Trypsinization and the Radiosensitivity of Mitotic and Log Phase Chinese Hamster V79 Cells Exposed to 250 kVp X-rays

Summary

We studied the influence of trypsin-induced morphological changes on the radiosensitivity of cells plated at either low (4–600/cm²) or high (2 × 10⁴/cm²) density and grown overnight before treatments. Trypsin treatment induced contraction and rounding of spread cells. The radiosensitivity of cells trypsinized and plated either: (1) immediately before [(a) D₀ = 1·7 Gy for cells at low-, and (b) 1·5 Gy at high-density] or (2) immediately after (D₀ = 1·6 Gy, high-density cells) irradiation was higher than that of (3) cells at high density, irradiated and delayed plated [cells remained spread until the completion of potentially lethal damage repair (PLDR) and trypsinization, D₀ = 2·2 Gy], and (4) cells at low density which were neither delayed plated nor trypsinized (i.e. remained spread) after irradiation (D₀ = 2·4 Gy). These data show that PLDR is reduced in trypsin-treated cells of both high (compare 1b and 2 with 3) and low (compare 1a with 4) density cultures; the latter comparison provides a direct measure of the trypsin effect. Since the comparison between conditions 1 and 2 vs. 3 and 4 is of round vs. spread cells, PLDR appears to be influenced by the cell's morphological state. Kinetic studies showed that when cells were incubated in growth medium to recover from trypsin-induced effects before irradiation, the radiation sensitivity of spread cells (plated in situ), but not of those remaining rounded (in suspension until plating and irradiation), decreased and became equal to that of delayed plated high-density cells. Neither irradiated cells treated with hypertonic saline, nor mitotic cells, showed the trypsin effect. From these results we suggest that: (1) trypsin-induced cell contraction affects the ability of cells to repair radiation damage, (2) spread cells are better able to repair PLD than rounded cells, (3) immediate plating survival of cells in high-density cultures may not represent their intrinsic radiosensitivity and (4) cell-to-cell contact is not necessary for log phase cells to repair PLD.