Effect of Hydroxyurea on Radiation-Induced Division Delay in CHO Cells

The effects of the DNA synthesis-inhibitor hydroxyurea (HU) on radiation-induced division delay in CHO cells were studied using the mitotic selection procedure for cell cycle analysis. After the addition of 1.0 mM HU, the mitotic yield (number of mitotic cells selected from an asynchronous population) remained unaltered for a time and then decreased eventually to zero with a point of action at the S/ G₂ boundary, i.e., cells in S were halted while cells in G₂ and M proceeded through mitosis and were selected, but at a slower rate than normal. Pulsed treatment with HU for up to 2 hr caused a decline in mitotic yield, but after the pulse-length-dependent S delay, S-phase cells entered G₂ and progressed to mitosis. Treatment with HU pulses (≤2 hr) either before or after X irradiation did not alter the number of cells refractory to radiation-induced division delay nor the duration of the delay. This indicates that DNA synthesis was not necessary for recovery from radiation-induced division delay, and that the first cells to recover from radiation-induced delay were in G₂ at the time of irradiation. Finally, utilizing HU pulses to partially synchronize cells in G₂, no differential division delay was observed for cells irradiated within the G₂ phase. However, if a cell in G₂ was exposed to HU for prolonged periods (i.e., treatment before irradiation and continuing during division delay and after mitotic recovery), considerable retardation of progression occurred.