Use of flow microfluorometry in detailed analysis of effects of chemical agents on cell cycle progression.

A technique has been developed for determining effects of chemotherapeutic agents on cell cycle progression in synchronized line CHO Chinese hamster cells grown in vitro . The proctocol combines autoradiographic and cell-enumeration techniques with analysis of DNA distribution patterns by means of the Los Alamos flow microfluorometer. CHO cells were accumulated in a state of G1 arrest by growth in isoleucine-deficient medium; then the cells were resuspended in fresh, complete medium containing isoleucine plus a test agent to reinitiate cycle traverse in the presence of the drug. Ten hr later, flow microfluorometric analysis of the population distribution of DNA contents for the cultures yielded the fractions of cells in G1, S, and G2 or M. For agents with reversible effects, confirmatory cell cycle data were obtained after resuspension of the cells in fresh, drug-free medium containing thymidine-3H; and aliquots were removed at intervals for determination of traverse capacity, even in slowly or nonprogressing populations. Four agents with differing effects on cell cycle progression were examined, namely, hydroxyurea, cytosine arabinoside, bleomycin, and camptothecin. Both hydroxyurea (10-3 m) and cytosine arabinoside (5 µg/ml) grossly decreased the rate of progression of cells into S phase, resulting in an accumulation of cells at the G1-S boundary. Neither agent completely prevented cells from initiating DNA synthesis. Bleomycin (100 µg/ml) allowed initiation and completion of genome replication to occur at a nearly normal rate, but cells accumulated in G2 and most cells lost the capacity to enter mitosis. Camptothecin (1 µg/ml) reduced the overall rate of cycle progression and allowed a few cells to replicate a complete complement of DNA. The relationship of these in vitro findings to the chemotherapy regimen design is discussed.