J. Cell Set. 13, 429-439 (i973) 429Printed in Great Britain

LETHALITY OF ADENOSINE FOR CULTURED

MAMMALIAN CELLS BY INTERFERENCE

WITH PYRIMIDINE BIOSYNTHESIS

K. ISHII* AND H. GREENDepartment 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 escapenotice because some sera (such as calf or human) commonly used in culture media, containadenosine deaminase. In the absence of serum deaminase, adenosine produced inhibition ofgrowth of a number of established cell lines at concentrations as low as 5 x io~* M, and killedat 2 x io~5 M. This effect required the presence of cellular adenosine kinase, since a mutantline deficient in this enzyme was 70-fold less sensitive to adenosine. The toxic substance istherefore derived from adenosine by phosphorylation, and is probably one of the adenosinenucleotides.

The toxic effect of adenosine in concentrations up to 2 x io~* M was completely preventedby the addition of uridine or of pyrimidines potentially convertible to uridine, suggesting thatthe adenosine was interfering with endogenous synthesis of uridylate. In the presence ofadenosine, 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 ofadenosine results from inhibition by one of its nucleotide products of the synthesis of uridylateat the stage of phosphoribosylation of orotate.

INTRODUCTION

Though adenosine is not an intermediate on the endogenous pathway of purinebiosynthesis, it can be efficiently utilized through the purine salvage pathways as thesole purine source in cultured mammalian cells whose endogenous purine synthesisis blocked by aminopterin (Green & Ishii, 1972). The route of its utilization underthese conditions is predominantly through deamination to inosine and successiveconversion to hypoxanthine and IMP. We report here that at least part of the reasonfor this is that calf and other mammalian sera contain sufficiently active adenosinedeaminase to deaminate, under cell culture conditions, most added adenosine withinsome hours. If this is avoided by the use of serum lacking the deaminase, a part of theadded adenosine is utilized through phosphorylation and even at quite low concen-tration has marked inhibitory effects on the cells, attributable to interference withpyrimidine 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 nopurines or pyrimidines) supplemented with 10% serum.

The lines used were 3T3 and 3T6 (Todaro & Green, 1963); 3T6-TG8, lacking hypoxanthinephosphoribosyl 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 celldensity 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 anarbitrary scale ( + , + +, and + + +). Killing of the cells was indicated by cell detachmentfrom the monolayers.

Adenosine deaminase activity of sera

[uC]adenosine (0-2-2-5 /*Ci) was diluted with unlabelled adenosine to IO~*-IO~3 M in 0-3 mlof a solution containing serum-free medium, 50 mM phosphate buffer, pH7-i, and 10%serum. The Km of calf serum adenosine deaminase is 3-3 x io"6 M (Cory, Weinbaum &Suhadolnik, 1967). During incubation at 37 °C, 50-/1I samples were taken at intervals andadded to 12 ml of cold o-oi N H C I ; I ml of the solution was immediately applied to a15 x o-6 cm column containing 0-4 ml of Dowex-soW-X8, 200-400 mesh, H+ form, previouslywashed with distilled water. The column was eluted first with 14-4 ml of 0-5 M LiCl solutionin 001 N HC1, which removed inosine and hypoxanthine; adenosine was then eluted with48 ml of 0-2 M LiOH solution. The eluates were collected in fractions and 1 ml of eachcounted 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 mediumwas supplied with or without adenosine at io~* M. After 3 h incubation, the medium wasrenewed 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 wascollected, and cold perchloric acid (PCA) was added to 5 %. The cell layers were washed withserum-free medium, and 4 ml of cold 5% PCA were added. After 10 min, the cell layerswere detached with a rubber policeman. Each extract was centrifuged at 3000 rev/min andthe pellet was washed with cold PCA. The washing and first supernatant were combinedand neutralized with potassium carbonate. After standing overnight at 4 °C the precipitatewas 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 waswashed with io~3 M formic acid and the adsorbed nucleotides eluted twice with 50% ethanolcontaining 0-5 N ammonium hydroxide. The eluates were combined, evaporated to dryness,disolved in water and applied to thin-layer chromatographic plates. Chromatography in thefirst 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 thenucleosides and nucleotides of the other bases. Chromatography in a second dimension, usingisopropanol:water:ammonia (85:15:1-3) (Wyatt, 1955) moved the nucleosides and basesaway from the nucleotides.

