Selenium compounds activate ATM-dependent DNA damage response via the mismatch repair protein hMLH1 in colorectal cancer cells

Abstract

Epidemiological and animal studies indicate that selenium supplementation suppresses risk of colorectal and other cancers. The majority of colorectal cancers are characterized by a defective DNA mismatch repair (MMR). Here, we have employed the MMR-deficient HCT 116 colorectal cancer cells and the MMR-proficient HCT 116 cells with hMLH1 complementation to investigate the role of hMLH1 in selenium-induced DNA damage response, a tumorigenesis barrier. The ATM (ataxia telangiectasia mutated) protein responds to clastogens and initiates DNA damage response. We show that hMLH1 complementation sensitizes HCT 116 cells to methylseleninic acid, methylselenocysteine, and sodium selenite via reactive oxygen species and facilitates the selenium-induced oxidative 8-oxoguanine damage, DNA breaks, G(2)/M checkpoint response, and ATM pathway activation. Pretreatment of the hMLH1-complemented HCT 116 cells with the antioxidant N-acetylcysteine or 2,2,6,6-tetramethylpiperidine-1-oxyl or the ATM kinase inhibitor KU55933 suppresses hMLH1-dependent DNA damage response to selenium exposure. Selenium treatment stimulates the association between hMLH1 and hPMS2 proteins, a heterodimer critical for functional MMR, in a manner dependent on ATM and reactive oxygen species. Taken together, the results suggest a new role of selenium in mitigating tumorigenesis by targeting the MMR pathway, whereby the lack of hMLH1 renders the HCT 116 colorectal cancer cells resistant to selenium-induced DNA damage response.

FIGURE 1.

Sensitivity of HCT 116 cells and hMLH1-complemented HCT 116 cells to the selenium compounds and antioxidants. A–C, HCT 116 cells and HCT 116+hMLH1 cells were cultured in six-well plates and treated with Na₂SeO₃, MSeA, or MSeC. D–F, HCT116+hMLH1 cells were pretreated with antioxidants NAC or Tempo, followed by treatment with the selenium compounds. Cell survival was assessed by colony formation assay. The number of colonies in the condition without selenium treatment was set as 100%. Values are mean ± S.E. (n = 3). *, p < 0.05 compared with HCT 116+hMLH1 cells (A–C) or with antioxidant treatments (D–F).

FIGURE 2.

Complementing HCT 116 cells with hMLH1 potentiates selenium-induced DNA oxidation and breaks. HCT 116 and HCT 116+hMLH1 cells were cultured on coverslips and treated with Na₂SeO₃ (2 μm) for 6 and 12 h, followed by immunofluorescence analyses of 8-oxoG and γH2AX. Ratios of nuclei (DAPI staining) showing 8-oxoG staining (A and B) and ratios of cells expressing γH2AX (C and D) to cells expressing total H2AX are presented with their respective S.E. values (n = 3). *, p < 0.05. A and C, representative pictures showing immunofluorescent signals of DAPI (blue), total H2AX (green), and γH2AX or 8-oxoG (red).

FIGURE 3.

Complementing HCT 116 cells with hMLH1 restores G₂/M arrest after selenium exposure. HCT 116 and HCT 116+hMLH1 cells were treated with Na₂SeO₃ (2 μm), harvested at the indicated time points, and analyzed for cell cycle profiles by flow cytometry (n = 3). Cells in G₀/G₁ (A), S (B), and G₂/M (C) phases were quantified and presented. Values are mean ± S.E. (n = 3). *, p < 0.05.

FIGURE 4.

Effects of ATM kinase activity and ROS on the hMLH1-dependent G₂/M arrest after selenium exposure. HCT 116+hMLH1 cells were pretreated with 10 μm KU55933 (A) and 10 mm NAC or 2 mm Tempo (B) for 24 h, followed by Na₂SeO₃ (2 μm) exposure for 12 h. Cell cycle profiles were analyzed as described in the legend to Fig. 3.

FIGURE 5.

hMLH1 mediates Na₂SeO₃-induced ATM pathway activation in G₂/M. Undisturbed or G₂/M-arrested (300 ng/ml Noc, 16 h) cells were treated with Na₂SeO₃ (2 μm) for 0–12 h prior to immunofluorescence analyses. Ratios of cells expressing pATM Ser-1981 (A and B) or pDNA-PKcs Thr-2647 (C and D) to cells expressing total ATM or total DNA-PKcs are presented with their respective S.E. values. *, p < 0.05.

FIGURE 6.

Selenium treatment increases hMLH1-hPMS2 association in a manner dependent on ATM and ROS. A, HCT 116 and HCT 116+hMLH1 cells were treated with Na₂SeO₃ (2 μm) for 24 h. B–D, HCT 116+hMLH1 cells were pretreated with NAC (10 mm), Tempo (2 mm), or KU55933 (10 μm) for 24 h, followed by Na₂SeO₃ (2 μm) exposure for 24 h. The lysates were immunoprecipitated (IP) using anti-IgG or anti-PMS2 antibodies, followed by immunoblotting using the indicated antibodies. The amount of hMLH1 associated with anti-hPMS2 immunoprecipitates was estimated based on the band intensity of the inputs. The related ratios in cells without selenium treatment were set as 1 and were presented as S.E. values. *, p < 0.05; **, p < 0.01 compared with no Na₂SeO₃ treatment. Inputs contained 5% of the amount of lysates used for immunoprecipitation.