Concurrent Development of Resistance to 6-Azauridine and Adenosine in a Mouse Cell Line' S . H A S H M I , S. R . MAY, R . S. KROOTH AND 0 . J . MILLER Depicrtment of Hzimirn G e n e t i c s u n d D e v e l o p m e n t , a n d Obstetrics cind G y n e c o l o g y , College of Physicicins i m d Surgeons, Columbiu U?ziversity

ABSTRACT A variant of the hypoxanthine-guanine phosphoribosyltransferase deficient, and adenine phosphoribosyltransferase deficient mouse A 9 cell line has been obtained by selecting cells which are resistant to Gazauridine. These cells are not only resistant to 6-azauridine (5 X M), but also to adenosine ( 1 0 - 3 M). Resistance persists indefinitely even in the absence of both compounds. The resistant cells are killed by 5-fluorouridine (10 - 6 M), indicating that the part of the salvage pathway for pyrimidine ribonucleotide biosynthesis which is relevant to the action of 6-azauridine is intact. The heritable change producing concurrent resistance to 6-azauridine and adenosine probably involves the d e novo pyrimidine biosynthetic pathway.

Efforts to develop mammalian cell lines which simultaneously express more than one alteration in their nutritional requirements, or their sensitivities to drugs and metabolic analogs, have usually involved sequential selection for each trait. For example, Kao and Puck ('68) have selected a subline of a proline requiring variant of Chinese hamster cells which requires glycine in addition to proline. The subline was isolated following treatment of a proline auxotroph with a mutagenic agent. The cells were then grown in a selective system designed to favor cells auxotrophic for glycine. Without the use of a mutagenic agent, we have isolated a variant mouse cell line which is resistant to 10-4 M 6-azauridine a concentration that readily kills the parental population from which the variant was selected. A concurrent phenotype of the variant cell line is virtually total resistance to the cytotoxic effects of adenosine in medium supplemented with 10% horse serum (which has been shown to lack adenosine deaminase activity by Ishii and Green, '73). The parental line is highly sensitive to adenosine in this medium. The simultaneous development of resistance to both agents suggests the involvement of a common element in the mechanism of resistance. Hence the cell line we have isolated may prove useful in clarifying the mode of action of these compounds, and in studies on the interrelationship between purine and pyrimidine metabolism. J . CELL. PHYSIOL.,86: 191-200.

Drug resistant mammalian cell lines (Littlefield, '64; Medrano and Green, '74; inter aliae) have also proven useful for the isolation of somatic cell hybrids, because they sometimes provide a basis for hybrid cell selection. The variant line we shall describe was obtained in the course of a search for new drug-resistant cell phenotypes that might be useful in hybrid cell selection systems. MATERIALS AND M E T H O D S

The 6-azauridine resistant variant was isolated from the A9 cell line (Littlefield, '64). A9 is a n L cell derivative deficient in the enzymes hypoxanthine-guanine phosphori bosyltrans fer ase (Littlefield, '64) and adenine phosphoribosyltransferase (Cox et al., '72). Cultures were grown in Eagle's Minimal Essential Medium (Grand Island Biological Co., Grand Island, N.Y.), containing 10% whole fetal calf serum, except where specified. In experiments employing dialyzed sera, the dialysis bags were made from cellulose tubing (Arthur Thomas Co., Phila., Pa.). The serum was dialyzed for 24 hours against running deionized water, and was intermittently and gently agitated during the course of the dialysis to ensure adequate equilibration with the deionized water. Received Sept. 10, '74. Accepted Feb. 3, '75. 1 This work was aided by grants from the U.S. Public Health Service (GM 18153 and CA 12504) and the National Foundation-March of Dimes, R.S.K. and O.J.M. are Career Scientists of the Health Research Council of the City of New York.




