Our data have indicated that mammalian Sir2О± can deacetylate p53 both in vitro and in vivo ( Figure 2) more importantly, Sir2О± can attenuate p53-mediated transcriptional activation ( Figure 4). Repression of p53-Mediated Functions by Mammalian Sir2О± Requires Its Deacetylase Activity Thus, these data clearly indicate that maximum induction of p53 acetylation requires inhibitors for both types of deacetylases (HDAC1 and Sir2О±), and that endogenous Sir2О± plays an important role in the regulation of the p53 acetylation levels induced by DNA damage. In contrast, 3-AB treatment had no effect on the level of p53 acetylation (lane 5 versus 3, Figure 3D), indicating that PARP-mediated poly-ADP ribosylation has no effect on p53 acetylation. Strikingly, a super induction of p53 acetylation was evident when the cells were treated with both TSA and nicotinamide (lane 4 versus 3, Figure 3D). Since no significant p53 acetylation was detected in the cells treated with a proteasome inhibitor ALLN (lane 6, Figure 3D), indicating that the observed stimulation of p53 acetylation is induced by DNA damage, not through p53 stabilization. Therefore, the enhancement of the acetylation level of p53 by TSA during DNA damage response may be due mainly to its inhibitory effect on endogenous HDAC1-mediated deacetylase activity (lane 3 versus 2, Figure 3D). Maximum Induction of p53 Acetylation Levels in Normal Cells Requires Inhibition of Endogenous Sir2О± Activity Thus, these data indicate that Sir2О±-mediated deacetylation of p53 can be inhibited by nicotinamide both in vitro and in vivo. Notably, this Sir2О±-mediated effect was completely abrogated by nicotinamide treatment (lane 8 versus 6, Figure 3C). However, the same DNA damage treatment failed to stimulate the p53 acetylation in the pBabe-Sir2О± infected cells even in the presence of TSA (lane 4 versus 2, upper panel, Figure 3C), indicating that Sir2О± expression results in deacetylation of endogenous p53. In the mock-infected cells, as expected, the acetylation level of p53 was significantly enhanced by DNA damage (lane 2 versus 1, upper panel, Figure 3C). Similar protein levels of p53 activation were induced in both types of cells after etoposide treatment for 6 hr (lanes 3, 4 versus lanes 1, 2, lower panel, Figure 3C). We first examined the protein levels of p53 activation in response to DNA damage in these cells by Western blot analysis. Mouse embryonic fibroblast (MEF) cells, which express the wild-type of p53, were infected with either a pBabe retrovirus empty vector or a pBabe retrovirus containing Sir2О±. Interestingly, this interaction was strongly detected in the cells after DNA damage treatment ( Figure 1D), suggesting that the possible regulation of p53 by mammalian Sir2О± may be still effective after DNA damage.ДЇurthermore, we examined the effect of Sir2О± expression on endogenous levels of acetylated p53. More importantly, both human and mouse p53 were clearly detected in the respective О±-Sir2О± immunoprecipitations from cell extracts, but not in the control immunoprecipitations with the preimmune serum, indicating that p53 interacts with mammalian Sir2О± in normal cells. Western blot analysis revealed that this antibody immunoprecipitated both Sir2О± and hSIRT1 (lower panels, Figures 1B and 1C). Cell extracts from both human (H460) and mouse cells (F9), which express wild-type p53 proteins, were immunopreciptated with О±-Sir2О±, or with the preimmune serum. Next, we used this antibody to investigate whether p53 interacts with Sir2О± or hSIRT1 in normal cells. and W.G., unpublished data, also see Figures 1B and 1C). By Western blot analysis, this antibody can detect both mouse Sir2О± and human SIRT1 proteins, but not other human Sir2 homologs (J.L.

), we developed a polyclonal antibody against the C terminus (amino acid 480вЂ“737) of mouse Sir2О±.

Negative control