Uptake of Thymidine and Synthesis of Deoxyribonucleic Acid in Mouse Ascites Cells

Abstract

RECENT studies of the synthesis of deoxyribonucleic acid have suggested that the radiation-sensitive period may occur at a stage in the cell-cycle before the polymerization of new deoxyribonucleic acid begins, that is, during the G1 period^1–3. It therefore appeared to be of interest to study, in more detail, the assimilation of the precursors into cells and their subsequent incorporation into deoxyribonucleic acid. With this object we have examined the uptake of thymidine labelled with tritium into mouse Ehrlich and Landschutz ascites cells in vitro, measuring the total activity present in the cell and the activity incorporated into deoxyribonucleic acid, as a function of time. The distribution of the precursor among the cells of the population was determined by autoradiography. 2 ml. aliquots of suspension containing about 10⁸ cells were incubated at 37° in a medium containing 50 per cent ascitic fluid and 50 per cent fortified Hanks' medium⁴ (5 gm. glucose/l.). At the end of the incubation the cells were washed five times with ice-cold Hanks' medium and finally suspended in 4 ml. of the medium. Autoradiographs were prepared in the usual manner, the cells being air dried, fixed with 2 per cent acetic acid or with 45 per cent acetic acid in alcohol and washed before the application of the stripping film. Deoxyribonucleic acid was prepared from 2 ml. of the cell suspension using the detergent method of Kay, Simmons and Dounce⁵, the final product being dissolved in 2 ml. of water. The solutions of deoxyribonucleic acid and the cell suspensions were homogenized by treatment with ultrasonics before plating on aluminium planchettes. Determinations of tritium activity were made using a windowless flow counter working in the proportional region with an efficiency of about 15 per cent. Deoxyribonucleic acid contents of the cell suspensions and acid solutions were determined using the modified diphenylamine method of Burton⁶.