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Purine and Pyrimidine Nucleotide Patterns of Normal, Differentiating, and Regenerating Liver and of Hepatomas in Rats

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Purine and Pyrimidine Nucleotide Patterns of Normal, Differentiating, and Regenerating Liver and of Hepatomas in Rats [CANCER RESEARCH 40, 1286-i 291 . April 1980] 0008-5472 /80/0040-0000$02.00 Purine and Pyrimidine Nucleotide Patterns of Normal, Differentiating, and Regenerating Liver and of Hepatomas in Rats Robert C. Jackson,1 May S. Lui, Theodore J. Boritzki, Harold P. Morris,2 and George Weber3 Laboratory for Experimental Oncology, Indiana University School of Medicine, Indianapolis, Indiana 46223 (R. C. J.. M. S. L., T. J. B., G. W.J,and the Department of Biochemistry, Howard University Medical College. Washington, 0. C. 20001 (H. P. M.J ABSTRACT nucleotide pattern are linked with transformation on pnogres sion in hepatomas of different growth mates.The pattern of The pattern of punine and pynimidine nucleotides was eluci deoxynucleoside tniphosphate pools in rapidly growing differ dated in freeze-clamped preparations of normal liven; in hepa entiating and regenerating liver and in hepatoma 3924A shows tomas of very slow (961 8A), intermediate (8999), and rapid that the concentrations of these key precursors of DNA biosyn (3924A) growth rates; in regenerating liven; and in liven of thesis are particularly increased in the tumor, and this may neonatal rats. The nibonucleotide content was measured by confer selective advantages on the neoplastic cells. high-pressure liquid chromatography, and that of the deoxyni bonucleoside tniphosphates was determined by enzymic as INTRODUCTION says. In the liver, the contents of deoxythymidine tniphosphate, In neoplastic transformation and progression, an integrated deoxycytidine tniphosphate, deoxyadenosine tniphosphate, and program of increases in enzyme activities has been observed deoxyguanosine tniphosphate were very low (1 to 5.1 nmol/g (21 , 22). Particularly striking elevations were found in the wet weight). The concentrations of deoxynibonucleoside tn activities of nibonucleotide reductase (3), IMP dehydnogenase phosphates were increased relative to liver in all hepatomas, (1 1), CTP synthetase (26), and thymidine kinase (21 , 22). and the rise correlated with the tumor growth rates. Adenosine However, not all the enzymes of nucleotide biosynthesis have tniphosphate (ATP) concentration and total adenine nucleotide increased activity after transformation; adenylate kinase, for amounts gave a negative correlation with tumor growth rate. example, is decreased (2). The regulation of nucleotide biosyn The ATP level in hepatoma 3924A was decreased to 43% of thesis is so complex that it cannot be expected that a simple the normal livenconcentration. By contrast, contents of cytidine correlation should exist between enzyme activities and the pool tniphosphate and of total cytosine nucleotides were increased levels of the products of the enzymes. Because of the many in all 3 liver tumors. The xanthosine monophosphate content of feedback-regulatory effects, an understanding of nucleotide hepatoma 3924A was 233% of that of normal liver and gua metabolism requires data concerning cellular nucleotide con nosine monophosphate concentration in all three hepatomas centrations under various metabolic conditions. The most ef was above the liven values. There were no other consistent fective use of antimetabolite drugs will likewise require infor changes in the guanine and unacil nucleotide concentrations in mation concerning concentrations of competing substrates and the hepatomas. feedback effectors. A preliminary report from this laboratory In the 24-hr regenerating liver, the cytidine tniphosphate and (8) compared nibonucleotide contents of normal rat liven and xanthosine monophosphate levels were higher than normal the rapidly growing hepatoma 3924A. The present study has liven values. Unlike the liven tumors, the regenerating liver had extended this work to hepatomas of slow and medium growth increased unidine nucleotides and normal ATP concentration. rate and to the nonmalignant proliferating hepatic cell popula In the liver of 6-day-old rats, with the exception of a 27% tions of regenerating and neonatal liven.The pattern of deoxy increase in cytidine tniphosphate level, the nibonucleotide con nibonucleotides has also been elucidated. Punine and pynimi tents did not differ from those of adult rat liver. dine nucleotides have numerous metabolic roles apart from In normal rat liver, a period of ischemia of up to 10 mm their function as substrates for nucleic acid biosynthesis. In resulted in extensive dephosphorylation of ATP and other particular, the adenine nucleotides act as the ubiquitous donors nucleoside tniphosphates, with the degraded material accu and acceptors of phosphate groups in energy metabolism. mulating as monophosphates and