Shirakawa Cell Cycle - d - Arabinofuranosylcytosine in the β The Locus of Action of 1 - Updated

son Hospital. The cultures were maintained in McCoy's 5A medium reconstituted from powder (Grand Island Biological Company) not containing antibiotics. The medium was supple mented with 20% fetal calf serum (Hyland). The cultures were fed daily and subcultured at least every two days. son Hospital. The KB cells were maintained in minimal essen tial medium (Eagle's) obtained in powdered form from Grand Island Biological Co. The medium was supplemented with 10% calf serum (Hyland) and streptomycin and neomycin, 250 mg/liter. The cells were fed every other day and subcultured at least twice a week. The KB cells were cultured periodically for the presence of myoplasma. The cells were always found to be free of this organism. Growth and Cloning Experiments Replicate cultures for growth curves were set up in either 2-oz. prescription bottles (Duraglas, Owens-Illinois, Co.) or plastic T-15 flasks (Falcon Plastics). For each time point, du plicate cultures were rinsed in Hank? medium lacking magne sium and calcium (rinse medium) to remove nonadherent cells and were trypsinized using 0.075% trypsin (Worthington) in rinse medium. The cells were counted using a Model F Coulter Counter after an appropriate dilution with physiologic saline. Recovery experiments were performed in two ways. Cultures were exposed in situ in 2-oz. prescription bottles to 1 pg/mi of ara-C for 1, 4, 8, and 16 hours, rinsed with Hanks' balanced salt solution, and fed with fresh medium not containing drug. The cultures were refed every 24 hours. Alternately cells were exposed to ara-C, 1 @.tg/mlfor 1, 4, 8, and 24 hours in 8-oz. prescription bottles. The cultures were then rinsed and subcul tured into 2-oz. bottles for subsequent growth determination by cell counting. Cloning was performed on cultures exposed 1, 4, 8, and 16 hours to ara-C at concentrations of 1, 5, 10, 50, and 100 @.Lg/ml or alternately to concentrations of 1 and 10 i.tg/ml for time periods of 1, 4, 8, and 16 hours. After the appropriate expo sure interval, the cultures were washed twice with rinse mcdi um, trypsinized, and diluted with fresh medium (1:1,000 to 1 : 10,000) and plated in plastic T-1 5 flasks. Each flask con tamed 200 to 1 ,000 cells in 3 ml of medium. Five replicate cultures were gassed with 95% air-5% CO2, tightly stoppered, and left undisturbed at 36.5°C for 7†" 9 days. At the end of this time, the medium was carefully removed by …