187614a0Nature1874737196008136146150028-0836196010.1038/187614a0ukNatureNatureNATUREnatureNature is a weekly international journal publishing the finest peer-reviewed research in all fields of science and technology on the basis of its originality, importance, interdisciplinary interest, timeliness, accessibility, elegance and surprising conclusions. Nature also provides rapid, authoritative, insightful and arresting news and interpretation of topical and coming trends affecting science, scientists and the wider public./nature/journal/v187/n4737issueJournal homeArchiveCurrent issueAdvance online publicationPrivacy policySubscribeNature Publishing GroupCurrent issue187614a0Uptake of Thymidine and Synthesis of Deoxyribonucleic Acid in Mouse Ascites Cells
AU  - CRATHORN, A. R.
AU  - SHOOTER, K. V.Chester Beatty Research Institute, Royal Cancer Hospital, Fulham Road, London, S.W.3.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 period1-3. 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[deg] 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.Fig. 1. Incorporation of tritium-labelled thymidine into cells and deoxyribonucleic acid, a, 0 -001 /*M, 2 A*C. 3H-thymidine.
The general pattern of the results obtained for both types of cells is illustrated in Fig. 1 . Thymidine is rapidly taken up by the cells, but the rate of incorporation into deoxyribonucleic acid is much slower. The amount of thymidine which the cells can take up is limited. For example, when 0-001 \j.M of thymidine is added to a suspension containing about 108 cells, nearly all of it is taken up by the cells in 1 hr., but when ten times this amount of thymidine is added the amount taken up in 1 hr. is only slightly increased. The amounts of tritium-labelled thymidine in the cells and in the deoxyribonucleic acid are not increased appreciably by longer periods of incubation (up to 3 hr.). Addition of the other three nucleosides does not influence the amounts taken up. The failure of the cells after 1 hr. to take up more thymidine or to synthesize more deoxyribonucleic acid is not due to a general stopping of the metabolic processes of the cells since the rate of incorporation of orotic acid into the ribonucleic acid continued at a constant rate for at least 2J hr. Neither can the effect be attributed to damage caused by radiation from the labelled thymidine present in the cells, since the amount of precursor taken into the cells and incorporated into the deoxyribonucleic acid is the same if 0-01 \iM of thymidine is added with an activity of 5 [ic. or 0-5 [ic. It appears that when the cells are incubated in this medium some changes occur or some metabolite is lacking which prevents them from either accumulating thymidine or synthesizing deoxyribonucleic acid for more than a short time.
We have also attempted to ascertain the nature and distribution of the precursor which is present in the cells but not polymerized into deoxyribonucleic acid. Nuclei were prepared using the method of Allfrey et al.7, and the non-nuclear fraction was found to contain scarcely any activity. The auto-radiographs also showed that the precursor was concentrated in the nuclei of the cells. Very little activity was lost from the nuclei on standing in Hanks' medium and no exchange was detected when inactive thymidine was added to the medium. It wag, however, found that treatment of the cells or nuclei with ultrasonics and centrifuging for 20 min. at 100,000< 7 left considerable amounts of activity in the supernatant fraction. Chromatography of these supernatant fractions (^sobutyric acid/ammonia solvent) obtained from cells incubated for 1, 5 and 16 min. showed that the activity of tritium was accounted for as 15 per cent thymidine di- and tri-phosphates (these two compounds were assayed together), 25 per cent thymidine monophosphate and 60 per cent thymidine in all cases. These results suggest that thymidine is taken up by the cells and transferred to the nucleus where an equilibrium is rapidly set up between thymidine, thymidylic acid and the di-and tri-phosphate derivatives. Although these compounds cannot be simply washed out from the cells or nuclei, it appears that they are not firmly bound within the nucleus since they can be liberated by destroying the nuclear structure. It seems probable that some part of the precursors may be even more firmly bound within the nucleus since the activity of the supernatant fractions obtained after ultrasonic treatment, added to the observed activity of the deoxyribonucleic acid, accounts, in some cases, for only 60 per cent of the total activity of the cells.
