Oncogenic ras induces premature senescence in endothelial cells: role of p21(Cip1/Waf1).
Recent studies have shown that the presence of tumor suppressors such as p53 or p16 account for the lack of transformation in primary cells. To investigate a potential role of active Ras in atherosclerosis, we infected bovine aortic endothelial cells with a replication-deficient, recombinant adenovirus containing the activated H-Ras61L gene. Ras overexpression led after 72 hours to G1- and G2/M-cell cycle arrest due to induction of p21(Cip1/Waf1). Treatment of Ras-infected endothelial cells with 40 ng/ml TNF-alpha for 20 hours augmented apoptosis 8-fold in comparison to Ad-Con (control virus with empty expression cassette) infected cells (36.2% vs. 4.3%, p < 0.001), while Ras itself did not cause any cell death. Furthermore, more than 58% of Ras-infected cells stained positive for senescence-associated beta-galactosidase activity as opposed to 2% in control vector-infected cells (p < 0.001), strongly suggesting a senescent phenotype in the Ras-infected population. We found further features of senescence in Ras-transduced endothelial cells, such as growth arrest and the lack of AP-1 serum inducibility. Finally, we evaluated the role of p21(Cip1/Waf1) in this process of senescence. Adenoviral overexpression of p21 led to growth arrest by induction of G1- and G2/M-cell cycle arrest. In addition, p21-overexpressing endothelial cells were highly sensitive for TNF-alpha induced-apoptosis. Surprisingly, senescence-associated beta-galactosidase activity was not apparant in p21-infected endothelial cells, suggesting further signaling events necessary for the senescent morphology of endothelial cells. Our results demonstrate a novel way to render primary endothelial cells senescent by overexpressing oncogenic Ras. Increased sensitivity of senescent endothelial cells for cytotoxic stimuli seemed to be due to Ras-induced upregulation of p21(Cip1/Waf1). Future studies have to investigate a potential role of Ras in human vascular biology.
Spyridopoulos
Ioakim
I
Dept of Cardiology and Cardiovascular Research, Medizinische Klinik III, Tübingen, Germany. ioakim_s@hotmail.com
Isner
Jeffrey M
JM
Losordo
Douglas W
DW
D000256:Adenoviridae / Q000235:genetics; D000818:Animals; D001011:Aorta / Q000166:cytology; D017209:Apoptosis / Q000502:physiology; D001161:Arteriosclerosis / Q000473:pathology / Q000503:physiopathology; D002417:Cattle; D002478:Cells, Cultured; D016922:Cellular Senescence / Q000502:physiology*; D050759:Cyclin-Dependent Kinase Inhibitor p21; D016213:Cyclins / Q000378:metabolism*; D004730:Endothelium, Vascular / Q000166:cytology*; D015870:Gene Expression / Q000502:physiology; D066298:In Vitro Techniques; D016283:Proto-Oncogene Proteins p21(ras) / Q000235:genetics* / Q000378:metabolism
D050759:Cyclin-Dependent Kinase Inhibitor p21; D016213:Cyclins; D016283:Proto-Oncogene Proteins p21(ras)