DNA damage response following X-irradiation in oral cancer cell lines HSC3 and HSC4.

ABSTRACT

OBJECTIVE: The objective of this study was to characterize the DNA damage response in two human oral cancer cell lines following X-irradiation. DESIGN: To visualize radiation-induced cell cycle alterations, two human oral cancer cell lines, HSC3 and HSC4, expressing fluorescent ubiquitination-based cell cycle indicator (Fucci) were established in this study. G2 arrest kinetics following irradiation were obtained from two-color flow cytometric analysis and pedigrees of Fucci fluorescence. DNA double strand break repair kinetics were obtained from immunofluorescence staining for phosphorylated histone H2AX, p53-binding protein 1, phosphorylated DNA-dependent protein kinase catalytic subunit, and breast cancer susceptibility gene 1. RESULTS: Both cell lines showed apparent G2 arrest after 10 Gy of irradiation, but it was more enhanced in the HSC3-Fucci cells. Radiosensitivity was higher in the HSC3-Fucci cells than in HSC4-Fucci cells. Pedigree analysis of Fucci fluorescence revealed that the HSC3-Fucci cells exhibited a significantly longer green phase (normally indicating S/G2/M phases, but here reflective of G2 arrest) when irradiated in the red phase (G1 phase) than HSC4-Fucci cells irradiated in either red or green phases. Non-homologous end joining was marginally suppressed during the G1 phase and markedly more likely to be impaired during the S/G2 phases in HSC3-Fucci cells. When G2 arrest was abrogated by checkpoint kinase 1 or Wee1 inhibitors, only HSC4-Fucci cells exhibited radiosensitization. CONCLUSIONS: We characterized DNA damage response in HSC3-Fucci and HSC4-Fucci cells following irradiation and the former demonstrated inefficient non-homologous end joining, especially during the S/G2 phases, resulting in enhanced G2 arrest. These findings may have clinical implications for oral cancer.