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Increased Cytidine 5'-Triphosphate Synthetase Activity in Rat and Human Tumors¹

Laboratory for Experimental Oncology, Indiana University School of Medicine, Indianapolis, Indiana 46223 [H. K., J. C. W., G. W.], and the Department of Biochemistry, Howard University School of Medicine, Washington, D. C. 20001 [H. P. M.]

The behavior of the activity of the rate-limiting enzyme of cytidine 5'-triphosphate (CTP) biosynthesis, CTP synthetase (EC 6.3.4.2), was elucidated in the rat in normal liver, in 14 hepatomas of vastly different growth rates, and in rapidly growing differentiating and regenerating liver. CTP synthetase activity was also determined in kidney cortex and kidney tumors in rats and in kidney and renal cell carcinomas in humans.

CTP synthetase activity was measured by an isotope method in the supernatant fluid prepared by centrifugation of 15% homogenates at 100,000 x g for 60 min. On storage at 4° and -20°, the enzyme activity in the hepatomas was less stable than in the liver. The affinity of CTP synthetase for uridine 5'-triphosphate, adenosine 5'-triphosphate, guanosine 5'-triphosphate, and glutamine in the crude extracts of normal liver and rapidly growing hepatoma 3924A yielded apparent K_m's of 0.6, 0.7, 0.25, and 0.1 mM, respectively. The liver and hepatoma 3924A enzymes were saturated at uridine 5'-triphosphate, adenosine 5'-triphosphate, guanosine 5'-triphosphate, and glutamine concentrations of 2, 3, 1, and 1.6 mM, respectively. The pH profiles of the liver and the hepatoma enzymes yielded an optimum at pH 7.4. A standard assay was developed in which CTP synthetase activity was linear during 60-min incubation and was proportionate with amounts of protein added over a range of 0.2 to 3 mg.

Organ distribution studies in the rat showed that CTP synthetase specific activity was over 4-fold higher in thymus and testis than in liver. Activities in spleen, adipose tissue, bone marrow, lung, brain, renal cortex, heart, and skeletal muscle were 207, 166, 117, 83, 83, 71, 29, and 29%, respectively, of that of the liver. CTP synthetase activity was present only in traces in the superficial and deep layers of the mucosa of the small intestine.

In starvation, liver CTP synthetase activity was preferentially depleted, and, on refeeding, the activity was restored more slowly than the protein content.

In hepatomas of slow and medium growth rates, CTP synthetase activity increased to 1.6- to 4.6-fold, and in the rapidly growing tumors the activity was elevated to 5- to 10-fold the values of normal rat liver. There was a significant correlation between the increase in synthetase activity and the growth rate of the hepatomas. In transplantable rat renal cell carcinomas, CTP synthetase activity was increased to 4.3- to 5.2-fold that of normal kidney cortex. In five cases of human renal cell carcinomas, CTP synthetase specific activity was increased to 1.6- to 3.8-fold the activity in the histologically normal cortex of kidneys from the same patients.

In tissues of similar growth rates, the increase in the CTP synthetase activity was much higher in hepatomas (10-fold) than in regenerating liver (2.4-fold), and the activity was low in the differentiating liver.

The increased activity of CTP synthetase in tumors appears to be characteristic of neoplasms, and in the hepatoma spectrum it was linked with transformation and progression. The increased capacity of CTP synthetase should confer selective advantages to proliferating cells and marks out this enzyme as an important target in the design of chemotherapy.

¹ This investigation was supported by USPHS Grants CA-13526, CA-05034, and CA-10792.

² To whom requests for reprints should be addressed.

Received 1/21/80. Accepted 7/24/80.

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