Arsenite-induced Radiosensitization of Glioma Cells Is Dependent on p53 Deficiency

YASUHARU NINOMIYA; TATSUHIKO IMAOKA; KAZUHIRO DAINO; SHIZUKO KAKINUMA; TETSUO NAKAJIMA

doi:10.21873/anticanres.16422

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Figure 1.
Surviving fraction of p53-proficient (U87MG-neo) and p53-deficient (U87MG-E6) glioma cells. Cells were pretreated for 1 h with 1.25 μM arsenite and then exposed to various doses of X-rays and heavy ion beams. Cell survival was measured in a colony formation assay. Each data point represents the mean value (n≥3); bars, SD.
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Figure 2.
Relationship between arsenite-induced radiosensitization and the increase in the percentage of U87MG-E6 cells in the late S/G₂/M phase at 36 h post-treatment with arsenite plus radiation. (A) Flow cytometry results showing the cell cycle distribution of U87MG-neo and U87MG-E6 cells irradiated with X-rays in the presence/absence of arsenite; cells were gated on the M1 (Sub-G₁), M2 (G₁/early S phase), M3 (late S/G₂/M phase), and M4 (>4N: hyperploidy) regions. The arrow shows a change in the increase in the percentage of cells in the late S/G₂/M phase. (B) Percentage of cells in the G₁/early S phase and in the late S/G₂/M phases, and the percentage of cells showing hyperploidy. Data are expressed as the mean±SD (n≥3). *p<0.05 (Student’s t-test).
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Figure 3.
Induction of γH2AX foci in U87MG-neo and U87MG-E6 cells at 24 h post-treatment with arsenite and/or irradiation. (A) Flow cytometry gating of γH2AX-positive cells was set at the cross-point of nonirradiated control (broken line) and the combination of arsenite plus irradiation (solid line). A typical result for U87MG-neo cells is shown. (B) Summary of flow cytometry analyses of the percentage of γH2AX-positive U87MG-neo and U87MG-E6 cells irradiated in the presence/absence of arsenite. The vertical axis shows the percentage of positive cells. (C) Summary of flow cytometry analyses of the percentage of γH2AX-positive U87MG-neo and U87MG-E6 cells irradiated in the presence/absence of arsenite. The vertical axis shows the percentage of positive cells, values after subtraction of the value of the nontreated control group. Data are expressed as the mean±SD (n≥3). *p<0.05, **p<0.01 (Student’s t-test).
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Figure 4.
Proposed model mechanism underlying radiosensitization of p53-deficient glioma cells by arsenite. In our previous study (15), we found that arsenite induced radiosensitization by generating reactive oxygen species (ROS), which inhibited BRCA2-mediated repair of DNA damage (broken lines). The present study (solid lines) shows that radiation increases DNA double strand breaks (DSB) and γH2AX foci (which are suppressed by p53) and increases the percentage of cells in the late S/G₂/M phases (which is suppressed by p53). Therefore, deficiency of p53 increases radiosensitivity.