A study of the change in DNA synthesis of S phase cells treated with N-methyl-N-nitrosourethane: A study using Amoeba proteus as a single cell model

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The present experiments using Amoeba proteus as a single cell model show that DNA synthesis continues during and after exposure of S phase cells to N-methyl-N'-nitrosourethane (MNU). At sublethal dose levels which caused long division delays, division and growth abnormalities and mutations, the amount of [3H] thymidine ([3H]Tdr) incorporated was decreased by 20–30%; at dose levels which killed all S phase cells it was inhibited by up to 90%. There was a direct correlation between the dose of MNU used and the degree of inhibition of [3H]Tdr incorporated. The effect was rapid, mainly taking place within 20 min of treatment.Amoeba heterokaryons (HKs) were used to examine the rate of DNA synthesis of treated and untreated nuclei in the same cytoplasm, i.e. where the nuclei would have the same [3H]Tdr intake, the same thymidine kinase (TK) activity and the same endogenous precursor pools. Direct comparison of the nuclear DNA synthetic activity in this way revealed less difference between treated and untreated nuclei than comparisons made using the nuclear grain counts from treated and untreated amoebae. This suggested that much of the decrease in [3H]Tdr incorporation by MNU-treatedS phase cells was due to a change in the cytoplasm and/or the cell membrane, rather than to nuclear damage. Thus MNU-treated nuclei were able to synthesize DNA at a near normal rate when they could draw on the resources of untreated cytoplasm, while the rate of DNA synthesis of control nuclei decreased when they occupied cytoplasm which had been exposed to high doses of MNU. These studies suggest that nuclear sites of damage were only involved when lethal doses of MNU had been used.