Resistance to 5-fluoroorotic acid and pyrimidine auxotrophy: a new bidirectional selective system for mammalian cells.

We have isolated a clone of murine erythroleukemic cells which will yield a population when a single cell is suspended in a medium composed of dialyzed sera and small molecules. We report that it is feasible--in one experiment--to screen more than 10 of these cells for growth under selective media containing 10(-4) M 5-fluoroorotic acid and 10(-4) M uridine. Cells capable of sustained growth in such media were eventually recovered. Clones of these cells, unlike clones of the parental population, required uridine for growth and contained only 0.39% as much orotate phosphoribosyltransferase, less than 0.5% as much 5-fluoroorotate phosphoribosyltransferase, and 0.013% as much orotidine-5'-monophosphate decarboxylase activity as the parental clones. The parental and variant clones had similar levels of activity for six other enzymes that participate in pyrimidine metabolism.

Effects of natural pyrimidines and of certain related compounds on the spontaneous activity of the mouse.

Uracil and perhaps other natural pyrimidines may effect the level of arousal of the mammalian brain since: 1) heterocyclic 6-membered rings, which resemble uracil, form part of the structure of many hypnotics; and 2) 6-azauracil (and its riboside) have shown to be hypnotic for several mammalian species, including man. The parenteral administration of uridine, 6-azauridine, cytidine or thymidine depressed the spontaneous activity of adult male C-57 mice. 6-Azauridine was much less potent than the other ribosides tested. Cytosine, barbituric acid, 2-thiobarbituric acid, 2,4-dihydroxypyridine and a variety of pyrimidine catabolites had no effect on activity. Thymine, uracil, 6-azauracil, barbital and phenobarbital increased activity at lower doses and decreased activity at higher ones. 6-Azauracil and uracil were about equally potent as stimulants of activity, but 6-azauracil had about twice the potency of uracil as a depressant of activity. Thymine, which was more active than uracil, had about 10% the potency of barbital, both as a stimulant and as a depressant of activity. For thymine and the two barbiturates the ED50 (for depression of activity) was of the same magnitude as the LD50, while the dose which caused 50% stimulation of activity was about an order of magnitude less than the LD50. These results suggest that the barbiturates might affect arousal by simulating the structure of thymine or uracil at some receptor.

Evidence for a striking increase in acetylcholinesterase activity in cultured human fibroblasts which are trisomic for chromosome two.

Acetylcholinesterase (AchE) is reported to have a narrowly restricted distribution among human tissues. Three strains of human fibroblasts which are trisomic for chromosome 2 had an average level of AchE activity over 28 times higher than the average level in 19 control strains of human fibroblasts. In contrast, the mean pseudocholinesterase activity of the trisomy-2 strains did not differ appreciably, or significantly, from the mean for the control strains. The 19 control strains included 10 strains trisomic for autosomes other than 2, and 9 euploid strains. Our estimate of the mean AchE activity in the control strains did not differ significantly from zero and might, in any case, have originated from a minute amount of activity contributed to the cells by an esterase in our culture medium. Despite the striking elevation of AchE activity in fibroblasts trisomic for chromosome 2, extracts of these cells had only about 1.5% of the specific AchE activity (per microgram DNA) present in extracts of human cerebral cortex. None of the 22 strains studied had detectable activity for two other enzymes which, like AchE, have a restricted distribution among human tissues: xanthine oxidase and choline acetyltransferase.

Increased intracellular phosphoribosylpyrophosphate and accelerated orotic acid decarboxylation in a mouse cell line resistant to purine and pyrimidine ribonucleosides.

A line of mouse fibroblasts (A9AU-1), originally selected for growth in the presence of 6-azauridine, has been found to be resistant to cytotoxic concentrations of adenosine, guanosine, and thymidine. A9AU-1 cells convert orotic acid to uridine 5'-monophosphate at twice the rate of the A9P line from which the A9AU-1 clone was selected. The resistant cells also excrete purines, synthesized de novo, into the medium at an increased velocity. The average intracellular 5-phosphoribosyl-1-pyrophosphate (PRPP) concentration of the resistant line is 45% higher than that of the parental line. The elevated PRPP concentration is likely to be responsible for both the apparent acceleration of pyrimidine synthesis and the increased excretion of purines into the growth medium; it might also account, by one of the several possible mechanisms, for the resistance of the cells to cytotoxic concentrations of the various nucleosides.

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-azauridine. These cells are not only resistant to 6-azauridine (5 X 10(-4) M), but also to adenosine (10(-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 de novo pyrimidine biosynthetic pathway.

Studies on the regulation of the three enzymes of the Leloir pathway in cultured mammalian cells. II. A search for quantitative interrelationships between the three enzyme activities.

Studies on a normal human diploid cell strain revealed that the specific activity of the cell protein, for each of the three enzymes of the Leloir pathway, changed significantly as the cells grew. The kinetics of change in specific activity varied according to the enzyme being studied, and the kinetics for each enzyme varied from experiment to experiment. Within each experiment, there was no consistent correlation between specific activity for any one enzyme and specific activity for the other two. The ratios between the specific activities did not tend to remain constant as the absolute levels of specific activity changed. Hence, the activities did not behave coordinately. The kinetics of change in these ratios varied from experiment to experiment. The failure of galactose to stimulate increased cellular activity for the three enzymes (shown in the preceding paper), and the absence of a coordinate relationship between the activities, represent a striking difference between the behavior of these enzymes in human diploid cell strains and their behavior in E. coli.

Studies on the regulation of the three enzymes of the Leloir pathway in cultured mammalian cells. I. Effect of substitution of galactose for glucose as the sole hexose in the medium in human diploid cell strains and in a rat hepatoma line.

In human diploid cell strains, the substitution of galactose for glucose as the sole hexose in the medium had no measurable effect on the specific activity of the cell protein for any of the three enzymes of the Leloir pathway. These enzymes are galactokinase, alpha-D-galactose-1-phosphate: UDP glucose uridyl transferase and UDP galactose 4-epimerase. A cell strain from a patient with galactosemia had no detectable activity for the transferase. The substitution of galactose for glucose in the medium of these cells (which has been shown to cause the cells to accumulate galactose-1-phosphate) also failed to affect cellular activity for the three enzymes. Similarly, the three activities failed to respond to the substitution of galactose for glucose in cultures of a rat hepatoma line. Cells of this line have been shown by others to perform a number of the tissue-specific functions of liver. The failure of galactose to stimulate increasd cellular activity for the three enzymes represents a striking difference between the behavior of these enzymes in human diploid cell strains and their behavior in E. coli.