nucleosides. However, hep Malignant tissues have long been known to have abnormal atoma 3924A (and the other hepatomas to a much lesser energy metabolism, characterized by decreased respiration extent) possessed the ability to maintain ATP levels near normal and increased dependence upon glycolysis (25). In the present values for 10 mm of ischemia. This capability was lost following study, we have used the ischemic condition to examine the iodoacetate treatment, suggesting that the maintenance of a relationship between the altered energy metabolism of tumors normal ATP level in hepatoma 3924A during ischemia was and the behavior of the nucleotide pools. associated with the high activity of anaerobic glycolysis in this tumor. MATERIALS AND METHODS The results indicate that aspects of the pynimidineand pumine Punine and pynimidine nucleotides, calf thymus DNA, and ‘Supported by Grant CA-i 81 29 from the National Cancer Institute, NIH. poly(deoxyadenylate-deoxythymidylate) were purchased from 2 Supported by Grant CA-i 0729 from the National Cancer Institute, NIH. Sigma Chemical Co., St. Louis, Mo. Tnitium-labeled dATP and 3 Supported by Grants CA-13526 and CA-05034 from the National Cancer Institute, NIH. To whom requests for reprints should be addressed. dGTP were obtained from the Radiochemical Centre, Amen Received October 8, 1979: accepted January 10, 1980. sham, England and DNA polymenase(Micrococcus luteus) from 1286 CANCERRESEARCHVOL. 40 Downloaded from cancerres.aacrjournals.org on September 26, 2021. © 1980 American Association for Cancer Research. Nucleotide Patterns in Hepatomas Miles Laboratories, Elkhart, Ind. High-pressure liquid chroma detectors at 254 and 280 nm and electronically integrated tognaphy columns were purchased from Whatman Inc., Clifton, using a Vanian Model CDS-111C integrator which had been N. J. Reagent chemicals were from Fisher Scientific Co., Fain calibrated against known quantities of standard nucleotides. Lawn, N. J., or from J. T. Baker Chemical Co., Phillipsburg, N. J., analytical grades being used where available. RESULTS Animals and Tumors. Hepatoma 3924A was maintained as a bilateral s.c. transplant in male ACI/N rats and was trans Ribonucleotides in Hepatomas. In these experiments, we planted at 1-month intervals. Hepatomas 96i 8A and 8999 used the freeze-clamp procedure to prepare nucleotide pool weresimilarlymaintainedinmaleBuffalorats;hepatoma96i 8A extracts from the livers of Buffalo and ACI/N rats, and from 3 was transplantedat 10-month intervals,and hepatoma8999 transplanted hepatoma lines: 96i 8A, a slowly growing tumor wastransplantedat 3.5-monthintervals.Forregeneratingliven (transplant interval, 10.5 months) grown in Buffalo rats; 8999, experiments, 200-g male ACI/N rats were used, and partial a hepatoma of medium growth rate (transplant interval, 3.5 hepatectomy was performed by the standard procedure (6). months), also grown in Buffalo rats; and 3924A, a rapidly For experiments with neonatal liven, Wistar matswere used. growing hepatoma (transplant interval, 1 month).maintained in Animals were kept in separate cages with water and Purina ACI/N rats. Ribonucleotide contents of these tissues are shown Laboratory chow available ad libitum. The animals were kept in in Table 1. Certain systematic trends are apparent in these a 12-hr light-i 2-hr dark cycle, and all experimental samples data. The concentrations of ATP, and of total adenine nucleo weretaken between9:30 and 10:30 a.m. tides, were significantly lower in all the tumors than in the Tissue Extraction. Rats were lightly anesthetized with ether; normal liver samples. Moreover, a negative correlation with livers and hepatomas were exposed and freeze-clamped tumor growth matewasapparent. The ATP content of hepatoma (within 2 sec of severing the blood vessles) between liquid 961 8A was 73% control, decreasing to 57% in 8999, and 43% nitrogen-cooled tongs (24, 27, 28). The frozen tissue was in 3924A. The contents of GTP and GDP were not significantly pulverized in a liquid nitrogen-cooled mortar, and 0.5 to 2.0 g different in the hepatomas from the control liven values, but of powder were extracted in aqueous perchlonic acid (0.7 M) increased levels of GMP and its immediate precursor, XMP, or in 60% aqueous methanol for nucleotide assays. were found in all 3 tumors. These increases, however, did not Deoxyribonucleoside Triphosphate Assay. Freeze appear to correlate with hepatoma growth rate. Unidinenucleo clamped powders were extracted in 60% aqueous methanol, tides were present in the hepatomas in about the same con and extracts were worked up by the procedure of Tynsted(20), centrations as in normal rat liven. CTP, however, showed sig as modified by Hamnapand Paine (5). dCTP and dGTP were nificantly elevated concentration in all 3 tumor lines, and the assayed using the enzymatic method of Solten and Handschu CTP content gave a positive correlation with hepatoma
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