Aberrant DNA damage response and DNA repair pathway in high glucose conditions

Abstract

Background: Higher cancer rates and more aggressive behavior of certain cancers have been reported in populations with diabetes mellitus. This association has been attributed in part to the excessive reactive oxygen species generated in diabetic conditions and to the resulting oxidative DNA damage. It is not known, however, whether oxidative stress is the only contributing factor to genomic instability in patients with diabetes or whether high glucose directly also affects DNA damage and repair pathways.

Results: Normal renal epithelial cells and renal cell carcinoma cells are more chemo- and radiation resistant when cultured in high concentrations of glucose. In high glucose conditions, the CHK1-mediated DNA damage response is not activated properly. Cells in high glucose also have slower DNA repair rates and accumulate more mutations than cells grown in normal glucose concentrations. Ultimately, these cells develop a transforming phenotype.

Conclusions: In high glucose conditions, defective DNA damage responses most likely contribute to the higher mutation rate in renal epithelial cells, in addition to oxidative DNA damage. The DNA damage and repair are normal enzyme dependent mechanisms requiring euglycemic environments. Aberrant DNA damage response and repair in cells grown in high glucose conditions underscore the importance of maintaining good glycemic control in patients with diabetes mellitus and cancer.

Keywords: ATR; Chemo resistant; DNA damage response; Diabetes; checkpoint kinase 1.

Figure 1

Radiation and chemo-resistance in high glucose conditions. Mouse renal tubular epithelial cells (A) and human renal cells (HK2) and renal cell carcinoma cells (RCC) (786-O) (B) were subjected to different doses of UV radiation or treated with the topoisomerase inhibitor etoposide. Cell survival was examined 24 hours later. After UV radiation, cells were recovered in either 7.8 mM glucose with 30 mM mannitol (MG) or 37.8 mM glucose (HG). For etoposide treatment, cells were maintained in MG or HG media. Human and mouse cells become more resistant to DNA damaging agents when cultured in high glucose conditions. Greater chemo-resistance was observed in 786-O RCC cells when compared to HK2 normal renal cells in the low glucose conditions. While in the high glucose, HK2 cells also showed chemo-resistance, similar to 786-O RCC cells.

Figure 2

DNA damage response in cells in high glucose conditions. Mouse renal tubular epithelial cells (passage 3) were treated with UV irradiation (0.1 mJ/cm²). The cells were thereafter collected at different time points, and in media containing different concentrations of glucose [low glucose (LG, 7.8 mM); high glucose (HG, 37.8 mM)] or in low glucose plus 30 mM mannitol (MG). Total proteins were extracted from cell lysates and subjected to SDS-PAGE analysis (A). The DNA damage response proteins, Nek1, 53BP1, claspin, Rad51, Mre11, and γH2AX, were analyzed. Analysis of p48 expression was included to serve as a protein loading control. Nek1 abundance was increased in both MG and HG low conditions, even before any UV irradiation (A, B). The abundance of claspin and Rad51 wanes over the 24 hours after UV irradiation, specifically in the high glucose condition (A). In the LG and MG conditions, γH2AX, which accumulates at sites of damaged DNA, increased in the hours after UV irradiation, as expected (A). Only in the cells recovered in the HG condition, however, was γH2AX expression significantly upregulated even before any UV treatment (A, lanes 1, 8, and 15, and C).

Figure 3

CHK1 signaling is impaired in high glucose conditions. Mouse renal tubular epithelial cells (passage 3) were treated with UV irradiation (0.1 mJ/cm²); cells were collected and proteins from lysates were subjected to SDS-PAGE analysis in a manner identical to that described for Fig. 2. Activation by specific phosphorylation of the DNA damage response proteins, ATR, Rad17, CHK1, and ATM, was examined after UV irradiation and after the cells were the maintained in different glucose or glucose plus mannitol concentrations. Here GAPDH was included as the protein loading control. The timing of ATR activation (P-ATR) is normal, irrespective of the glucose condition, as is the timing of Rad17 activation (P-Rad17) (A). Activation of ATM (P-ATM) is also intact in LG, MG, and HG conditions after UV irradiation (B). Histograms show that P-ATR (activated ATR) expression is high at baseline (before UV irradiation) in both high glucose (HG) and low glucose plus 30 mM mannitol (MG) conditions (C). A graph of the expression level of P-CHK1 (activated CHK1) versus time after UV irradiation shows that CHK1 fails to be activated at all, specifically in the HG condition (D). Histograms quantitating relative P-ATR expression show that culturing cells in HG or MG medium, without any UV irradiation, did not activate ATM compared to culture in LG medium (E).

Figure 4

Increased mutation rate in high glucose conditions. Renal tubular epithelial cells from Big Blue® mice were cultured in DMEM/F12 media containing low (7.8 mM) or high (37.8mM) glucose concentrations. At different passages, cells were harvested and mutation rates were analyzed by Big Blue® assay (counting blue colonies). The mutation rate was significantly higher in the cells maintained from primary culture in medium containing a high glucose concentration. Representative plates (A) and histograms quantitating blue colonies (B) are shown.

Figure 5

Reduced DNA repair ability in high glucose conditions after UV irradiation. Renal tubular epithelial cells were cultured in media containing a low (7.8 mM) concentration of glucose. After they were washed three times with PBS, the cells were UV irradiated at low dose (0.1 mJ/cm²) and refed with 7.8 mM glucose (LG), 7.8mM glucose plus 30 mM mannitol (MG), or 37.8mM glucose (HG). At the indicated times, cells were harvested and subjected to comet assays (A). Cells cultured for 24 hours in different concentrations of glucose, but not treated with UV were also analyzed by comet assays; in these control conditions, no significant differences were observed between cells in different cultured in LG, HG, or MG, either at 0 or 24 hour time points (B). DNA repair ability as assessed by comet tail length (C) and tail moment (D), however, was significantly different in cells maintained in the various glucose concentrations. Based on the comet assays, cells cultured in low glucose repair their DNA faster or better than those cultured in the high glucose. * p<0.001

Figure 6

High glucose increases cell growth rate. Mouse renal epithelial cells (2×10⁵) were cultured from primary conditions by plating them onto 60-mm dishes, and then maintained and passed using a 3T3 protocol. Cells at different passages were examined for the saturation density (A) and colony formation in soft agar (B). The cells in the high glucose condition grew to have higher saturation densities and became transformed after only a few passages, as demonstrated by growth as colonies in soft agar.