To determine the survival of each line in 6-azauridine (Sigma Chem. Co., St. Louis, Mo.), replicate monolayer cultures, in flasks (Falcon Plastics, Oxnard, Calif.) containing 5 x 10:’cells/ml of either the A9 parent or the variant clone, were grown in concentrations of the drug varying from 10-1’ M to 10-1 M (as well as in control media free of 6-azauridine). Similar experiments were concurrently performed on a thymidine-deficient subline of mouse 3T3 cells. This line is known as 3T3-4E (Matsuya and Green, ’69). Each culture flask contained a total of 15 ml of medium. After 48 hours these cells were removed by trypsinization and the number of cells in each culture was determined with a haemocytometer. The proportion of viable cells was calculated by mixing an aliquot of the cell suspension in balanced salt solution containing 0.4% trypan blue, and determining the percentage of nonstained cells. In the studies on adenosine cytotoxicity, adenosine (Sigma Chem. Co., St. Louis, Mo.) rather than 6-azauridine, was added to the medium at a final concentration ranging from 10-1; M to 10-1 M . In these experiments the medium was supplemented with horse serum instead of fetal calf serum, since fetal calf serum has been shown to contain high levels of adenosine deaminase activity, which rapidly degrades adenosine (Ishii and Green, ’73). The other experimental details are the same as those described above. In some experiments, replicate cultures were concurrently grown in medium containing uridine (10 -;{ M) or uridine plus adenosine, to determine whether uridine prevented the cytotoxic effect of adenosine. The proportion of viable cells was determined on all cultures after 72 hours of incubation in experimental media at 37°C. The cell lines tested included the 6-azauridine resistant line, the parental A9 line, GM43 (a human diploid strain obtained from the Institute for Medical Research, Camden, N.J.), HeLa (a heteroploid human line) and 3T3-4E (a heteroploid mouse line). To determine the cloning efficiency of the parental A9 and variant lines, tissue culture cloning plates (no. 3040, Falcon Plastics) were seeded either with A9P or variant cells at a dilution such that each cloning well contained, on the average, a

single cell. The position of all wells with but one cell was noted. The wells were then filled with either medium containing no additives, medium with lo-.’ M 6-azauridine or medium with 1 0 - 4 M adenosine. A week later, the number of wells innoculated with a single cell, in which distinct clones were growing, was counted. To obtain the cloning efficiency, the number of such wells was divided by the number of wells initially noted to contain only one cell. The cytotoxic effects of 5-fluorouridine (Sigma Chem. Co., St. Louis, Mo.) on the parental and the variant cells were determined by the addition of 10-6 M to 10 -;] M of this pyrimidine analog to flasks containing 5 X 10;’ cells/ml. The proportion of viable cells remaining after 48 hours was measured by the trypan blue exclusion method described above. The methods for the preparation of chromosome spreads and for the development of quinacrine fluorescent karyotypes are detailed in Allderdice et al. (’73). RESULTS

Exponentially growing A9 cells were cultivated in medium containing either 10 7 M 6-azauridine or no 6-azauridine. 6-azauridine at a concentration of 1 0 - 7 M had no effect on either cell growth or viability. The level of 6-azauridine in the medium was then increased, at weekly intervals, until a concentration was reached which noticeably affected the cells. At 3 X lo-,; M cell death became apparent. After a week of incubation in medium containing this concentration of 6-azauridine, the overwhelming majority of cells had either lysed or detached from the growth surface. Two small colonies appeared, and one of these, designated A9AU-I, was isolated and expanded for further study. The other colony was lost due to contamination during the course of subsequent expansion. Subsequent experiments aimed a t the isolation of additional A9 colonies which grow in 3 X lo-,; M 6-azauridine have thus far failed. The cells of this colony proved highly resistant to 6-azauridine (fig. 1). The LDSo of 6-azauridine after 48 hours for the variant A9AU-1 colony was 4 X lo-” M; the LDSl,for the parental A 9 cells and for 3T3 cells after the same time period was less






