Purpose: The present study was designed to investigate the ability of N-[4-(5-bromo-2-pyrimidyloxy)-3-methylphenyl]-(dimemethylamino)-benzoylphenylurea (dimemethylamino benzoylphenylurea; BPU) to sensitize cells to radiation and to examine the relationship between phenotype versus survival, DNA damage, apoptosis, or cell cycle progression in non-small cell lung cancer (NSCLC) cell lines.
Methods: Asynchronous cultures of three NSCLC (phenotype) lines, A549 (adenocarcinoma), NCI-H226 (squamous) and NCI-H596 (adenosquamous) were used. Cells were treated for 24 h with BPU at various concentrations (0-10 microM) to obtain drug doses for inhibiting cell survival by approximately 50% (IC50). Cells were X-irradiated without BPU or after 24 h BPU treatment at IC50. Radiation doses ranged from 0 to10 Gy. Cell survival was determined by a colony-forming ability assay. The effect of BPU on the cell cycle distribution and induction of apoptosis were measured by flow cytometry-based assays. The effect of BPU on radiation-induced DNA damage and repair was analyzed according to nuclear gammaH2AX immunofluorescence of cells exposed to X-rays alone or after BPU. Anti-gammaH2AX antibody staining, a surrogate determinant of double stranded DNA breaks, was measured using flow cytometry.
Results: BPU (1.5 microM) for 24 h produced approximately 50% cell survival. BPU and X-irradiation were synergistic in the three cell lines at survival levels of 20-50%. Flow cytometry analysis of replicate experiments with BPU (1.5 microM for 24 h) showed that BPU blocked cell progression at S and/or G2/M. The incidence of apoptosis in BPU-treated versus control cells ranged from approximately 0.3 to approximately 8%. Twenty-four hour after X-irradiation cells pre-treated with BPU and X-irradiated after drug exposure showed gammaH2AX levels approximately two times higher than did the cells exposed to X-rays only.
Conclusions: The study identified BPU as a novel radiation sensitizer. The analysis of phosphorylated histone H2AX as a surrogate marker of DNA double strand breaks suggested a positive association between radiosensitization and the inhibition of X-irradiation-induced DNA damage repair by BPU.