Selection of mitotic Chinese hamster ovary cells from microcarriers Cell cycle-dependent induction of mutation by 5-bromo-2′-deoxyuridine and ethyl methanesulfonate

Abstract

Large quantities of mitotic cells may be collected by mitotic detachment from a population of Chinese hamster ovary cells growing on positively charged dextran microcarriers in suspension culture. Exponentially growing cells are treated for 2.5 h with colcemid and mitotic cells are detached from the microcarriers by increasing the stirring speed. A yield of 4–6% of the total population is obtained and, of the cells collected, 85–95% are arrested in metaphase. Using this means to synchronize cells we have determined the cells cycle dependence of the toxic and mutagenic effects of 5-bromo-2′-deoxyuridine (BUdR) and ethyl methanesulfonate (EMS). Mutation was measured at two independent loci resistance to 6-thioguanine and resistance to ouabain. Both mutagens were more toxic during S phase as compared to G 1 or G 2 or mitosis. BUdR induced significant mutation only during S phase. The maximum induction of 6-thioguanine resistance was observed in cultures treated 10 h after plating of mitotic cells (2 h into S phase), while the maximum induction of ouabain resistance was observed cultures treated 10–12 h after plating of mitotic cells (2–4 h into S phase). EMS induced significant mutation at all points in the cell cycle. Mutation induction reached a minimum during S phase by the magnitude of difference between any two points in the cell cycle was found to be less than two-fold.