J. Cell Set. 13, 429-439 (i973) 429 Printed in Great Britain LETHALITY OF ADENOSINE FOR CULTURED MAMMALIAN CELLS BY INTERFERENCE WITH PYRIMIDINE BIOSYNTHESIS K. ISHII* AND H. GREEN Department of Biology, Massachusetts Institute of Technology, Cambridge, Massachusetts, 02139, U.S.A. SUMMARY Adenosine at low concentration is toxic to mammalian cells in culture. This may escape notice because some sera (such as calf or human) commonly used in culture media, contain adenosine deaminase. In the absence of serum deaminase, adenosine produced inhibition of growth of a number of established cell lines at concentrations as low as 5 x io~* M, and killed at 2 x io~5 M. This effect required the presence of cellular adenosine kinase, since a mutant line deficient in this enzyme was 70-fold less sensitive to adenosine. The toxic substance is therefore derived from adenosine by phosphorylation, and is probably one of the adenosine nucleotides. The toxic effect of adenosine in concentrations up to 2 x io~* M was completely prevented by the addition of uridine or of pyrimidines potentially convertible to uridine, suggesting that the adenosine was interfering with endogenous synthesis of uridylate. In the presence of adenosine, the conversion of labelled aspartate to uridine nucleotides was reduced by 80-85 %> and labelled orotate accumulated in both the cells and in the culture medium. The lethality of adenosine results from inhibition by one of its nucleotide products of the synthesis of uridylate at the stage of phosphoribosylation of orotate. INTRODUCTION Though adenosine is not an intermediate on the endogenous pathway of purine biosynthesis, it can be efficiently utilized through the purine salvage pathways as the sole purine source in cultured mammalian cells whose endogenous purine synthesis is blocked by aminopterin (Green & Ishii, 1972). The route of its utilization under these conditions is predominantly through deamination to inosine and successive conversion to hypoxanthine and IMP. We report here that at least part of the reason for this is that calf and other mammalian sera contain sufficiently active adenosine deaminase to deaminate, under cell culture conditions, most added adenosine within some hours. If this is avoided by the use of serum lacking the deaminase, a part of the added adenosine is utilized through phosphorylation and even at quite low concen- tration has marked inhibitory effects on the cells, attributable to interference with pyrimidine synthesis. • Present address: Institute for Virus Research, Kyoto University, Sakyo-Ku, Kyoto 606, Japan. 43 o K. Ishii and H. Green MATERIALS AND METHODS Cell culture Cells were cultivated as monolayers in the Dulbecco-Vogt medium (which contains no purines or pyrimidines) supplemented with 10% serum. The lines used were 3T3 and 3T6 (Todaro & Green, 1963); 3T6-TG8, lacking hypoxanthine phosphoribosyl transferase (Long et al. 1973); 3T6-TM, lacking adenosine kinase (Chan, Ishii, Long & Green, 1973); 3T6-DF8, lacking adenine phosphoribosyl transferase (Kusano, Long & Green, 1971); and HeLa. Effects of adenosine on cell growth were tested on exponentially growing cultures at a cell density below 5 x 103 per ml of medium. The cultures were observed over a period of 1 week, and growth was compared to that of the control in absence of adenosine according to an arbitrary scale ( + , + +, and + + +). Killing of the cells was indicated by cell detachment from the monolayers. Adenosine deaminase activity of sera u [ C]adenosine (0-2-2-5 /*Ci) was diluted with unlabelled adenosine to IO~*-IO~3 M in 0-3 ml of a solution containing serum-free medium, 50 mM phosphate buffer, pH7-i, and 10% 6 serum. The Km of calf serum adenosine deaminase is 3-3 x io" M (Cory, Weinbaum & Suhadolnik, 1967). During incubation at 37 °C, 50-/1I samples were taken at intervals and added to 12 ml of cold o-oi NHCI; I ml of the solution was immediately applied to a 15 x o-6 cm column containing 0-4 ml of Dowex-soW-X8, 200-400 mesh, H+ form, previously washed with distilled water. The column was eluted first with 14-4 ml of 0-5 M LiCl solution in 001 N HC1, which removed inosine and hypoxanthine; adenosine was then eluted with 48 ml of 0-2 M LiOH solution. The eluates were collected in fractions and 1 ml of each counted by liquid scintillation. Conversion of 14C-labelled aspartate to uridine nucleotides 3T6 cells were inoculated into 100-mm Petri dishes and grown in medium containing 10% horse serum. On the following day, when the cells were in exponential growth, fresh medium was supplied with or without adenosine at io~* M. After 3 h incubation, the medium was renewed with addition of adenosine as before. Uniformly labelled [14C]aspartate (167 mCi/mM) was added to a concentration of 5 /tCi/ml of medium. After 2 h incubation, the medium was collected, and cold perchloric acid (PCA) was added to 5 %. The