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Furine and Pyrimidine Enzymic Programs and Nucleotide Pattern in Sarcoma1

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Furine and Pyrimidine Enzymic Programs and Nucleotide Pattern in Sarcoma1 [CANCER RESEARCH 43. 1019-1023, March 1983] 0008-5472/83/0043-0000$02.00 Furine and Pyrimidine Enzymic Programs and Nucleotide Pattern in Sarcoma1 George Weber,2 Michael E. Burt, Robert C. Jackson, Noemi Prajda, May S. Lui, and Eiji Takeda Laboratory for Experimental Oncology, Indiana University School of Medicine, Indianapolis, Indiana 46223 [G. W., R. C. J., N. P., M. S. L., E. T.], and the Surgery Branch, National Cancer Institute, NIH, Bethesda, Maryland 20205 [M. E. B.] ABSTRACT 4.5- to 12-fold. The cytidine triphosphate concentration in creased 17-fold. In the muscle, the concentrations of cytidine The purpose of this study was to elucidate the enzymic diphosphate, inosine monophosphate, and xanthosine mono- program and nucleotide pattern of a chemically induced, trans- phosphate were too low for the sensitivity of our method to plantable sarcoma and to compare the biochemical makeup detect, but these pools were well measurable in the sarcoma. with that of normal and differentiating skeletal muscle in the The enzymic program of sarcoma was distinguished from rat. that of the muscle of 6-day-old rats by the quantitatively more The activities of 28 key enzymes of pyrimidine, purine, and pronounced alterations in the sarcoma. The activities of uridine carbohydrate metabolism were determined in the 100,000 x phosphorylase, hexokinase, 6-phosphogluconate dehydroge- g supernatant fluid or in purified extracts. The concentrations nase, and adenosine deaminase were higher in sarcoma but of the adenosine, guanosine, uridine, and cytidine mono-, di-, were lower or not significantly altered in 6-day-old muscle as and triphosphates were measured by high-pressure liquid chro- compared to activities of adult muscle. The activities of aden matography of samples prepared by the freeze-clamp method. osine monophosphate deaminase and xanthine oxidase de The results of enzymic and metabolite assays were given in creased in sarcoma, whereas they were unchanged or in nmol/hr/mg protein and nmol/g, respectively, and for com creased, respectively, in 6-day-old muscle. parability were also expressed as percentages of the muscle In the sarcoma, important segments of alterations in enzy- of adult rat as the reference normal tissue in this study. These mology of carbohydrate, purine, and pyrimidine metabolism percentages denote that the activities in sarcoma or in differ were identified that proved to be a program shared with that entiating muscle were higher or lower than those in the muscle observed in chemically induced and virus-derived transplanta- of adult rats. ble rat and avian hepatocellular carcinomas and in primary In pyrimidine metabolism, the specific activities of cytidine human liver, kidney, and colon neoplasms. There were also diphosphate reducíase, cytidine triphosphate synthetase, and sarcoma-specific markers in the enzymic programs and nu thymidine kinase increased in the sarcoma 60-, 78-, and 80- cleotide patterns that readily distinguish this tumor from other fold over those of the muscle. The activities of the key glycolytic examined neoplasms. enzymes, hexokinase and phosphofructokinase, increased 7- The present investigation revealed a formidable biochemical and 3-fold, whereas that of pyruvate kinase decreased to 35%. capacity for replication in the sarcoma cells. These biochemical The activities of glucose-6-phosphatase and fructose-1,6-di- studies also point out possible targets in the strategy of anti- phosphatase declined to 42 and 48%, respectively. The activ cancer drug treatment of sarcoma. ities of enzymes involved in pentose phosphate production and utilization increased, with that of the glucose-6-phosphate de- INTRODUCTION hydrogenase being elevated 288-fold. The activity of galacto- kinase was unchanged, whereas that of uridine diphosphoglu- Previous work in this laboratory demonstrated, in a spectrum cose pyrophosphorylase decreased to 22%. In purine metab of chemically induced, transplantable hepatocellular carcino olism, the activities of the first three enzymes of guanosine mas of the liver, a transformation- and progression-linked im triphosphate biosynthesis, inosine monophosphate dehydro- balance in the activities of key enzymes of carbohydrate, purine, and pyrimidine metabolism and in the pattern of ribo- genase, guanosine monophosphate synthetase, and guanosine monophosphate kinase, increased 22-, 2-, and 5-fold, respec nucleotides and deoxyribonucleoside triphosphates (3, 9, 10). tively. In contrast, the activities of adenylosuccinate lyase and Subsequent studies indicated the presence of the metabolic adenosine monophosphate deaminase decreased to 28 and imbalance in a series of carcinomas