Incubation with T3 results in a dose-dependent increase in growth rate of cultured GC cells, a GH-producing rat pituitary tumor cell line. The T3-induced increase in growth rate results mainly from shortening of the Gi period from 79.4 ± 4.3 (SD) h in cells grown in T3-depleted medium (-T3) to 10.0 ± 0.9 h. This effect can also be demonstrated in synchronized populations. Addition of T3 (0.3 nM) to cells synchronized in early Gi in the absence of T3 shortened the Gi period, estimated from graphic data, from more than 40-50 h to 13.4 ± 2.1 h (n = 7). To determine the mechanism of this T3 effect, GC cells were grown in Dulbecco's modified Eagle's medium containing 10% serum plus or minus T3 (0.3 nM) and synchronized at the beginning of the Gi period by mitotic selection. Mitotic cells (85-100%), obtained by controlled mechanical shaking, were isolated by centrifugation and replated. The end of Gi was determined by the onset of DNA synthesis with [3H]thymidine as assessed by autoradiography (percent labeled nuclei). L-T3-induced shortening of Gi was detectable at 0.05 nM T3, halfmaximal at physiological T3 (0.17 nM), and maximal between 0.3 nM and 1.0 nM T3. Addition of cycloheximide, 0.025 fig/ml or 1.0 Mg/ml, decreased protein synthesis by 50% and 90%, respectively, and attenuated the T3 effect on Gx by 80-90%. The attenuation of the T3 effect on Gi by cycloheximide at a dose which inhibited protein synthesis suggests that T3-induced shortening of Gi may require new protein synthesis. Since glucocorticoids decrease the effect of T3 on inducton of a-aminoisobutyric acid transport, their effect on T3-induced shortening of Gi was determined in Gi-synchronized GC cells and in asynchronous cultures. Cortisol, 100 nM, significantly decreased the growth rate of asynchronous GC cells and attenuated the effect of T3 in d-synchronized cells. Finally, T4 also decreased the length of Gi in a dose-dependent manner with a half-maximal effect at 40.0 nM. The half-maximal effect of T4 occurred at a nuclear iodothyronine concentration that was comparable to that achieved in incubations with 0.17 nM T3 (half-maximal dose). Thus, half-maximal shortening of Gi in synchronized GC cell cultures occurred at iodothyronine concentrations required for half-maximal occupancy of nuclear T3 receptors and for halfmaximal induction of GH synthesis, growth rate, a-aminoisobutyric acid uptake, and depletion of the nuclear T3 receptor. Our data suggest that stimulation of GC cell growth by T3 occurs by an action that is restricted to the early Gi period, which requires protein synthesis, and which may be mediated by the nuclear T3 receptor.
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