A TECHNIQUE FOR DETERMINING THE PROPORTION OF THE CLONOGENIC CELLS IN S PHASE IN EMT6 CELL CULTURES AND TUMORS

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Abstract
The survival of cultured EMT6 [mouse mammary tumor] cells was examined after treatment with hydroxyurea (HU) or high specific activity 3H thymidine (3H-TdR). The concentrations of the agents, duration of exposure to the agents and postexposure treatment of the cultures influenced cell survival. The effects of these factors are reported. Conditions were defined under which the proportions of cells killed by HU and 3H-TdR were the same and were also the same as the proportion of labeled cells seen on autoradiographs of cultures labeled with small doses of 3H-TdR. Under these conditions, 3H-TdR or HU could be used to determine the proportion of the clonogenic cells in the S phase. Single cell suspensions prepared from solid EMT6 tumors were treated in vitro with HU or 3H-TdR, using the conditions optimal for each agent with cultured cells. The proportion of the tumor cells killed by treatment with HU in vitro was the same as the proportion killed by HU in vivo and the proportion labeled by 3H-TdR in vivo, and incubation of tumor cell suspensions with HU in vitro appeared to provide a valid measurement of the proportion of clonogenic tumor cells in the S phase. Incubation of tumor cell suspensions with 3H-TdR in vitro was difficult to perform and the results were relatively unreliable because of severe problems with reutilization of 3H-TdR during incubation for colony formation.