cell layers were washed with serum-free medium, and 4 ml of cold 5% PCA were added. After 10 min, the cell layers were detached with a rubber policeman. Each extract was centrifuged at 3000 rev/min and the pellet was washed with cold PCA. The washing and first supernatant were combined and neutralized with potassium carbonate. After standing overnight at 4 °C the precipitate was centrifuged. The supernatant was acidified by the addition of formic acid to 2 x io~3 N (pH 3-5), and washed charcoal (2% w/v) added (Smith & Khorana, 1963). The charcoal was washed with io~3 M formic acid and the adsorbed nucleotides eluted twice with 50% ethanol containing 0-5 N ammonium hydroxide. The eluates were combined, evaporated to dryness, disolved in water and applied to thin-layer chromatographic plates. Chromatography in the first dimension using isopropanol:HC1:water (70:15:15) (Wyatt, 1955) separated the nucleo- sides and nucleotides of thymine, uracil and orotate, together with orotate itself, from the nucleosides and nucleotides of the other bases. Chromatography in a second dimension, using isopropanol:water:ammonia (85:15:1-3) (Wyatt, 1955) moved the nucleosides and bases away from the nucleotides. For measurement of radioactivity in the pyrimidine fractions, the plastic-backed cellulose layers were cut into 8-mm slices along the second dimension and counted in dioxane-based scintillator solution. The identity of the labelled products was established by comparison with the mobility of unlabelled standards located under u.v. illumination. In some cases radioautographs were prepared from chromatograms in which the radioactive pyrimidines and known unlabelled markers had migrated in the same track. Adenosine lethality and pyrimidine biosynthesis 431 100Q Time, h Fig. 1. Deamination of adenosine by calf serum. [14C]adenosine was added to produce concentrations of ICC'-IO"6 M to a mixture of serum-free medium, phosphate buffer (pH 71) and 10 % calf or horse serum. Incubation was carried out at 37 °C. Ordinate shows the amount of labelled adenosine remaining with increasing incubation time. O—O—O, horse serum with io"3 M adenoaine, A; #—#—0, calf serum with io~3, io~4 and io~6 M adenosine, B, C, D respectively. For further confirmation of the identity of labelled orotate, the labelled spot obtained in the second dimension chromatography (Fig. 2, p. 435) was eluted, evaporated and rechromato- graphed, either in a third solvent system containing n-butanohmethanol: ammonia: water (60:20:1:20) (Randerath & Randerath, 1967), or in a fourth solvent consisting of ethanol and 1 M ammonium acetate, 1:1 (P. Cashian, personal communication). The latter gave ex- cellent resolution of orotate from orotidine and of orotate from dihydroorotate. RESULTS The adenositie deaminase activity of mammalian serum The presence of an adenosine deaminase of low specific activity has been demon- strated in calf serum, and the enzyme has been purified (Cory et al. 1967). We have confirmed that relative to cell extracts the deaminase activity of calf serum is very low; for example, an extract of 3T6 cells contains per unit of protein about 300 times more deaminase activity than that of unfractionated calf serum. Yet considering the relative amounts of cells and medium employed in cell cultures and the time scale involved, the serum activity may be very appreciable. Fig. 1 shows the results of an experiment in which labelled adenosine was added to culture medium containing 10% calf serum or 10% horse serum, and the amount of adenosine remaining in the medium was followed with time (no cells were present). In the presence of calf serum, adenosine at the highest initial concentration (io~3 M) was half destroyed in about 8 h. At io"4 M, the half-life was about 90 min, and at icr5 M, about 35 min. Most of the radioactivity lost from adenosine was recovered as hypoxanthine, indi- cating that calf serum also contains an enzyme capable of deribosylating inosine, 432 K. Ishii and H. Green Table i. Effect of adenosine on 3T6 Medium supplemented with mmol/1. Calf serum Horse serum o-o + + + + + + O-OO2 + + + + + + 0-005 + + + + + o-oi + + + ± 0-02 + + + Killed O-2O + + + Killed i-o ± Killed 2-O Killed Killed Growth assessed on an arbitrary scale, +, + +, + + +, compared with control without added adenosine. Table 2. Inhibition by adenosine of growth of different cell lines in medium free from adenosine deaminase Lowest inhibitory adenosine concentration Cell line mol/1. (xio«) 3T6 5 3T3 2 HeLa 35 3T6-TG8(HPT-) 20 3T6-DF8(APT-) S 3T6-TM(AK-) 350 probably inosine phosphorylase. Inosine and hypoxanthine together accounted for all the radioactivity lost from adenosine. In contrast to calf serum, horse serum was found to be completely free from deaminase. Incubation of io~3 M adenosine with medium containing 10% horse serum led to no detectable loss of adenosine (Fig.