Inhibition of SIRT1 catalytic activity increases p53 acetylation but does not alter cell survival following DNA damage

Abstract

Human SIRT1 is an enzyme that deacetylates the p53 tumor suppressor protein and has been suggested to modulate p53-dependent functions including DNA damage-induced cell death. In this report, we used EX-527, a novel, potent, and specific small-molecule inhibitor of SIRT1 catalytic activity to examine the role of SIRT1 in p53 acetylation and cell survival after DNA damage. Treatment with EX-527 dramatically increased acetylation at lysine 382 of p53 after different types of DNA damage in primary human mammary epithelial cells and several cell lines. Significantly, inhibition of SIRT1 catalytic activity by EX-527 had no effect on cell growth, viability, or p53-controlled gene expression in cells treated with etoposide. Acetyl-p53 was also increased by the histone deacetylase (HDAC) class I/II inhibitor trichostatin A (TSA). EX-527 and TSA acted synergistically to increase acetyl-p53 levels, confirming that p53 acetylation is regulated by both SIRT1 and HDACs. While TSA alone reduced cell survival after DNA damage, the combination of EX-527 and TSA had no further effect on cell viability and growth. These results show that, although SIRT1 deacetylates p53, this does not play a role in cell survival following DNA damage in certain cell lines and primary human mammary epithelial cells.

FIG. 1.

EX-527 is a potent inhibitor of SIRT1 that also inhibits Sir2-dependent silencing in yeast. (A) Structure of EX-527. (B) EX-527 inhibits deacetylase activity of purified human SIRT1. Deacetylase activity of GST-SIRT1 in the presence of increasing concentrations of EX-527 was measured by the Fluor de Lys assay. Samples were assayed in triplicate and analyzed using Prism (Graphpad Software, Inc.). (C) The active optical isomer of EX-527, EX-243, and not the inactive optical isomer, EX-242, inhibits Sir2-dependent silencing in yeast. A yeast strain constructed with a URA3 gene inserted near a telomere was grown in the presence of compounds with or without 5-FOA. Growth was measured by light scattering at 600 nm.

FIG. 2.

Inhibition of SIRT1 catalytic activity increases p53 acetylation after DNA damage. (A) Effect of EX-527 on p53 acetylation after DNA damage. NCI-H460 cells were treated with etoposide in combination with either nicotinamide (Nicot.), EX-527, or dimethyl sulfoxide vehicle (DMSO) for 6 h. Blots were probed with an anti-acetylated p53 Lys 382 antibody (upper panel) or p53 antibody (Ab-7) that recognizes all forms of human p53 (lower panel). (B) Effect of EX-527 and a series of closely related compounds on p53 acetylation after DNA damage. Cells were treated with 1 μM concentrations of the indicated compounds for 6 h, and samples were prepared as described for panel A, except that cells were treated additionally with 25 nM TSA to inhibit class I/II HDAC activity. (C) Effect of enantiomers of EX-527 on p53 acetylation after DNA damage. Cells were treated with EX-242 or EX-243 for 6 h. p53 was immunoprecipitated and immunoblotted as described for panel A. +, present; −, absent.

FIG. 3.

Inhibition of SIRT1 catalytic activity enhances p53 lysine 382 acetylation after a variety of DNA-damaging agents and is not cell type specific. (A) NCI-H460 cells were treated with adriamycin, hydroxyurea, or hydrogen peroxide for 6 h. p53 was immunoprecipitated and immunoblotted with an anti-acetylated p53 Lys 382 antibody or p53 antibody (Ab-7). (B) U-2 OS and MCF-7 cells were treated with etoposide in the presence or absence of 1 μM EX-527 for 6 h. Immunoprecipitation and immunoblotting was performed as described for panel A. (C) HMEC were treated with etoposide or adriamycin in the presence or absence of 1 μM EX-527 for 6 h. Immunoprecipitation and immunoblotting of p53 was performed as described for panel A. Total SIRT1 levels were measured by immunoblotting using antibody 07-131 (Upstate Cell Signaling Solutions). +, present; −, absent.

FIG. 4.

p53-controlled gene expression is not altered by inhibition of SIRT1 in several p53-positive cell lines. (A) NCI-H460, MCF-7, U-2 OS, and HMEC were treated with various concentrations of etoposide for 6 h in the presence of 1 μM EX-527 or dimethyl sulfoxide vehicle (DMSO). Levels of p21 mRNA were measured using quantitative PCR and normalized to the expression levels of hypoxanthine phosphoribosyltransferase (HPRT). (B) NCI-H460 cells were treated with etoposide or adriamycin in the presence or absence of EX-527 for 6 h. Cell lysates were prepared for immunoblotting and were probed with antibodies directed against p21 and tubulin. +, present; −, absent.

FIG. 5.

Effect of inhibition of SIRT1 catalytic activity on cell viability and proliferation following DNA damage in cell lines. NCI-H460, U-2 OS, and MCF-7 cells were treated with increasing concentrations of etoposide or adriamycin in the presence or absence of 1 μM EX-527. NCI-H460 cells were additionally treated with 5 mM nicotinamide. (A, C, and E) Cell viability was measured at 48 h; (B, D, and F) cell proliferation at 72 h was measured by monitoring [¹⁴C]thymidine incorporation.

FIG. 6.

Effect of inhibition of SIRT1 catalytic activity on cell viability and proliferation following DNA damage in primary human cells. HMEC were treated with increasing concentrations of etoposide or adriamycin in the presence or absence of 1 μM EX-527. (A and C) Cell viability was measured at 48 h; (B and D) cell proliferation at 48 h was measured by monitoring [¹⁴C]thymidine incorporation.

FIG. 7.

Effect of inhibiting SIRT1 and HDAC1 on p53 lysine 382 acetylation and cell growth. (A) NCI-H460 cells were treated for 6 h with various concentrations of TSA and 1 μM EX-527 in the presence of 20 μM etoposide. Cell lysates were analyzed by immunoblotting with an anti-acetylated p53 Lys 382 antibody or p53 antibody (Ab-7). The graph shows the quantification of bands on the gel shown, but this is representative of the results from two such experiments. +, present; −, absent. (B to E) NCI-H460 cells were treated with increasing concentrations of etoposide or adriamycin and deacetylase inhibitors as indicated. Dimethyl sulfoxide (DMSO) was used as the vehicle control. (B and D) Cell proliferation at 72 h was measured by monitoring [¹⁴C]thymidine incorporation; (C and E) cell viability was measured at 48 h.

FIG. 8.

Effect of overexpression of SIRT1 on hydrogen peroxide-induced cell death. Cell viability was measured in IMR-90 (A) or MCF-7 (B) cells overexpressing SIRT1 or GFP treated with increasing concentrations of hydrogen peroxide for 24 h. Overexpression of SIRT1 in these cells was confirmed using immunoblot analysis (insets). Endogenous SIRT1 is not visible because very small amounts of protein (2 to 4 μg) were needed to obtain a clean signal from cells overexpressing SIRT1 protein.