Effect of medium replenishment or composition on [3H] thymidine incorporation in uv-irradiated CHO-K1 cells

Because culture medium contains uv-absorbing material, it is usually removed just before uv-irradiation of tissue culture monolayers. However, medium removal and replenishment with fresh medium alone (sham-irradiation) causes up to a 10-fold reduction in the rate of [3H]TdR incorporation in CHO-K1 cells which persists for several hours. This reduction, which is much smaller (<2-fold) if 2 mM CdR is present in the medium, results from dissimilarities in the specific activity of the [3H]TdR pulse-label in conditioned (spent) and in fresh medium; TdR in the former is converted by cells to thymine. When responses of uv-irradiated cells are normalized to responses of corresponding sham-irradiated cultures, considerable variation is observed in replicate experiments because fresh medium appears to induce transient metabolic imbalances in irradiated cells which are not readily controlled. This problem can, in part, be circumvented by replenishing treated cultures with the original spent medium; however, the presence of CdR in the growth medium still causes an anomalous 2-3-fold greater uv-induced reduction in [3H]TdR incorporation than is observed in the absence of CdR. 17 refs., 3 figs., 1 tab