Evidence That Repair and Expression of Potentially Lethal Damage Cause the Variations in Cell Survival after X Irradiation Observed through the Cell Cycle in Ehrlich Ascites Tumor Cells

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Abstract
The survival of synchronously growing Ehrlich ascites tumor cells (EAT cells) [mouse] was measured after X irradiation in various stages of the cell cycle. Cells at the beginning of S or in G2 + M phase showed a high level of killing; cells irradiated in G1 or in the middle of S phase were more resistant. These changes resulted from a change in the survival curve shoulder width (Dq) as cells passed through the cell cycle, and the mean lethal dose (D0) remained practically unchanged (0.8 .+-. 0.05 Gy [Gray]). When synchronization of the cell population was further sharpened using nocodazole, exponential survival curves were obtained at the beginning of S phase and at mitosis with a D0 = 0.8 Gy. When cells (in all stages) were incubated in balanced salt solution for 6 h after irradiation, repair of potentially lethal damage (PLD) was observed, resulting in an increase in Dq, while D0 remained constant. Treatment of the cells after irradiation with either caffeine (2-6 mM) or .beta.-arabinofuranosyladenine (.beta.-araA) (60-100 .mu.M) or hypertonic medium resulted in an expression of PLD and reduced the Dq of the survival curve, which approached or reached an exponential line with D0 = 0.8 .+-. 0.1 Gy. The rate of the loss of sensitivity of these treatments that assumably reflects the rate of repair of PLD, was measured. For caffeine (6 mM) treatment (S cells, 5 h), a repair time constant (t50 of .apprx. 1 h was found, similar to that observed for repair of PLD in growth medium containing 0.5 .mu.g/ml aphidicolin. With hypertonic treatment 2 repair components were detected, a fast one that restored the slope of the survival curve, and a slow one with a t50 of .apprx. 1 h that restored the shoulder of the survival curve. PLD induced by irradiating in G1 phase was repaired when cells were arrested for some hours either in G1 phase or in the subsequent mitosis but was not repaired if the cells were arrested in S phase. PLD induced in S or G2 + M phase was repaired only when the cells were arrested in the cell cycle before division. Results indicate that the shoulder width Dq of the survival curve in cells irradiated at various stages of the cell cycle results from repair of PLD. This repair of PLD probably takes place in the interval between irradiation and the next S phase or mitosis and is therefore minimal for cells irradiated at the G1/S border or in mitosis (Dq = 0). PLD still unrepaired when the cells reach these plhases is assumed to be expressed, as was found for PLD repaired in cells incubated in balanced salt solution, for some hours after irradiation. The variations observed in cell survival through the cell cycle might reflect variations in the final amount of PLD either repaired or expressed as the cells progress through the various stages of the cell cycle.