10 100

70 50



10 100 70 50



10 I I





Molar conc. 6 - a z a u r i d i n e Fig. 1 6-azauridine killing curves of A9AU-1, A9P and 3T3-4E cell lines. Note that the LD,, of ASAU-1 cells is 4 X 10-3 M. At this concentration there is less than 20% cell survival in parental A9 and 3T3-4E cell lines. The LD5,, of 3T3-4E and A9P is approximately 3 X 1 0 - 4 M

than 1 0 % of this value (fig. 1). The high level of resistance appeared to be an autonomous cell attribute since clones of ASAU-1 cells were readily isolated in the presence of 1 0 - 4 M 6-azauridine (vide inf r a ) . When the cells of the colony were

grown for a number of generations in 6azauridine-free media, and were then challenged with 6-azauridine, they remained resistant. Similarly, the resistance could be demonstrated when live cells were frozen and then thawed and expanded €or some


S H A S H M I , S. R. MAY, R


time in 6-azauridine-free media. The resistant phenotype was not associated with any discernible modification of cell or colonial morphology or any change in the growth properties of the cells. Discrimination between the sensitive and resistant phenotype became more sharp if the duration of incubation in the presence of 6-azauridine was prolonged. When 7.5 X 104 non-resistant cells of the various lines studied were incubated in the presence of 10 4 M 6-azauridine for seven or more days, no survivors capable of further growth were recovered. On the other hand, the A9AU- 1 clones grew normally and could be readily propagated in this concentration of the drug. In addition to being resistant to 6-azauridine, the A9AU-1 cells and clones derived from them proved highly resistant to the toxic effects of adenosine in media containing horse sera. Adenosine a t 10-2 M decreased the frequency of viable A 9 A U - 1 cells observed after 48 hours by only 5%, whereas more than 50% of the parental A 9 cells were killed by this concentration of adenosine (fig. 2). We also noted that the parental A 9 P cells were significantly more resistant to adenosine than cells of the 3T3-4E, GM 43 and HeLa lines. A s shown in table 1, the toxicity of adenosine for A 9 P , at least up to a concentration of 1 0 - J M , was markedly decreased if the -





-0 50

.-> >


A9 P


* m +





Wfect of crdrnosinr on the ~ 7 c i b z l i t yof ASP ( i n d A9AU-1 cells zn the p r e w n c e ctnd cibsenct, of u r i d i n e




Fig. 2 Survival of parental A 9 and ASAU-1 cell lines i n medium containing M E M a n d 1 0 % undialyzed horse sera, to which varying concentrations of adenosine were added. Note that there i s about 5cC A9AU-1 cell death a t 1 0 - 3 M, whereas this concentration is the LD,,, for the A S P line.


Percent survival Medium contains

+ +

MEM dialyzed horse serum dialyzedhorse MEM serum 10-XM adenosine dialyzed horse MEM serum 10-3, adenosine 10-3 M uridine







88.5%1.4 N=522

8 5 . 0 k 1.9 N=353

51.3 & 2.1 N=585

88.4 f 1 . 3 N=602

84.3%1.9 N=369

88.6C1.3 N=641

N is the total number of cells counted. 1 The above data were obtained by pooling the results of at least three independent experiments on both lines in all three media.

medium also contained lo-:$ M uridine. Adenosine was not measurably toxic to A 9 A U - 1 cells at this concentration. With adenosine, as with 6-azauridine, the discrimination between the sensitive and the resistant phenotype became more sharp if the incubation of the cells with adenosine was prolonged beyond 72 hours. The experiments for the determination of the cloning efficiency of A 9 P and A 9 A U - 1 demonstrated that single cells of both lines generated colonies with high frequency in media containing only MEM and calf sera. Similar cloning efficiencies were obtained for the A 9 A U - 1 cells in the presence of 1 0 - 4 M 6-azauridine. However the A 9 P cells yielded no colonies in media containing 6-azauridine (table 2). We were repeatedly unable to obtain clones of the A 9 P cell line in MEM 10% undialyzed horse serum, although mass cultures grew quite well in this medium. However, in the case of both A 9 P and A 9 A U - I , clones were readily obtained in MEM 10% dialyzed horse serum. A9AU-1, but not A 9 P , cells generated clones in the presence of lo--' M adenosine in medium containing dialyzed horse sera (table 2). Unlike 6-azauridine and adenosine, 5fluorouridine proved lethal to both A 9 A U - 1 and A 9 P cells, even at concentrations as low as l o - " M . Comparisons between the chromosome consititution of the parental A9 and A 9 A U - 1 cells were made using the specific quinacrine fluorescent banding patterns (figs.






