X-irradiation induces ER stress, apoptosis, and senescence in pulmonary artery endothelial cells

PURPOSE: The use of clinical radiation for cancer treatment is limited by damage to underlying normal tissue including to the vascular endothelium. We investigated the mechanisms of X-ray-induced cell damage to endothelial cells.

METHODS: We evaluated necrosis, apoptosis, cellular senescence, and the contribution of endoplasmic reticulum (ER) stress in pulmonary artery endothelial cells (PAEC) irradiated with X-rays (2-50 Gray [Gy]).

RESULTS: Clonogenic assays showed that 10 Gy induced ∼99.9% loss of cell viability. No necrosis was detected using lactate dehydrogenase assays, but a low population underwent extrinsic and intrinsic apoptosis, as indicated by the activation of caspases 3, 8, and 9 as well as by neutral comet assay. A majority of PAEC underwent accelerated senescence, as indicated by morphological changes, increased 21 kD cyclin-dependent kinase inhibitor (p21/waf1), decreased sirtuin 1 (SIRT1), and elevated senescence-associated β-galactosidase (SA-β-gal). ER stress was detected by assays for glucose-regulated protein 78 (GRP78), CCAAT/enhancer-binding protein homologous protein (CHOP), and growth arrest and DNA damage-inducible protein 34 (GADD34) mRNA, and transient phosphorylation of eukaryotic translation initiation factor 2 alpha (eIF2α). The ER stress inhibitor salubrinal blocked ∼50% of apoptosis with no effect on senescence.

CONCLUSIONS: X-rays primarily induced cellular senescence with limited levels of apoptosis in endothelial cells. ER stress contributed to apoptosis but not to senescence.