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. 2012 Aug;31(8):392-8.
doi: 10.5732/cjc.012.10089. Epub 2012 Jun 14.

Nampt is involved in DNA double-strand break repair

Bingtao Zhu  1 , Xiaoli Deng, Yifan Sun, Lin Bai, Zhikai Xiahou, Yusheng Cong, Xingzhi Xu
Affiliations

Nampt is involved in DNA double-strand break repair

Bingtao Zhu et al. Chin J Cancer. 2012 Aug.

Abstract

DNA double-strand break (DSB) is the most severe form of DNA damage, which is repaired mainly through high-fidelity homologous recombination (HR) or error-prone non-homologous end joining (NHEJ). Defects in the DNA damage response lead to genomic instability and ultimately predispose organs to cancer. Nicotinamide phosphoribosyltransferase (Nampt), which is involved in nicotinamide adenine dinucleotide metabolism, is overexpressed in a variety of tumors. In this report, we found that Nampt physically associated with CtIP and DNA-PKcs/Ku80, which are key factors in HR and NHEJ, respectively. Depletion of Nampt by small interfering RNA (siRNA) led to defective NHEJ-mediated DSB repair and enhanced HR-mediated repair. Furthermore, the inhibition of Nampt expression promoted proliferation of cancer cells and normal human fibroblasts and decreased β-galactosidase staining, indicating a delay in the onset of cellular senescence in normal human fibroblasts. Taken together, our results suggest that Nampt is a suppressor of HR-mediated DSB repair and an enhancer of NHEJ-mediated DSB repair, contributing to the acceleration of cellular senescence.

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Figures

Figure 1.
Figure 1.. Nampt associates with CtIP and DNA-PKcs/Ku80.
A, mass spectrometric analysis reveals that DNA-PKcs and Ku80 are present in the Nampt immunocomplex. The endogenous Nampt immunocomplex was pulled down from 50 mg of total cell lysates derived from log-phase HeLa cells using 50 µg of affinity-purified Nampt rabbit polyclonal antibodies or normal rabbit IgG as a negative control. Discrete bands were excised for mass spectrometric analysis. The numbers on the right side of the panel indicate the number of identified polypeptides divided by the number of predicted polypeptides equals the percent coverage of the polypeptides. B, endogenous Nampt associates with endogenous CtIP and DNA-PKcs/Ku80. Endogenous Nampt in HeLa cell lysate was immunoprecipitated with an anti-Nampt antibody, and the precipitated complex was resolved with 4%–8% gradient SDS-PAGE and immunoblotted with antibodies as indicated. *non-specific band. C, HA-Nampt pulls down endogenous CtIP and DNA-PKcs/Ku80. HA-Nampt is stably expressed in HeLa cells. Total cell lysate was extracted and immunoprecipitated with an anti-HA antibody and blotted with antibodies as indicated.
Figure 2.
Figure 2.. Nampt suppresses HR-mediated DSB repair while promoting NHEJ-mediated DSB repair.
A and B, inhibition of Nampt expression increases HR-mediated DSB repair efficiency. DR-GFP U2OS cells were transfected with a mixture of 4 siRNA oligos (siNampt) (A) or individual siRNA oligos (si1-4Nampt) (B). Transfectants were additionally transfected with an I-SceI expression construct 24 h after siRNA transfection. Cells were harvested 48 h after the second transfection to quantify GFP-positive cells by FACS. C and D, expression of an siRNA-resistant form of Nampt rescues the HR-mediated DSB repair increase that resulted from the depletion of endogenous Nampt. DR-GFP U2OS cells were stably transfected with an empty HA vector or an HA-resNampt expression construct whose expression product is resistant to depletion by si4Nampt. Expression of endogenous Nampt was inhibited by si4Nampt (C). The relative HR-mediated DSB repair efficiency was determined by analyzing GFP-positive cells by FACS (D). E, depletion of Nampt expression decreases NHEJ-mediated DSB repair efficiency. EJ5-GFP 293 cells were first transfected with siNampt, si1-4Nampt, or a non-targeting control siControl, and 24 h later with an expression construct for I-SceI. Cells were harvested 72 h after the second transfection for immunoblotting with the antibodies as indicated (bottom panel) and flow cytometric analysis of GFP-positive cells (top panel). All values are presented as mean ± standard deviation (SD) of at least three independent experiments. *P < 0.05, **P > 0.05, vs. control cells.
Figure 3.
Figure 3.. Depletion of Nampt expression promotes cell proliferation and suppresses cell senescence.
A and B, inhibition of Nampt expression promotes cell growth. Nampt expression was inhibited by transfection with siNampt, and the cell number was determined every 24 h in U2OS cells (A) and WI38 cells (B). Six duplicate wells were used at each time point. C-E, inhibition of Nampt expression delays cell senescence. WI38 cells at passage 39 were infected with shNampt or shControl, cultured for 14 days, and stained for β-gal. Representative cells are shown in (C), and quantitative data are shown in (D). The status of Nampt expression is shown in (E). All values are presented as mean ± SD of at least three independent experiments. *P < 0.05, vs. control cells.
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