S . H A S H M I , S . R . M A Y . R. S. KROOTH A N D 0. J . MILLER

Fig. 3

Quinacrine fluorescent karyotype of a parental A S cell

accelerates the pathway so that a given level of inhibition still permits the generation of sufficient product for the cells' needs. Another possible change in the de novo sequence could involve reduced sensitivity of an enzyme or enzyme aggregate (Krooth and Sell, '70; Shoaf and Jones, '73) to the inhibitory effects of the active

derivatives of adenosine and 6-azauridine, respectively. ( 3 ) the change might result in more efficient utilization or retention of pyrimidines so that the cell continues to proliferate despite a significant deceleration of de novo pyrimidine synthesis. This could involve the enzymes which catabolize UMP


C l o n i n g efficiericirs of the two lines Medium contains

+ +

MEM lor.; fetal calf serum MEM loc; fetal calf serum 10-4 M 6-azauridine 1 0 V dialyzed MEM horse serum MEM 10'; dialyzed horse serum 1 0 - 4 M adenosine

+ +








84.3 k 2.2 N = 280

82.6 i2.3 N =285

66.0 k2.8 N = 275 N =281 56.9 i- 3.8 41.3 ? 3.7 N = 169 N=179 0.00


N = 175

17.7 *2.9 N=175

N i s the number of wells containing single cells. 1 The above data were obtained by pooling the results of three independent experiments concurrently performed on both lines and i n all four media.

3 , 4). The parental A9 possessed 46-58 chromosomes per cell of which 13-18 were biarmed and the rest telocentric. The chromosome number of A9AU-1 cells varied from 50-57 with 10-16 biarmed chromosomes per cell. Most of the biarmed and telocentric markers could be recognized by their individual banding patterns and were common to both the parental and the 6-azauridine resistant line. Chromosomes 15, 16, 17 and the X were present only as parts of biarmed marker chromosomes in both A9P and A9AU-1. It was not possible to associate the resistant phenotype with a specific chromosomal alteration. Such associations, even when they exist, tend to be obscured by the chromosomal heterogeneity which is characteristic of heteroploid lines like A9. However, despite some numerical variability in the chromosomes within and between the two lines, our analysis revealed that the karyotypes of both lines were very similar, supporting the contention that the A9AU-1 line was derived from the parental A9 population. Moreover, the chromosome constitution of the parental and variant cells resembled quantitatively and qualitatively the karyotype that has been described for A9 cells (Allderdice et al., '73). DISCUSSION

Basis for the cytotoxicity of 6-azauridine and adenosine In mammalian cells, and all other organisms which have been studied in this respect, the pathway for the synthesis of pyrimidine ribotides is bipartite, consisting of a longer de nouo biosynthetic branch


and a shorter salvage sequence (fig. 5). The de nouo pathway converts ATP, bicarbonate and glutamine, through a series of six enzymatic steps, into uridine-5'-monophosphate (UMP). The salvage pathway converts uracil into uridine and uridine into UMP by means of the sequential action of two enzymes: nucleotide pyrophosphorylase and uridine kinase (Canellakis, '57). The pyrimidine analog, 6-azauridine, imposes a nutritional requirement for uridine on mammalian cells (Pinsky and Krooth, '67). It is known that 6-azauridine5'-monophosphate (&azaUMP), the ribotide of 6-azauridine, competitively inhibits orotidine-5'-monophosphate decarboxylase (ODCase), the final enzyme in the de nouo pathway leading to UMP (Pasternak and Ilandschumacher, '59; Saenger and Suck, '73). Adenosine also imposes a strict nutritional requirement for uridine on mammalian cells, as shown by Ishii and Green ('73). Their data suggest that, by an unknown mechanism, a phosphorylated derivative of adenosine blocks the conversion of orotic acid to orotidine-5'-monophosphate, the penultimate reaction in the de nouo sequence leading to UMP (fig. 5).

