A simple, rapid protocol is described for separation of chemical agents into different classes, dependent upon their effects on cell-cycle traverse in line CHO Chinese hamster cells. The technique is a functional assay in that it measures the ability of synchronized cells to traverse the cell cycle on the basis of determination of the labeled and mitotic fractions in autoradiographs prepared from cultures receiving thymidine-3H after addition of test agent in either G1 or S. Equivalent results were obtained with G1 cells prepared by either the isoleucine deficiency technique or in cells prepared by mitotic selection and allowed to enter G1 prior to addition of drug. With the test protocol it was possible to distinguish reliably among at least seven classes of agents, including those agents inhibiting primarily the G1 to S transition, those affecting primarily the initiation and completion of DNA synthesis, and those affecting either G2-specific, mitotic, or cycle-wide processes. Agents tested included hydroxyurea, puromycin, dactinomycin, and neocarzinostatin (all agents with known specific effects on the cell cycle) and bleomycin, ellipticine, and l-O-ethylthreonine (all little studied, potentially useful antitumor compounds). Bleomycin inhibited G2 processes specifically, with essentially no effect upon genome replication. Ellipticine inhibited both initiation of DNA synthesis and completion of G2 but did not prevent continued synthesis of DNA in cells already in S at time of drug addition. At 8 × 10-4 m l-O-ethylthreonine, the primary effect was an inhibition of the G1 to S transition, although at higher concentrations both genome replication and completion of G2 were inhibited.
This study was supported by Contract NIH-CR-(71)-56 from Drug Research and Development, Chemotherapy, National Cancer Institute, USPHS, NIH, under interagency agreement with the U. S. Atomic Energy Commission.