Attenuation of constitutive DNA damage signaling by 1,25-dihydroxyvitamin D3

Abstract

In addition to its traditional role in the regulation of calcium homeostasis and bone metabolism, vitamin D also exhibits immunomodulatory, anti-proliferative and cancer preventive activities. Molecular mechanisms that confer the chemo-preventive properties to vitamin D are poorly understood. We previously reported that constitutive phosphorylation of histone H2AX on Ser139 (γH2AX) and activation of ATM (Ser1981 phosphorylation), seen in untreated normal or tumor cells predominantly in S phase of the cell cycle, is to a large extent indicative of DNA replication stress occurring as a result of persistent DNA damage caused by endogenous oxidants, by-products of oxidative metabolism. In the present study we observed that exposure of mitogenically stimulated human lymphocytes, pulmonary carcinoma A549 and lymphoblastoid TK6 cells to 1,25-dihydroxyvitamin D3 (1,25-VD) reduced the level of constitutive expression of γH2AX and ATM-S1981P. We also observed that the H2O2-induced rise in the level of γH2AX in lymphocytes was attenuated by 1,25-VD. Whereas in lymphocytes 1,25-VD reduced by 50-70% the level of endogenous oxidants as determined by their ability to oxidize 2,7-dichlorodihydrofluorescein (DCFH) in A549 and TK6 cells the attenuation of DNA damage signaling by 1,25-VD was seen in the absence of detectable reduction in DCFH oxidation. These findings suggest that while the anti-oxidant activity of 1,25-VD may contribute to a reduction in the intensity of DNA replication stress in lymphocytes, other factors play a role in the 1,25-VD effects seen in A549 and TK6 cells. The data are consistent with the recent report on the interaction between DNA damage signaling (ATM activation) and 1,25D receptor (VDR) phosphorylation that lead to enhancement of DNA repair efficiency, and provide further support for the chemo-preventive and anti-aging properties of this vitamin/hormone.

Figure 1. Effect of the treatment of A549 cells with 1,25-VD on the level of expression of γH2AX with respect to the cell cycle phase

Exponentially growing human pulmonary carcinoma A549 cells were untreated (Ctrl) or treated with 2 or 10 nM 1,25-VD for 24 or 48 h and expression of γH2AX was detected immunocytochemically using phospho-specific Ab targeting the phosphorylated S139 epitope; the intensity of fluorescence was measured by laser scanning cytometry (LSC) [70]. The numbers above the respective arrows indicate the percent reduction in intensity of immunofluorescence of the mean values of cell populations in G₁, S and G₂M phases of the cell cycle (gated as shown in the Ctrl panel) in the 1,25-VD-treated cells with respect to the untreated cells (Ctrl), in the same phase of cell cycle. Inserts show cellular DNA content histograms from the respective cultures. The skewed dash line in Ctrl panel shows the maximal level of fluorescence intensity of the cells stained with isotype control Ab.

Figure 2. Effect of treatment of A549 cells with 1,25-VD on the level of expression of ATM phosphorylated on Ser1981 in relation to the cell cycle phase

Expression of ATM-S1981^P in A549 cells treated with 2 nM or 10 nM 1,25-VD for 24 or 48 h was detected immunocytochemically using phospho-specific Abs and measured by LSC. The numbers above the arrows indicate the percent reduction in intensity of immunofluorescence of the mean values of cell populations in G₁, S and G₂M phases of the cell cycle in the 1,25-VD-treated cells with respect to the untreated cells (Ctrl) in the same phase of cell cycle. The skewed dash line in Ctrl panel shows the maximal level of fluorescence intensity of the cells stained with isotype control Ab.

Figure 3. Effect of treatment of TK6 cells with 1,25-VD on the level of constitutive expression of γH2AX and ATM-S1981^P

Exponentially growing TK6 cells were untreated (Ctrl) or treated with 10 nM 1,25-VD for 24 h. Expression of γH2AX and ATM-S1981^P was detected in individual cells immunocytochemically, cellular fluorescence was measured by flow cytometry. The numbers above the arrows show the percent reduction in intensity of immunofluorescence of the mean values of cell populations in G₁, S and G₂M phases of the cell cycle in the 1,25-VD-treated cells with respect to the untreated cells (Ctrl) in the same phase of cell cycle.

Figure 4. Effect of treatment of mitogenically stimulated proliferating human lymphocytes untreated or treated with H₂O₂ and 1,25-VD on the level of expression of γH2AX

Human peripheral blood lymphocytes mitogenically stimulated with PHA for 48 h were untreated (Ctrl) or treated with 10 nM 1,25-VD for 24 h (A). Cells in another set of cultures (B) were either untreated with 1,25-VD but exposed to 200 μM of H₂O₂ for 1 h (H₂O₂) or were pretreated with 1,25-VD for 24 h and then treated with 200 μM H₂O₂ for 1 h (+H₂O₂). Expression of γH2AX was detected immunocytochemically and cell fluorescence was measured by flow cytometry. Through gating analysis (as shown in Figs. 1-3) the mean values (+SD) of fluorescence intensity for cells in G₁, S and G₂M phases of the cell cycle were obtained and plotted as bar graphs. Numbers above the arrows show the percent decline of the mean values of the 1,25-VD-treated cells compared to the cells not treated with 1,25-VD in the respective phases of the cycle.