Cell Cycle Changes after Glioblastoma Stem Cell Irradiation: The Major Role of RAD51

Abstract

"Glioma Stem Cells" (GSCs) are known to play a role in glioblastoma (GBM) recurrence. Homologous recombination (HR) defects and cell cycle checkpoint abnormalities can contribute concurrently to the radioresistance of GSCs. DNA repair protein RAD51 homolog 1 (RAD51) is a crucial protein for HR and its inhibition has been shown to sensitize GSCs to irradiation. The aim of this study was to examine the consequences of ionizing radiation (IR) for cell cycle progression in GSCs. In addition, we intended to assess the potential effect of RAD51 inhibition on cell cycle progression. Five radiosensitive GSC lines and five GSC lines that were previously characterized as radioresistant were exposed to 4Gy IR, and cell cycle analysis was done by fluorescence-activated cell sorting (FACS) at 24, 48, 72, and 96 h with or without RAD51 inhibitor. Following 4Gy IR, all GSC lines presented a significant increase in G2 phase at 24 h, which was maintained over 72 h. In the presence of RAD51 inhibitor, radioresistant GSCs showed delayed G2 arrest post-irradiation for up to 48 h. This study demonstrates that all GSCs can promote G2 arrest in response to radiation-induced DNA damage. However, following RAD51 inhibition, the cell cycle checkpoint response differed. This study contributes to the characterization of the radioresistance mechanisms of GSCs, thereby supporting the rationale of targeting RAD51-dependent repair pathways in view of radiosensitizing GSCs.

Keywords: DNA repair protein RAD51 homolog 1 (RAD51); Glioma Stem Cells; cell cycle; ionizing radiation.

Figure 1

Analysis of cell cycle progression of radiosensitive and radioresistant unsynchronized Glioma Stem Cell (GSC) lines after 4Gy IR exposure (merged data are shown). (A) Proportion of cells in G2 phase after 0, 24, 48, and 72 h following IR. Histograms represent mean data ± the standard error of the mean (SEM, Mann–Whitney test). (B) Mean variations of G1, S, and G2 phases following IR. (* p < 0.05; ** p  < 0.01; Kruskal–Wallis test). (C) Comparative proportion of cells in G2 phase at T0 before IR between radioresistant and radiosensitive cell lines; Histograms represent the mean data ± SEM. (D) Mean variation of G2 phase at 24, 48, and 72 h post-irradiation in radiosensitive and radioresistant GSCs.

Figure 2

Analysis of cell cycle progression in GSC-1 and GSC-14 lines and the effect of IR. (A) Mean variation of cell cycle phases in GSC-1 and GSC-14 after synchronization. GSCs were synchronized by a double thymidine block, released, and collected at the indicated time points (Unsyn: unsynchronized, Syn: synchronized). (B) Proportion of synchronized cells in G2 phase following 4Gy IR. Cells were collected at the indicated time points. (Unsyn: unsynchronized without IR, Syn: synchronized without IR, Syn+IR: synchronized with IR). Histograms represent the mean data ± SEM obtained for both cell lines.

Figure 3

G2 phase variation in GSC lines according to the Verhaak classification scheme (merged data are shown). (A) Comparative proportion of cells in G2 phase at T0 before IR. (B) Mean variation of G2 phase in GSCs following 4Gy IR. Cells were collected at the indicated time points. Histograms represent the mean data ± SEM. Proneural (n = 4), Classical (n = 2), and Mesenchymal (n = 4).

Figure 4

Effect of RI-1 (10 µM) inhibitor on cell cycle progression of the GSC-1, GSC-6, GSC-11, and GSC-14 lines with or without IR exposure (merged data are shown). (A,B) Mean variation of G2 phase of radiosensitive GSC-1 and GSC-11 cells (A) and radioresistant GSC-6 and GSC-14 cells (B) after 4Gy exposure with or without RI-1. Cells were collected at the indicated time points (IR: irradiation, INH: RI-1 inhibitor). Histograms represent the mean data ± SEM.

Figure 5

Comparison of gene expression enriched in radiosensitive GSCs to radioresistant GSCs. (A) Significant enrichment of a signature of 15 genes involved in DNA damage response (p value = 0.003, False Discovery Rate (FDR) = 0.072. Normalized Enrichment Score (NES) = 1.849). (B) Significant enrichment of a signature of 71 genes involved in DNA repair (p value < 0.001, FDR = 0.0369, NES = 1.847). (C) Signature of 32 genes involved in the DNA damage checkpoint (p value = 0.01, FDR = 0.0839, NES = 1.691). (D) Signature of 46 genes involved in DNA repair (p value = 0.013, FDR = 0.0838, NES = 1.661).