For measurement of radioactivity in the pyrimidine fractions, the plastic-backed celluloselayers were cut into 8-mm slices along the second dimension and counted in dioxane-basedscintillator solution. The identity of the labelled products was established by comparisonwith the mobility of unlabelled standards located under u.v. illumination. In some casesradioautographs were prepared from chromatograms in which the radioactive pyrimidinesand 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 produceconcentrations of I C C ' - I O " 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. Ordinateshows the amount of labelled adenosine remaining with increasing incubation time.O—O—O, horse serum with io"3 M adenoaine, A; # — # — 0 , calf serum withio~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 inthe 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 ethanoland 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 haveconfirmed that relative to cell extracts the deaminase activity of calf serum is verylow; for example, an extract of 3T6 cells contains per unit of protein about 300times more deaminase activity than that of unfractionated calf serum. Yet consideringthe relative amounts of cells and medium employed in cell cultures and the timescale involved, the serum activity may be very appreciable. Fig. 1 shows the resultsof an experiment in which labelled adenosine was added to culture medium containing10% calf serum or 10% horse serum, and the amount of adenosine remaining inthe medium was followed with time (no cells were present). In the presence of calfserum, adenosine at the highest initial concentration (io~3 M) was half destroyed inabout 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

mmol/1.

o-oO-OO20-005

o-oi0-02O-2Oi - o2-O

Growth assessed on anadded adenosine.

Medium

Calf serum

+ + ++ + ++ + ++ + ++ + ++ + +

±Killed

arbitrary scale, +, + +,

supplemented with

Horse serum

+ + ++ + ++ +±

KilledKilledKilledKilled

+ + +, compared with control without

Table 2. Inhibition by adenosine of growth of different cell lines inmedium free from adenosine deaminase

Lowest inhibitory adenosine concentrationCell line mol/1. (xio«)

3T6 53T3 2HeLa 353T6-TG8(HPT-) 203T6-DF8(APT-) S

3T6-TM(AK-) 350

probably inosine phosphorylase. Inosine and hypoxanthine together accounted forall the radioactivity lost from adenosine.

In contrast to calf serum, horse serum was found to be completely free fromdeaminase. Incubation of io~3 M adenosine with medium containing 10% horseserum led to no detectable loss of adenosine (Fig. 1) or appearance of labelled inosine.

Other sera tested and found to possess deaminase activity were foetal calf serum,human serum and y-globulin-free calf serum. The activity of the calf serum enzymewas unaffected by heating at 60 °C for 30 min.

The effect of adenosine on cultured cells in the absence of serum deaminase

Several established lines were found to grow as well in medium supplementedwith 10% horse serum as with 10% calf serum. However, when adenosine wasadded to the cell cultures in medium containing horse serum, it was found to bequite toxic at very low concentration. Table 1 shows a comparison of the effects ofadenosine on 3T6 in medium containing the 2 kinds of serum. In 10% calf serumthere was no effect on cell growth at low or moderate adenosine concentrations,while in medium containing 10 % horse serum there was definite inhibition of growth

Adenosine lethality and pyrimidine biosynthesis 433

at 0-005 mil and killing of the cells at 0-02 mM. To obtain any effect on cells inmedium containing calf serum, adenosine had to be added at 1 HIM or higher.

A comparison of the relative sensitivity of a number of lines to adenosine inmedium containing horse serum is shown in Table 2. 3T3 and HeLa cells wereslightly more and slightly less sensitive, respectively, than wild type 3T6. Sublinesof 3T6 lacking the enzyme hypoxanthine phosphoribosyl transferase (3T6-TG8) orlacking adenine phosphoribosyl transferase (3T6-DF8) were approximately as sensi-tive to adenosine as the wild type. However, a 3T6 subline deficient in adenosinekinase (3T6-TM) was much more resistant to adenosine, as an approximately 70-fold higher concentration was required to produce inhibition of growth. This suggestsstrongly that the toxic effect of low adenosine concentrations requires direct con-version of the adenosine to AMP, a reaction which cannot be carried out to anydegree by the T36-TM (AK~) line (Chan et al. 1973). Of course, adenosine can beconverted to AMP indirectly even in this line through the pathway adenosine -> ino-sine -y hypoxanthine -> IMP ->• adenylosuccinate -> AMP. However, as inosine andhypoxanthine are not toxic to 3T6 or 3T6-TM in concentrations up to 2 mM, theinhibitory effects of adenosine in low concentrations appear to depend on the directconversion of adenosine to AMP by adenosine kinase. Other bases and nucleosidestested at 1 mM and found to have no inhibitory effect on the growth of 3T6 includeduridine, cytidine, xanthine, xanthosine and guanosine; and guanine at 0-4 mM.Adenine was toxic to 3T6 cells at 1 mM, but not at lower concentrations. If theenzyme adenine phosphoribosyl transferase, which converts adenine to AMP is lessactive than adenosine kinase, which converts adenosine to AMP, the AMP levels inthe cell would not be driven up as readily by adenine as by adenosine.