Possible mechanisms for the resistant phenotype The development of a phenotype, concurrently resistant to the two structurally unrelated compounds, 6-azauridine and adenosine, might involve the following mechanisms: (1) a n hereditary change in some common element, which is required for the entry of these compounds into the cell, or for their conversion into the corresponding inhibitory ribotides. The enzyme concerned with the conversion of 6-azauridine to 6azaUMP is almost certainly uridine kinase. The conversion of adenosine to its active phosphorylated form is catalyzed by adenosine kinase, and perhaps subsequently acting enzymes. In addition, the enzymes which generate ATP should also be considered in this group, since ATP serves as a co-substrate for both uridine and adenosine kinases. (2) a change which renders the cell or the de nouo pathway less sensitive to the action of the inhibitory ribotides. For example, the change might be one which



Fig. 4 Quinacrine fluorescent karyotype of a cell of the 6-azauridine/adenosine resistant ASAU-1 line.

to uridine and uracil, or the kinases which convert U M P to UDP and UTP for co-enzyme and polynucleotide synthesis. There might also be as yet undiscovered enzymes or other cellular elements which influence

the loss of uridine and uracil from the cell (Chan et al., '74). If the defect involves pyrimidine biosynthesis, our data seem to locate the alteration of the ASAU-1 cell line within the de










orotidine -51orotate t 1

t t AT P HCOS glutamine


ur idine - 5't t t RNA

5-f I uoroUM P



Fig 5 The synthesis of uridine-5'-monophosphate in mammalian cells Stipled arrows show the metabolic conversions a n d sites of inhibition of adenosine a n d 6-azauridine

novo pathway rather than in a cell per- uridine kinase. Thus the killing of A9AU-1 mease or the salvage pathway, thereby cells by low levels of 5-fluorouridine tends eliminating the first possibility. The to argue against the involvement of a pyrASAU-1 cells are permeable to 6-azauri- imidine permease or the pyrimidine saldine and adenosine (Hashmi and May, vage pathway in the concurrent resistances unpublished). The possibility of a perme- of this cell line. ability barrier being involved as reported The resistance of the variant line to the by Peterson et al. ('74) in the case of their cytotoxicity of adenosine may well signify puromycin/actinomycin D resistant vari- that the biochemical alteration in these ant, is ruled out in the ASAU-1 cells. The cells is located in the d e novo biosynthetic reasons for the exclusion of the salvage pathway. We cannot of course categorically pathway are twofold: the susceptibility of rule out the third possibility that reduced the variant cell line to the cytotoxic effect catabolism, or loss of pyrimidines, or imof 5-fluorouridine, and the resistance of proved utilization of pyrimidine ribotides these cells to 6-azauridine. As noted, 6- might enable the cell to survive in the azauridine is taken up by the cell and is presence of adenosine. However, we susconverted to its active form by uridine pect that the 10-3 M adenosine we have kinase. Likewise, 5-fluorouridine is con- employed may, essentially, inhibit all d e verted by this same enzyme to 5-fluorouri- novo pyrimidine synthesis in sensitive cells. dine-5'-monophosphate (Reichard a n d The reason for this suspicion is that the Skold, '59; Champe and Benzer, '62). This conversion of orotate to OMP is inhibited latter compound is incorporated into RNA in 3T3 cells by 85% after only four hours M adenosine and and the resulting fraudulent RNA then in media containing causes cell death (Ibid). Hence the sus- horse sera (Ishii and Green, '73). Another ceptibility of the ASAU-1 cell line to 5- possibility is that the cellular level of ATP fluorouridine strongly suggests that the is reduced sufficiently to prevent the invariant cells are likely to be permeable to tracellular accumulation of toxic levels of pyrimidines and to retain uridine kinase the active phosphorylated derivatives of activity. Recently, a variant cell line re- adenosine and 6-azauridine. The synthesis sistant to the cytotoxic effect of 5-fluo- of these derivatives depends upon ATP. rouridine has been isolated from the mouse However, here one has to postulate that fibroblast line, 3T3-4E (Medrano and the reduction of ATP is just enough to preGreen, '74). Enzymatic studies revealed vent the cytotoxicity of both of these inthat these cells were indeed deficient in hibitors but not enough to impair signifi-



