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Behavior of Opposing Pathways of Thymidine Utilization in Differentiating, Regenerating, and Neoplastic Liver1

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Behavior of Opposing Pathways of Thymidine Utilization in Differentiating, Regenerating, and Neoplastic Liver1 CANCER RESEARCH 31, 550-556, May 1971) Behavior of Opposing Pathways of Thymidine Utilization in Differentiating, Regenerating, and Neoplastic Liver1 John A. Ferdinandus, Harold P. Morris, and George Weber2 Department of Pharmacology, Indiana University School of Medicine, Indianapolis, Indiana 46202 /J. A. F., G. W.¡,and Department of Biochemistry, Howard University College of Medicine, Washington, D. C. 2000 ÃŒ[H.P. M.¡ SUMMARY expression in cancer cells (20, 24). The identification of the pattern should provide the investigator with an understanding The behavior of opposing pathways of thymidine utilization of the progressive metabolic changes that relate to neoplastic was compared in proliferating normal liver (from developing proliferation rate. The Molecular Correlation Concept rats and from partially hepatectomized rats) and in neoplastic developed in this laboratory postulated the existence of a liver (spectrum of hepatomas of different growth rates). The meaningful biochemical pattern, which may be understood studies were carried out by assaying simultaneously the when a number of preconditions are satisfied (21). Among incorporation of labeled thymidine into DNA (synthetic these preconditions is the need to obtain an insight into the pathway) and the degradation to C02 (catabolic pathway) in regulation of opposing synthetic and catabolic pathways and tissue slices in an in vitro system. key enzymes and to elucidate their relationship with tumor In the liver of newborn rats, the incorporation of thymidine growth rate. For example, it was recognized that the activity into DNA is very high and gradually decreases during of the key glycolytic enzymes increased and concurrently the differentiation to very low levels in the adult. The catabolic activity of the key gluconeogenic enzymes decreased in utilization of thymidine is high in the newborn rat liver, and it hepatomas parallel with the increase in the hepatoma growth further increases during differentiation, being approximately rate (20-25). twice as high in the adult as in the newborn. The rise in the This paper describes the behavior of the opposing pathways activity of the catabolic pathway is blocked by administration of synthetic utilization and catabolism of thymidine in of actinomycin. differentiating, regenerating, and neoplastic liver. The results Partial hepatectomy results in marked changes in the demonstrate that the incorporation of thymidine into DNA behavior of thymidine metabolism. The activity of the increases and concurrently the degradation to CO2 decreases synthetic pathway increases, while the activity of the catabolic parallel with hepatoma growth rate. The ratio of the activities pathway decreases. The increased activity of the synthetic of the synthetic:catabolic pathways correlates closely with pathway decreases to normal range and the decreased activity hepatoma growth rate. These observations are further evidence of the catabolic pathway returns to high levels about 96 hr in support of the Molecular Correlation Concept. after the operation. In the hepatoma spectrum, the activity of the synthetic pathway increases and that of the catabolic pathway decreases parallel with the increase in tumor growth rate. As a result, MATERIALS AND METHODS there is a pronounced increase in the ratio of synthetic: catabolic utilization of thymidine, which shows a close The animals were kept in separate cages and illuminated correlation with hepatoma growth rate. The close linking of daily from 6 a.m. to 7 p.m. Purina laboratory chow and water hepatoma growth rate with the behavior of the activities of were available ad libitum to the experimental animals. opposing pathways of thymidine utilization and of the key Studies on Regenerating Liver. For studies on regenerating enzymes of the pathways provides further evidence in support purchased from Harían Industries, Cumberland, Ind. The of the Molecular Correlation Concept. litters were allowed to stay in the same cage with the mother for 18 days after birth; then they were placed in individual cages. INTRODUCTION Studies on Regenerating Liver. For studies on regenerating Investigations in this laboratory have been directed to liver, male, albino Wistar rats weighing 180 to 200 g were uncovering the underlying pattern in the alteration of gene obtained from Harían Industries. The rats were partially hepatectomized under light ether anesthesia by removal of 1Supported by USPHS Grant CA-05034, an American Cancer Society 66% of the liver (3). The remaining liver lobes were examined Grant, a Damon Runyon Memorial Fund, Inc., grant (G. W.), and at different time intervals after operation. Sham-operated USPHS Grant CA-10729 (H. P. M.). 