The entry of cells into mitosis was monitored by shaking off mitotic cells from monolayer cultures. The location in G2 of the X-ray transition point (TP), beyond which the cells were not delayed by X-irradiation, moved closer to mitosis as the dose was increased, i.e., at 10 min before prophase for 100-200 rad, and at 19 min for 30-60 rad. Treatment with cycloheximide (CH, 20 μg/ml) at the time of irradiation (50-100 rad) shifted the TP for X-irradiation by 5-9 min toward mitosis. Calculations from these shifts in TP with radiation dose indicated that the logarithm of both the fraction of cells in G2 not delayed by irradiation and the fraction beyond the TP for CH (located at 26 min before prophase for 5-50 μg/ml) not delayed by irradiation decreased linearly with dose ($\tilde{D}_{0}$ of 30 rad). Furthermore, inhibition of protein synthesis by CH, with or without a simultaneous treatment with actinomycin D (AMD), prevented repair of damage causing mitotic delay for a period of time equal to the duration of the CH treatment. Treatment with AMD (2-5 μg/ml) at the time of irradiation, however, had effects opposite to those of CH, in that (1) the TP was shifted away from mitosis by 6-8 min, and (2) mitotic delay (100 min for 100 rad) for cells trapped between the TP's for AMD (2 μg/ml) and radiation was reduced by an amount which increased with the radiation dose, i.e., 20 min for 50 rad to 85 min for 150 rad. But the effects of AMD were greatly dependent on concentration because at 5 μg/ml, repair was completely inhibited. Also, as the concentration was increased from 2 to 15 μg/ml, the TP for AMD shifted from near the S/ G2 transition (80 min before prophase) and approached asymptotically the TP for 100-200 rad. Other relevant observations from a previous study were: (1) Mitotic delay is a maximum in early G2 and then decreases as the cells are irradiated in late G2. (2) Cells irradiated in late S or early G2 sustain considerable delay in late G2. From these observations, it was postulated that (1) X-irradiation or high concentrations of AMD interact in a stochastic manner with a critical structure, which is essential for division and is assembled in G2 from particular protein molecules synthesized during G2; (2) the size of the critical structure, and consequently the length of mitotic delay, decrease during late G2 and approach zero at the TP for X-irradiation because the critical structure is converted into a final structure, essential for division, but refractory to damage from AMD or X-irradiation; (3) during the 16-min interval between the TP's for CH and X-irradiation (100 rad), synthesis of the particular protein molecules ceases, but final assembly continues from a pool of protein molecules and from remnants of the critical structure; (4) synthesis of the particular protein is required for repair of X-ray damage in the critical structure, and the cells are delayed in late G2 until the critical structure is completely repaired and then converted into the final structure which is essential for division; and (5) AMD at lower concentrations accelerates repair because it stimulates the synthesis of this particular protein. For a heat treatment of 46.5°C for 5-7 min (with or without X-irradiation or AMD), the TP was located in prophase, i.e., 4 min before metaphase, and the heat-induced delay probably results from effects on the mitotic spindle.

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