Table 1. COMPARISON OF THE TRITIUM ACTIVITY OP CELLS AND DEOXYRIBONUCLEIC ACID AND THE DEGREE OF LABELLING OBSERVED IN AUTORADIOGRAPHY EXPERIMENTS
 	 	C.p.m./Atgm. 	 	Fraction
 	C.p.m.//;gm. 	deoxyribo- 	 	of cells
 	deoxyribo- 	nucleic acid in 	Average 	labelled
Minutes 	nucleic acid 	the isolated 	spots per 	in auto-
incubated 	in the cell 	deoxyribo- 	cell in auto- 	radiograph
at 37  C. 	suspension 	nucleic acid 	radiograph 	(per cent)
15 	115 	32 	7-1 	32
60 	119 	79 	5-5 	29
It has usually been considered that the activity observed, in the nucleus, by autoradiographic procedures after appropriate washing and fixing techniques can be ascribed to the thymidine incorporated into deoxyribonucleic acid8. The results of this work show that this is not necessarily valid. It can be seen, for example, in Table 1, that after 60 min. incubation the average amount of tritium-labelled thymidine assimilated per cell is little different from that observed after 15 min. incubation, though more than twice as much of the thymidine has been incorporated into deoxyribonucleic acid. The data from the autoradiographs suggest that the average activity per cell is roughly the same for the two periods of incubation and clearly bears no relationship to the activity of the deoxyribonucleic acid alone. It would therefore appear that the fixing procedure does not remove a significant part of the labelled thymidine, which is incorporated into the cell but not polymerized into deoxyribonucleic acid. To confirm this conclusion a suspension of washed cells was centrifuged and the cells resuspended in (a) 2 per cent acetic acid and (b) 45 per cent acetic acid in alcohol. After standing for 5 min. at room temperature the suspensions were centrifuged and the activity of the supernatant fractions assessed. In both cases the total activity of the fixing fluids was less than 10 per cent of the total activity of the cell suspension. Table 1 also shows that only one-third of the cells in the suspension have assimilated labelled thymidine. It would therefore appear that under the conditions used in our experiments, the autoradiographic process detects not only the cells which are actively polymerizing new deoxyribonucleic acid but also those which contain precursors in a form not easily removed by the washing and fixing procedures (cf. Harris)9. Other workers have attempted to assess mitotic activity from counts of the number of labelled cells in autoradiographs and have found an apparent index considerably greater than that obtained from experiments using colchicine to inhibit cell division10. The results described above could provide an explanation for this anomaly.
We are indebted to Prof. J. A. V. Butler for encouragement in this work. We also wish to bhank B. Hunter and B. Wilson for technical assistance. This investigation has been supported by grants to the Chester Beatty Research Institute (Institute of Cancer Research : Royal Cancer Hospital) from the Medical Research Council, the British Empire Cancer Campaign, the Jane Coffin Childs Memorial Fund for Medical Research, the Anna Fuller Fund and the National Cancer Institute of the National Institutes of Health, U.S. Public Health Service.Lajtha, , L. G., Oliver, , R., Kumatori, , T., and Ellis, , F., Rad. Res., 8, 1 (1959).ISIHolmes, , B. E., Ciba Foundation Symp. [ldquo]Ionizing Radiations and Cell Metabolism[rdquo], 225 (1956).Bollum, , F. J., Anderegg, , J. W., McElya, , A. B., and Potter, , V. R., Cancer Res., 20, 138 (1960).PubMedISIChemPortPaul, , J., [ldquo]Cell and Tissue Culture[rdquo] (Livingstone, 1956).Kay, , E. R. M., Simmons, , N. S., and Dounce, , A. L., J. Amer. Chem. Soc., 74, 1724 (1952).ArticleISIChemPortBurton, , K., Biochem. J., 62, 315 (1956).PubMedISIChemPortAllfrey, , V. G., Mirsky, , A. E., and Osawa, , S., J. Gen. Physiol., 40, 451 (1956-57).Pelc, , S. R., and La Cour, , L. F., Experientia, 15, 131 (1959).ArticlePubMedISIChemPortHarris, , H., Biochem. J., 72, 54 (1959).PubMedISIChemPortHughes, , W. L., Bond, , V. P., Brecher, , G., Cronkite, , E. P., Painter, , R. B., Quastler, , H., and Sherman, , F. G., Proc. U.S. Nat. Acad Sci., 44, 476 (1958).