cantly the other biosynthetic pathways in which ATP is involved. Such a possibility seems on the face of it a little unlikely. A t the moment, therefore, the second mechanism listed above seems to us to be the most reasonable possibility.

higher concentrations of 6-azauridine, cell selection appeared to occur in a single step: the parental population was killed and the resistant colony emerged following one week of incubation in 0.03 mM 6az auridine.

Nature of the hereditary change It is important to ascertain whether the concurrent resistance to 6-azauridine and adenosine in the A9AU-1 cell line is the result of a mutation or represents some kind of heritable epigenetic change (Harris, '71). We believe our data suggest that a gene mutation may be responsible for the distinctive phenotype of the A9AU-1 cell line. This view is presently supported by three lines of evidence. First, the cell phenotype, both with respect to 6-azauridine and adenosine, appears to be stable during growth in the absence (as well as in the presence) of the two agents. Second, the resistant phenotype is a clonal attribute of the A9AU-1 subline, since all of the subclones isolated to date from the original variant line are resistant. Third, our inability to recover similar co-resistant sublines from the A9 parental population from which the resistant variant was originally derived (or from other cell lines, such as 3T3, RAG, etc.), suggests that we are dealing with a rare event. Indeed, the frequency of occurrence of variants simultaneously resistant to adenosine and 6-azauridine was less than one in 3 X 106 cells, even though the line had not been cloned for more than a year. If a genetic mutation is indeed responsible for the difference between the parental and variant lines, it seems to us quite likely that only one locus is involved, despite the apparent pleiotropy. For one thing, a mutation at a single locus is, a priori, a more probable occurrence than mutations a t multiple loci. In addition, resistance to the two agents has never been observed to segregate in the course of propagation. Moreover, the selective medium in which the variant colony was isolated employed only one of the two agents to which resistance developed. Also, a derivative of adenosine and a derivative of 6-azauridine are known to inhibit the same biosynthetic sequence. Finally, although the resistant variant was isolated following growth of the parental line in progressively

Uses of t h e variant line In addition to its possible value in clarifying the relationship between purine and pyrimidine biosynthesis, a cell line resistant to 6-azauridine and adenosine may prove useful in the isolation of somatic cell hybrids, provided the resistant phenotype is expressed in the hybrid cells. Since the A9AU-1 line is deficient in both hypoxanthine-guanine phosphoribosyltransferase and adenine phosphoribosyltransferase, these cells can be eliminated in medium containing hypoxanthine, aminop terin and thymidine (Littlefield, '64). The other parental cell line used to form the hybrids can be selectively killed by the presence of 6-azauridine. Hybrids of this type, once isolated, may permit the assignment of the resistant phenotype to one or more specific murine chromosomes. In addition, complementation studies on hybrids formed between a variety of independently derived lines resistant to both agents might contribute to an enumeration of the genetic loci involved in pyrimidine metabolism. LITERATURE CITED Allderdice, P. W., 0. J . Miller, D. A. Miller, D. Warburton, P. L. Pearson, G. Klein and H. Harris 1973 Chromosome analysis of two related heteroploid mouse cell lines by quinacrine fluorescence. J. Cell Sc., 12: 263-274. Canellakis, E. S. 1957 Pyrimidine metabolism. 11. Enzymatic pathways of uracil anabolism. J . Biol. Chem., 227: 32Sb-338. Champe, S. P., and S . Benzer 1962 Reversal of mutant phenotype by 5-fluorouracil: an approach to nucleotide sequences in messenger-RNA. Proc. Nat. Acad. Sci., U.S.A.,48: 532-546. Chan, T., M. Meuth and H . Green 1974 Pyrimidine excretion by cultured fibroblasts: Effect of mutational deficiency in pyrimidine salvage enzymes. J. Cell. Physiol., 83: 263-266. Cox, R. P., M . R. Krauss, M. E. Balis and J. Dancis 1972 Communication between normal a n d enzyme deficient cells in tissue culture. Exp. Cell Res., 74: 251-268. Harris, M. 1971 Mutation rates in cells at different ploidy levels. J. Cell. Physiol., 78: 177184. Ishii, K., and H. Green 1973 Lethality of adenosine for cultured mammalian cells bv interference with pyrimidine biosynthesis. J. Cell Sci., 13: 4 2 W 3 9 . Kao, F., and T. Puck 1968 Genetics of somatic