All tests of the effect of purines and pyrimidines on cell lines were carried out atcell concentrations below 5 x io3 per ml of medium. At higher cell concentration, thetoxic effects of adenosine could be observed on the day following its addition, butthe cells often recovered and grew progressively, though they were delayed comparedwith controls. Their recovery was probably due to destruction of the adenosine bythe cellular adenosine deaminase, for in separate experiments it was found that inculture medium supplemented with 10% horse serum, and containing 4X io6 3T6cells/ml, adenosine added to o-i mM was half destroyed in 2-5 h.

Protection from adenosine inhibition and lethality by pyrimidines

Because of the possibility that the toxic effects of adenosine might result fromsome type of imbalance in the supply of purines or pyrimidines, other bases andnucleosides were examined for their ability to counteract the effect of adenosine.Table 3 shows that the toxic effect of adenosine in medium containing horse serumwas completely abolished at all concentrations up to 0-2 mM by the addition of1 mM uridine. The same was true for all other lines tested (3T6-DF8, HeLa). Atconcentrations of 1 mM, cytidine, deoxyuridine and deoxycytidine were as effectiveas uridine (Table 4). The pyrimidine bases of these nucleosides can very probablybe converted into uridine through the action of deaminases and phosphorylasesknown to be present in mammalian cells (Creasy, 1963; Pontis, Degerstedt & Reichard,

28 CE L 13

434 K- Ishii and H. Green

Table 3. Protection of T,T6 from adenosine toxicity by uridine

Growth of 3T6Adenosine,

mmol/1. — Ur +Ur(imM)

o-o + + + + + +o-ooi + + + + + +O-OO2 + + + + + +0-005 + + + + +o-oi ± + + +0-02 Killed + + +0-05 Killed + + +0 1 Killed + + +0-2 Killed + + +0-5 Killed +i-o Killed +2-0 Killed ±

Growth assessed on an arbitrary scale ( + , + + , + + +) against control culture withoutadenosine.

Table 4. Ability of other pyrimidines to protect 3 T6 cells againstthe lethal effects of adenosine (o-i mM)

I IIProtection No protection

UridineCytidineDeoxyuridineDeoxycytidineUracil

CytosineOrotidineOrotate

HypoxanthineInosineGuanineGuanosineThymidineDeoxy guanosine

All compounds were tested at 1 mM, except guanine which was at 0-4 mM, and deoxyguano-sine, at o-2 mM. Those in group I were equally effective at 1 mM, but at lower concentrationsuridine was more effective than the others. Compounds in group II failed to prevent killingof the entire cell population.

1961). Uracil was also effective but cytosine was not. Orotate, orotidine and all of thepurines tested were ineffective (Table 4). The toxicity of adenosine concentrationsof 0-5 mM or higher could not be prevented in 3T6-TM by any of the compounds,including uridine.

Effect of adenosine on biosynthesis of uridine nucleotides

In view of evidence suggesting that purines and pyrimidines may utilize the samepermeation system for entry into the cell (Hakala & Kenny, 1972), the possibility wasconsidered that uridine might protect against the toxic effects of adenosine by com-

Adenosine lethality and pyrimidine biosynthesis 435

05

OriginSlice no.

