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-Ras^61L 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-α 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 β-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-α induced-apoptosis. Surprisingly, senescence-associated β-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. Received: 5 June 2001, Returned for revision: 28 June 2001, Revision received: 6 July 2001, Accepted: 31 July 2001