mammalian cells. V I I . Induction and isolation of nutritional mutants i n Chinese hamster cells. Proc. Nat. Acad. Sci., U.S.A.,6'0: 127.51281, Krooth, R. S.. and E. K. Sell 1970 'The action of Mendelian genes in h u m a n diploid cell strains. J. Cell. Physiol., 76. 311-330. Littlefield, J . 1964 Three degrees of' guanylic acid-inosinic acid pyrophosphorylase deficiency in mouse fibroblasts. Nature, 203; 1142-1144. Matsuya, Y . . and H. Green 1969 Somatic cell hybrid between the established h u m a n line D98 (presumptive He1,a) a n d 3T3. Science, 163: 697698. Medrano, L., and H. Green 1974 A uridine kinase-deficient m u t a n t of 3T3 and a selective method for cells containing the enzyme. Cell, 1 : 23-26. F'asternak, C . A,. and R. E. Handschumacher 1959 The biochemical activity of 6-azauridine: Interference with _ pyrimidine metabolism i n . transplantable mouse tumors. J . Biol. Chem., 234: 2992-2997.

KROOTH AND 0. J. MILLER Peterson, R. H. F.. J. A. O'Neil and J . L. Biedler 1974 Some biochemical properties of Chinese hamster cells sensitive and resistant to actinomycin D. J . Cell Biol., 63: 773-779. Pinsky, L.. and R. S . Krooth 1967 Studies on the control of pyrimidine biosynthesis in human diploid cell strains. I. Effect of 6-azauridine on cellular phenotype. Roc. Nat. Acad. Sci. U.S.A., 57: 9 2 S 9 3 2 . Reichard, P . . a n d 0. Skold 1959 Possible enzymatic mechanism for the development of resistance against fluorouracil in ascites tumors. Nature, 183: 939-941. Saenger, W.. a n d D. Suck 1973 6-azauridine5'-phosphoric acid: unusual molecular structure and functional mechanism. Nature, 242: 610612. Shoaf', W. T., and M. E. Jones 1973 Uridylic acid synthesis in Ehrlich ascites carcinoma. Properties, subcellular distribution and nature of enzyme complexes of the six biosynthetic enzymes. Biochem., 12: 4039-4051.

Note A d d e d in Proof: Since the initial submission of this paper, McBurney and Whitmore (J. Cell. Physiol., 85: 87-99, '75), have described the isolation of two Chinese hamster substrains which are resistant to adenosine. One of the strains was deficient in adenosine kinase, and the other (which was not deficient in adenosine kinase) appeared to have reduced ability to retain molecules of adenosine. The sensitivity of these two substrains to 6-azauridine was not reported.

Concurrent development of resistance to 6-azauridine and adenosine in a mouse cell line.

A variant of the hypoxanthine-guanine phosphoribosyltransferase deficient, and adenine phosphoribosyltransferase deficient mouse resistant to 6-azauri...
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