Induction Of DNA Damage And Cellular Senescence By TNF-alpha In Human Fibroblasts

Cellular senescence refers to the permanent withdrawal from cell cycle and the gradual decline in normal physiological function. Although they still possess basic metabolic activities, they will eventually die. There exist two kinds of senescence: replicative senescence and stress-induced premature senescence. Senescence can be induced by telomere shortening, DNA damage, oncogen activation, inflammatory factors and other chemical stimuli. Inflammation-induced cellular senescence recently draws increased interest. In particular, TNF-a, a pleiotropic cytokine, was shown to cause cellular senescence in some experimental systems. TNF-a was reported to induce senescence through up-regulating the level of reactive oxygen species (ROS) and resulting in DNA oxidative damage. It was also demonstrated that TNF-a can directly affect p38/MAPK pathway and induce subsequent cellular senescence by activating the expression of p16INK4a in endothelial cells. However, many questions remain. How exactly does TNF-a induce senescence? Does TNF-a induce senescence in all cell types? What is the relationship between DNA damage response and MAPK pathways?In order to further elucidate the role of TNF-a in cellular senescence, we treated human fibroblasts with TNF-a at the concentration of lOng/ml and then observed SA-β-galactosidase activity by (3-gal staining. We found that the number of cells with positive SA-β-gal staining is increased by TNF-a treatment in a time-dependent way. Then we determined the expression of key factors in ATM and MAPK pathways by Western blotting and Real time RT-PCR. Our results showed that chronic TNF-a treatment led to an increased level of DNA double-strand breaks, as measured by the y-H2AX, in HNF cells, and an activation of Chk2. At the same time, TNF-a was also shown to activate p38/MAPK, and blocking the activation of p38/MAPK with SB203580can attenuate the increase of SA-p-gal positive cells caused by TNF-a. TNF-a treatment also caused the upregulation of p16INK4a and p21Cip1in HNF, which could be offset by SB203580. However, contrary to reported effect of TNF-a on cell cycle progression in HUVECs cells, we observed that TNF-a treatment decreased the proportion of cells at G0/G1and significantly increased that in S phase. In addition, we found that TNF-a may induce the apoptosis of U2OS.Therefore, TNF-a can activate ATM and p38/MAPK pathways and induce cellular senescence in HNF. However, the relationship between ATM and p38/MAPK remains to be determined. TNF-a can up-regulate p16INK4a and p21Cip1expression, which are mediated by p38/MAPK, but not by p53, in inducing cellular senescence. Ours findings suggest that reducing inflammation may delay the onset of aging.