. A top down proteomic approach to profile all core histones was developed and applied to study histone acetylation changes after selective knocking down or inhibition of histone deacetylases. This method also allowed us to find decreased acetylation levels on all core histone proteins in senescent cells. An alternative method analyzing peptides generated by protease digestion provided a quantitative view of most of the methylation sites on histone H3. The changed pattern of methylation on Lys 27 and Lys 36 of histone H3 in senescent cells, along with hypoacetylation on all core histones, were consistent with chromatin in senescent cells adapting a more compact, heterochromatic state.The global formation of heterochromatin did not translate to globally suppressed levels of transcription and protein translation. A comprehensive quantitative proteomic study applying SILAC (stable isotope labelling of amino acids in cell culture) indicated that a large number of proteins were up-regulated (179 of 2206 proteins identified) in senescent cells. These proteins were categorized into several functional groups, functioning in RNA processing, protein synthesis and energy production.Upregulation of proteins involved in oxidative phosphorylation and down regulation of proteins involved in glycolysis implied specific alternations of primary metabolism in senescent cells. This metabolic shift is the opposite of the well-known ‘Warburg effect’ which is an observation that most cancer cells predominantly produce energy by a high rate of glycolysis followed by lactate fermentation instead of oxidative phosphorylation. The metabolic shift detected by large scale proteomics is likely controlled by the regulation of pyruvate dehydrogenase (PDH) activity. Data consistent with this hypothesis was collected in OIS as both the inactive form of PDH and the cognate kinase (PDK-1) that turns it off decreased. Increased activity of PDH drove an increased flux of pyruvate into the TCA cycle followed by oxidative phosphorylation instead of lactate production for more efficient ATP production during senescence. A combination of limited glycolysis and decreased PDK-1 expression was shown to trigger the senescence phenotype in normal IMR-90 cells. These findings suggest that altered cell metabolism is a central part of the senescence phenotype and also contribute to successful execution of the senescence program. How to control the molecular switch of cell metabolism could have therapeutic implications in cocktail cancer therapy.
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Quantitative proteomic analysis of cellular senescence: from systems biology to targeted signaling pathways
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