Inaccuracy of estimations of s phase fraction by reduction in cloning efficiency with hydroxyurea or tritiated thymidine.
The current hypothesis, that the fractional reduction of cloning efficiency in semi-solid culture systems induced by pretreatment of the cells with hydroxyurea (HU) or [3H]TdR equals the fraction of cells initially in S phase, is tested. A lymphoblastoid cell line, SK-L7, with known cell cycle kinetics was exposed to cytotoxic concentrations of HU or suicidal doses of [3H]TdR and then initiated in semi-solid and liquid culture. Although approximately 0.6 of the initial population was in S, 1-hr exposures of HU at concentrations of up to 10(-2) M failed to reduce subsequent cloning efficiency. The 1-hr exposure to HU did not reduce either the immediate cell number or the gross population doubling rate over 24 hr. A 24-hr exposure to 10(-3) M HU reduced the cloning efficiency by approximately 98%, confirming the drug's cytotoxic capability. [3H]TdR at doses of 100 microCi/ml for 20--40 min reduced the cloning efficiency by approximately 60 and 70%, respectively. Although no cytotoxicity immediately after exposure was observed in either case, gross population doubling rate in liquid culture was reduced. While HU failed to reduce subsequent cloning efficiency, [3H]TdR reduced cloning efficiency by approximately the fraction of initial cells in S. The above hypothesis, therefore, cannot be applied naïvely as a technique for quantitating the fraction of a clonogenic cell population in S phase.