Effects of actinomycin D and puromycin on the mitotic cycle in synchronized cell culture 1

Abstract

Treatment of Chinese hamster cells with 5-fluoro-2′-deoxyuridine (FUdR) in a concentration of 10 −6 M for 12 h resulted in an accumulation of the population in the S period but not at the G1-S boundary. After the addition of exogenous thymidine in a concentration of 10 −5 M a synchronous withdrawal of cells from the S period took place followed by a wave of mitoses. Cell cultures synchronized by FUdR blockage and thymidine reversal appear to be a suitable model for studying the action of regulating factors on the progression of cells through the mitotic cycle. Under the highest extent of synchrony this model allows to investigate the transition of cells from the S to the G2 period, the progression through G2 and the entry into mitosis. Using radioautographic technique with 3H-thymidine the steps sensitive to actinomycin D (0.1 μg/ml) and puromycin (20 μg/ml) were studied during the synchronized mitotic cycle. Continuous exposure to antibiotics prevented cells from entering the S period. Those cells which were in early S could not complete DNA synthesis in the presence of the drugs but cells in late S appeared to be sensitive only to actinomycin D. On the contrary, the initiation and maintenance of mitosis was completely blocked in the presence of puromycin while actinomycin D only delayed the processes.