Fig. 2. Chromatography of pyrimidine nucleotides and nucleosides labelled with[uC]aspartate (second dimension). A, cell layer; B, culture medium. # %t cellslabelled with aspartate during the period 2-5 h after addition of 01 mM adenosine;O—O—O, cells labelled with aspartate without added adenosine.

petitively inhibiting its entrance into the cell. An experiment was therefore performedto measure incorporation of [3H]adenosine present in the medium at a concentrationof o-i mM, in the presence and absence of 1 mM uridine. The incorporation of labelledadenosine into perchloric acid (PCA)-insoluble form was higher in the presence of1 mM uridine than in its absence at all times over the 24-h period examined. Thisfinding gave no support to the idea that uridine prevented the uptake of adenosine,and it seemed more likely that the toxicity of adenosine over the range reversible byuridine (see Table 3) was due to interference with pyrimidine synthesis.

As a test of this possibility, we determined the effect of added adenosine on theconversion of [14C]aspartate to acid-soluble uridine nucleotides. Adenosine (o-i mM)was added to a culture of growing 3T6 cells; 3 h later, fresh medium containing thesame adenosine concentration and 5 /iCi/ml of [14C]aspartate was added. Afterfurther incubation, the medium was removed, the cells were extracted with 5 %PCA and the uridine nucleotides separated by 2-dimensional thin layer chromato-graphy on cellulose. It was found that the radioactivity in the uridine nucleotideswas lowered to approximately 15 % of control levels (Fig. 2 and Table 5). Chromato-graphy also disclosed a labelled spot corresponding to orotate or orotidine in the

28-3

43° K. Ishii and H. Green

Table 5. Effect of adenosine on pyrimidine synthesis from [14C]aspartate*

Expt. 1

Expt. 2

Expt. 3 •

Cell layerMedium

Sum. Total

Cell layerMedium

SumTotal

Cell layerMedium

Sum, Total

• Summary

Control,cpm x io~*

Undinenucleotides Orotate

21-7

7'5292

348

1 3 28-o

2 1 2

2 5 6

IS-4196

3S'O457

of 3 experiments of the

0-4

S'2

56

0

44

44

O-2

IO5

10-7

type described

+ Adenosine,cpm x io"1

A.

Uridinenucleotides

6 357

12-0

466

2 2

96

n-84 5 2

2 6167

1935 7 3

in Fig. 2.

Orotate

5'4292

34'6

2'5309

33'4

2 2

35-8

38-0

extracts of cells grown in the presence of adenosine, while none was present in cellsgrown in medium to which no adenosine was added (Fig. 2 A). NO significant labelwas found in positions corresponding to uridine or thymidine in either extract,consistent with a relatively small pool of nucleosides compared with nucleotides.

Examination of the culture medium at the time the cells were harvested showedsmall amounts of label migrating in the region occupied by the uridine nucleotides,and the amounts were similar whether adenosine had been present or not; but inthe medium containing adenosine there was a large labelled spot corresponding toorotate or orotidine. The radioactivity in this spot exceeded that of the entire uridinenucleotide spot (Fig. 2B and Table 5). No discrete labelled spot with this mobility wasobtained from the medium of cultures grown without added adenosine. The identityof the compound was established by elution from the chromatogram, addition ofunlabelled orotate or orotidine and chromatography in the third or fourth solventsystems. The labelled compound whether isolated from the culture medium or thecell layer had a mobility which corresponded perfectly with the u.v. spot for orotate;no radioactivity could be found with the mobility of orotidine or dihydroorotate.

DISCUSSION

Purine nucleotides have been shown to affect the synthesis of pyrimidines inbacteria (Anderson & Meister, 1966) but their effects are thought to be exerted atan early stage of the pyrimidine biosynthetic pathway. In cultured fibroblasts, allo-purinol, an analogue of hypoxanthine, interfered with the late stages of the biosyn-thetic pathway (Kelley, Beardmore, Fox & Meade, 1971) and caused excretionof orotidine and orotate in man (Fox, Royse-Smith & O'Sullivan, 1970; Kelley &

Adenosine lethality and pyrimidine biosynthesis 437

Beardmore, 1970). Even at 1 mM, allopurinol did not have any toxic effect on thegrowth of cultured cells. Allopurinol ribonucleotide and xanthosine monophosphatewere found to be inhibitors of orotidylic decarboxylase in cell extracts (Kelley &Beardmore, 1970).