of kidney and colon in the 42%. The activities of adenosine deaminase and kinase in rat and mouse and, recently, in human renal and colon carci creased 1.8- and 3.5-fold. The activity of the rate-limiting nomas (1,2,12). The purpose of the present investigation was enzyme of de novo inosine monophosphate biosynthesis, ami- to answer the following questions. Is the biochemical imbalance dotransferase, increased 13-fold, whereas that of the rate- discovered in carcinomas applicable to sarcoma? Are the limiting purine-catabolic enzyme, xanthine oxidase, decreased enzymic indications of an imbalance in the cellular metabolic to 54%. programs verifiable by the pattern of nucleotides in sarcoma? In the sarcoma, the concentrations of adenosine tri-, di-, and Is the biochemical pattern of the sarcoma different from that of monophosphate decreased to 24, 49, and 76%. In contrast, differentiating muscle? Is it possible to identify a shared pattern the pools of guanosine tri-, di-, and monophosphate increased of biochemical imbalance in carcinomas and sarcoma? Are 9- to 11-fold, and those of the uridylates were also elevated there any sarcoma-specific biochemical alterations? 1This investigation was supported by USPHS Grants CA-13526, CA-05034. MATERIALS AND METHODS and CA-10792. 2 To whom requests for reprints should be addressed. For these studies, a methylcholanthrene-induced sarcoma line car Received July 22, 1982; accepted December 3, 1982. ried in male Fischer (F344) rats (Charles River Laboratories, Portage, MARCH 1983 1019 Downloaded from cancerres.aacrjournals.org on September 27, 2021. © 1983 American Association for Cancer Research. G. Weber et al. Mich.) and skeletal muscles from control, normal rats of the same sex, In the sarcoma, the activities of all synthetic enzymes ex strain, age, and weight were used. The tumor was a rhabdomyosar- amined were very markedly increased as compared to muscle, coma; the induction, maintenance, histology, and biological behavior ranging from a rise of 78- to 80-fold for thymidine kinase and are described in detail elsewhere.3 This is a fairly rapidly growing CTP synthetase to increases of 26- and 14-fold for uridine neoplasm which resembles the macroscopic appearance and growth kinase and uracil phosphoribosyltransferase. These elevations rate of hepatoma 3924A and reaches a diameter of 1.5 cm in about 14 were much more marked (about 10-fold higher) than were days, with death occurring 30 to 34 days after inoculation. The 12- to 18-day-old tumor has no necrosis, and it is well suited for the metabolic those observed in the rapidly growing liver tumors compared to the resting liver (16). studies that were conducted. The animals were inoculated s.c. by injection of 106 viable tumor In carbohydrate metabolism in the muscle, the activities of cells, and 1 week later the tumor-bearing and control rats were shipped the key glycolytic enzymes, hexokinase, phosphofructokinase, by air from the National Cancer Institute to Indiana University. All rats and pyruvate kinase, were higher than those in rat liver (11). were housed in individual cages in air-conditioned rooms, which were This is expected in view of the well-known glycolytic capacity illuminated daily from 6 a.m. to 7 p.m. Purina laboratory chow and of skeletal muscle. In the sarcoma, the activities of phospho water were available ad libitum. Rats were always killed between 9 and fructokinase and hexokinase increased 3- to 7-fold, which is in 10 a.m. line with observations made in hepatic carcinomas of similar Biochemical Methods. Animals were stunned, decapitated, and rapid growth rates (11 ). In contrast to results in hepatomas, bled; tumors were excised, and 10% homogenates were prepared in the appropriate media as reported earlier (1, 2, 13). For the freeze- the activity of pyruvate kinase, which is orders of magnitude higher than any other enzyme that we have studied, was clamp studies, the animals received light ether anesthesia, and tumors decreased to one-third of that observed in the control muscle. and muscles were removed as described elsewhere (1 7). Preparation of the extracts and methods for measurement of en In muscle, the gluconeogenic enzymes, glucose-6-phospha- zymes were described in detail elsewhere (1, 2, 5, 13). Preliminary tase and fructose-1,6-diphosphatase, were present only in low kinetic studies were carried out to ensure that the enzyme assays were activities, which were further decreased to 42 to 48% in the conducted under linear kinetic conditions, at optimum substrate and sarcoma. These results are in line with the decline in activities activator concentrations, and that the activities measured were pro of gluconeogenic enzymes first reported in hepatomas (11 ). portionate with the amount of enzyme added and reaction time elapsed. In pentose phosphate metabolism in the muscle, the activity
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