The Effects of Cell Culture Oxygen Levels on the Replicative Senescence Processes of Primary Human Fibroblasts
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A series of experiments were performed to assess changes during the induction of replicative senescence under conditions of low (3%) and high (20%) oxygen levels. Measurements were made at the transcriptional, protein, and metabolite levels. Microscopy wasalso utilized to monitor changes in mitochondrial morphology and volume. While previous studies have evaluated specific pathways and/or products; this work combines a more complete metabolomic, genomic, proteomic, and morphological picture of cells undergoing senescence and oxidative stress.
Considering the low cell population densities of primary adherent fibroblasts and the subsequent low concentrations of small polar metabolites involved in glycolysis and the TCA cycle, methodologies needed to be developed in order to optimize metabolite extraction and liquid chromatography-mass spectrometric analysis. Protein kinase and transcriptional microarrays were also performed in order to quantify the changes in activated/deactivated signaling cascades as well as gene expression and relate these findings to metabolomic data. Mitochondrial dynamics of cells at different age time points and under different oxygen conditions were also assessed including mitochondrial size, shape, membrane potential, and percent volume per cell volume using confocal microscopy.
The results obtained not only confirm the major pathways involved in senescence (p53/p21, PTEN/p27, and RTK/Raf/MAPK) but also provide evidence at both the transcriptional and protein levels for additional senescence-associated pathways. The majority of the changes observed were related to pathways involved in cellular stress, cell cycle control, and the survival response. Metabolic data suggested a â pooling effectâ of glycolysis and TCA precursor molecules due to attenuation in enzyme function; this theory was also supported by an observed up regulation of gene expression as a compensatory mechanism. Mitochondria exhibited changes in membrane potential as well as volume and percent volume per cell which suggested compensatory hypertrophy and/or attenuation of mitochondrial fission processes. When the aforementioned analyses are tied together, a â theoretical model of senescenceâ can be formulated and is characterized by increased metabolic protein and associated metabolite levels due to attenuation in their respective enzyme function, resulting in increases in expression of their associated genes as a compensatory mechanism.
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