The PI3K/Akt/mTOR Pathway Is Implicated in the Premature Senescence of Primary Human Endothelial Cells Exposed to Chronic Radiation

Abstract

The etiology of radiation-induced cardiovascular disease (CVD) after chronic exposure to low doses of ionizing radiation is only marginally understood. We have previously shown that a chronic low-dose rate exposure (4.1 mGy/h) causes human umbilical vein endothelial cells (HUVECs) to prematurely senesce. We now show that a dose rate of 2.4 mGy/h is also able to trigger premature senescence in HUVECs, primarily indicated by a loss of growth potential and the appearance of the senescence-associated markers ß-galactosidase (SA-ß-gal) and p21. In contrast, a lower dose rate of 1.4 mGy/h was not sufficient to inhibit cellular growth or increase SA-ß-gal-staining despite an increased expression of p21. We used reverse phase protein arrays and triplex Isotope Coded Protein Labeling with LC-ESI-MS/MS to study the proteomic changes associated with chronic radiation-induced senescence. Both technologies identified inactivation of the PI3K/Akt/mTOR pathway accompanying premature senescence. In addition, expression of proteins involved in cytoskeletal structure and EIF2 signaling was reduced. Age-related diseases such as CVD have been previously associated with increased endothelial cell senescence. We postulate that a similar endothelial aging may contribute to the increased rate of CVD seen in populations chronically exposed to low-dose-rate radiation.

Unique proteomic signature for radiation sensitive patients; a comparative study between normo-sensitive and radiation sensitive breast cancer patients

“…Deregulation greater than +1.3-fold or less than −1.3-fold was set as cut off. This overcomes the technical variability in our experiments (3.4%) and the biological significance of this cut-off is in good agreement with previously published data [15,18,19,22,25]. The CV% of spiked proteins in different replicates and complete tables of proteins and p-values are shown in Supporting Information Tables A-G.…”

“…The biological significance of this fold change cut off is in good agreement with the previously published data [18][19][20]. The ICPL method is well established and has in previous publications been extensively validated [14,15,17,21,22]. Due to limited amounts of material a validation with individual samples was not feasible in this study, however the two separate ICPL analyses showed good correlation.…”

“…Protein concentration was determined by Bradford assay following the manufacturer's instructions (Thermo Fisher). The duplex or triplex labeling was done as previously reported [14,15]. Schematic representation of labeling work flow from three biological replicates is shown in Fig.…”

“…Data processing for protein identification and quantification of ICPL pairs was performed using Proteome Discoverer version 1.3 (Thermo Fisher) as described before [15,17] Matrix Science) with the following parameters: a precursor mass error tolerance of 10 ppm and a fragment tolerance of 0.6 Da. One missed cleavage of enzyme ArgC was allowed.…”

Erratum to: From cell senescence to age-related diseases: differential mechanisms of action of senescence-associated secretory phenotypes

“…Enhanced protein synthesis during senescent arrest is maintained by mTOR activation and upregulated activity of phosphatidylinositol 3-kinase (PI3K), an upstream activator of mTOR (43)(44)(45). Moreover, GSK3-mediated augmentation of lipogenesis and glycogenesis has been reported to be critically linked 643 http://bmbreports.org BMB Reports with an increase in the overall mass of organelles (such as mitochondria, lysosome, Golgi, and ER) and cell granularity (46,47).…”