DNA repair after irradiation in glioma cells and normal human astrocytes

Abstract

We examined DNA damage responses and repair in four human glioma cell lines (A7, U87, T98G, and U373) and normal human astrocytes (NHAs) after clinically relevant radiation doses to establish whether we could identify differences among them that might suggest new approaches to selective radiosensitization. We used phosphorylation of histone H2AX visualized by immunocytochemistry to assess DNA double-strand break (DSB) formation and resolution. Fluorescence immunocytochemistry was used to visualize and quantify repair foci. Western blotting was used to quantify repair protein levels in the different cell lines before and after irradiation and during different cell cycle phases. Mitotic labeling was used to measure cell cycle parameters after irradiation. We found that the glioma cell lines repaired DSBs more slowly and less effectively than did NHAs in the clinically relevant dose range, as assessed by induction and resolution of H2AX phosphorylation, and this was most marked in the three TP53-mutated cell lines (T98G, A7, and U373). The glioma cells also expressed relatively high repair-protein levels compared with NHAs that were not altered by irradiation. High levels of the repair protein Rad51 in these cells persisted throughout the cell cycle, and a marked increase in Rad51 foci formation, which was not restricted to cells in G2/S phase, occurred at early time points after irradiation. TP53-mutated glioma cell lines demonstrated a very prominent dose-responsive G2 checkpoint and were sensitized to radiation by caffeine, which inhibits G2/S phase checkpoint activation. In conclusion, DNA repair events differed in these four glioma cell lines compared with NHAs. In particular, the three TP53-mutated glioma cell lines exhibited markedly increased Rad51 protein levels and marked, dose-dependent Rad51 foci formation after low radiation doses. This suggests that agents that disrupt Rad51-dependent repair or prevent G2 checkpoint activation may selectively sensitize these cells.

Inhibition of serine/threonine phosphatase PP2A enhances cancer chemotherapy by blocking DNA damage induced defense mechanisms

“…We assessed by Western blots the effects of LB-1.2, TMZ, DOX, LB-1.2 plus TMZ, and LB-1.2 plus DOX on the amount of pAkt, p53, and MDM2 in U87MG, a cell line with wild-type p53, and in U373, a cell line with mutant p53 (28). Exposure of U87MG cells to LB-1.2 alone for 24 h increased both pAkt-1 and pMDM2 and eliminated phosphorylated p53; TMZ alone and DOX alone decreased pAkt-1, increased p53, and had little effect on MDM2.…”

LB100, a small molecule inhibitor of PP2A with potent chemo- and radio-sensitizing potential

“…Similar downstream effects on protein expression and mitotic catastrophe were observed. 75 These findings suggest that chemo-sensitization to TMZ with LB102 is independent of p53 status in GBM cells.…”

Glioblastoma multiforme: the role of DSB repair between genotype and phenotype

“…While DNA-PKcs expression appears to protect glioblastoma cells from undergoing autophagy, the radioresistance does not necessarily imply efficient DSB repair of this tumor. By contrast, and as mentioned above, several radioresistant glioblastoma cell lines have been shown to inefficiently repair DSBs (Short et al, 2007). Inefficient DSB repair is likely due to the specific genetic background of this tumor: High-grade glioma frequently bear TP53 mutations (Ohgaki and Kleihues, 2007).…”

“…Recently, Glioblastoma multiforme: the role of DSB repair U Fischer and E Meese clinically relevant radiation doses were applied to four glioblastoma cell lines (U373, U87, A7 and T898G) and to normal human astrocytes for comparison. Glioblastoma cell lines repaired DSBs more slowly and less efficiently than the normal human astrocytes (Short et al, 2007). A less efficient repair process may appear inconsistent with high levels of repair protein in glioma cells: Rad51, which is central in HR repair (Raderschall et al, 2002) and DNA-PKcs are highly expressed in various glioblastoma cell lines.…”

“…Inefficient DSB repair is likely due to the specific genetic background of this tumor: High-grade glioma frequently bear TP53 mutations (Ohgaki and Kleihues, 2007). Glioblastoma cells with mutated TP53 show no significant G 1 checkpoint response but largely depend on G 2 activation for their radioresistant phenotype (Short et al, 2007).…”