Krooth (1964) has shown an inhibitory action of adenosine on the growth ofmutant human diploid fibroblasts obtained from persons with orotic aciduria. Owingto the very reduced activity of the last 2 enzymes in the pathway of pyrimidinesynthesis (OMP pyrophosphorylase and OMP decarboxylase) the cells suffered from,partial deficiency of uridine, and grew somewhat more rapidly in the presence ofadded uridine. The growth of these strains was inhibited by adenosine, and theinhibition was relieved by uridine. Measurement of OMP decarboxylase activity incells grown in the presence of adenosine showed slight reduction of the enzymeactivity in wild type cells and inhibition to about one third of the activity in mutantcells. Since the addition of adenosine nucleotides did not inhibit the activity of theenzymes in extracts, the effect of adenosine on the cells was considered to be on theenzyme synthesis. Adenosine did not kill the mutant fibroblasts but only inhibitedtheir growth reversibly. Wild type human diploid fibroblasts were completely un-affected (Krooth, 1964), but in view of our results this may only have been becauseadenosine deaminase was present in the serum supplement. In experiments on wildtype human diploid fibroblasts (strain SB), we found that after preliminary adaptationfor satisfactory growth in medium supplemented with horse serum, growth of thecells was strongly inhibited at adenosine concentrations of 0-02 mM and higher, andthere was considerable cell killing, though the cultures were not totally destroyed.

Our experiments on the effect of adenosine on pyrimidine synthesis in 3T6 indi-cates failure of reaction catalysed by OMP pyrophosphorylase. The effect of adenosinecould hardly be due solely to reduced synthesis of the enzyme, as was thought to bethe case for OMP decarboxylase in the experiments of Krooth, since the effect onnucleotide synthesis and the orotate excretion took place within 2-5 h after additionof adenosine. This was the earliest time tested since it is known that the cellularATP pool does not equilibrate with externally added adenosine for at least 1*5 h(Emerson, 1971). Our results are more consistent with an effect of an adenosinenucleotide on the activity of OMP pyrophosphorylase or possibly on the availabilityof phosphoribosyl pyrophosphate. In the presence of a lethal concentration of adeno-sine, most of the free pyrimidines were in the form of orotate liberated into themedium (Table 5). Taking this orotate into consideration, it should be noticed thatthere was no overall reduction in pyrimidine synthesis in the presence of adenosine;on the contrary, there was some increase, implying that the scarcity of uridinenucleotides activated the reactions leading to orotate. These results suggest thatorotic aciduria in man or animals might follow from a mutation affecting the level ofadenosine nucleotides.

The mammalian organism seems to have gone to considerable length to protectcells from adenosine. The intestinal mucosa is differentiated for the production of adeaminase (Imondi, Lipkin & Balis, 1970) which presumably acts on absorbedadenosine. The serum deaminase would destroy any adenosine produced by nucleo-

438 K. hhii and H. Green

tidase in cells and liberated into the blood. Fibroblast adenosine deaminase is extremelyactive, but in the absence of the serum enzyme, it is unable to protect small numbersof cells from the toxic effects of adenosine. It may be of interest that a fibroblastline (NBL-6, American Type Culture Collection) derived from the horse (the onlyspecies of the 3 examined whose serum did not contain adenosine deaminase) wasalso sensitive to adenosine killing in culture.

We have not identified the nucleotide which interferes with the phosphoribosylationof orotate. The concentrations of AMP, ADP, ATP, cyclic AMP and other adenosinenucleotides may be elevated in cells exposed to adenosine. Cyclic AMP and itsderivatives are known to suppress cell growth (Sheppard, 1971; Johnson, Friedman& Pastan, 1971) but are believed to do this at concentrations lower than those whichproduce toxic effects.

In the absence of the serum deaminase, adenosine inhibits cell growth at concen-trations comparable to those at which some analogues of adenosine are toxic. Theaction of those analogues which can be phosphorylated (Schnebli, Hill & Bennett,1967; Acs & Reich, 1967) may in part be due to effects similar to those we havedescribed here for adenosine itself.

These results also bear on the role of adenosine kinase. It seems clear that thefunction of this enzyme is not to act on exogenous adenosine but on adenosinegenerated within the cell through the action of nucleotidase. The kinase thereforehas a regulating function which is to prevent excessive deamination of adenosine,a process which would lead to purine loss from the cells (Green & Ishii, 1972;Chan et al. 1973). On the other hand, when cells are exposed to exogenous adenosine,the kinase can phosphorylate an excessive amount, the eventual nucleotide productbeing raised to lethal concentration.

This investigation was aided by grants from the National Cancer Institute.

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{Received 2 January 1973)