2To whom requests for reprints should be sent, at the Department of animals were used as controls. Tumor-bearing and Control Animals. Male Buffalo and Pharmacology, Indiana University School of Medicine, Indianapolis, ACI/N rats were used in these experiments. The tumor-bearing Ind. 46202. Received September 2, 1970; accepted January 4, 1971. and control rats were shipped by air express from Dr. H. P. 550 CANCER RESEARCH VOL.31 Downloaded from cancerres.aacrjournals.org on September 27, 2021. © 1971 American Association for Cancer Research. Opposing Pathways of Thymidine Metabolism Morris, Washington, D. C., to our laboratories at Indiana Experiments with Actinomycin. For these studies, University School of Medicine, Indianapolis, Ind. Normal rats actinomycin D was purchased as dactinomycin in vials of the same strain, sex, age, and weight were used for controls containing 500 ^g (Merck Sharp and Dohme, West Point, Pa.). and were sacrificed along with the tumor-bearing rats under Further conditions are described in "Results and Discussion" the same conditions. We used a number of tumor lines but and in Table 3, where the data obtained with this drug are concentrated on hepatomas, which included the slow-growing presented. 9618-A and 9618-B; 7800 and 5123-D, which now are Expression and Evaluation of Results. The incorporation of considered of intermediate growth rate; and rapidly growing thymidine into DNA and the degradation of thymidine to CO2 3924-A, 7288-C, 7777, 3683-F, and 9618-A2. The tumors were expressed as dpm/g of tissue/hr. The counts obtained for were transplanted bilaterally in a s.c. position, and they were incorporation of thymidine into DNA were corrected for allowed to grow to a diameter of about 1 inch before they self-absorption by extrapolating cpm/g of tissue to infinite were harvested. The biological and growth properties of the dilution (13). hepatoma spectrum were previously described (9). The results were subjected to statistical evaluation by means Experimental Procedures. The rats were stunned, of the t test for small samples. Differences between means decapitated, and exsanguinated. Livers and tumors were giving a probability of less than 5% were considered to be rapidly removed and placed in beakers, which stood on significant. crushed ice. Tissues were carefully dissected free of necrotic, hemorrhagic, and nontumorous material. For routine studies on thymidine metabolism, the tissues RESULTS AND DISCUSSION were sectioned into 1- to 2-cm cubes, and slices were cut to a uniform thickness of about 1 to 2 mm by a Stadie-Riggs slicer. Behavior of the Activity of Opposing Pathways of Synthetic Tissue slices, 50 mg, were placed in separate 25-ml Erlenmeyer and Degradative Utilization of Thymidine in Differentiation. flasks, each containing 5 ml of Krebs-Ringer phosphate buffer For an understanding of the pattern of thymidine metabolism (pH 7.4), 12.5 mM glucose, 20 mM glycylglycine, and 0.5 ¡id during development, the behavior of the activity of the of thymidine-2-14C (specific activity, 43.7 mCi/mmole; New synthetic and degradative pathways was examined in the liver England Nuclear, Boston, Mass.). Each flask was capped with a of rats of different age groups from the early postnatal time to rubber stopper (No. 8826 rubber stoppers, 13 mm; Arthur H. adulthood. The results in Table I indicate that the Thomas Co., Philadelphia, Pa.) from which was suspended, by incorporation of thymidine into DNA is high in the liver of a straight pin, a glass fiber filter strip (934AH glass fiber filter. newborn rats, and during subsequent development the activity 2.4 cm; H. Reeve Angel and Co., Inc., Clifton, N. J.) cut to 1 x of the thymidine biosynthetic pathway decreases to very low 2.4 cm. levels in the liver of the adult. The incorporation of thymidine Flasks were incubated at 37°and shaken at 72 rpm in a into DNA is 30 times higher in the 1-day-old rat than in the Dubnoff metabolic shaker. The reaction was terminated after adult. incubations of 0, 30, and 60 min by injections of 50 jul of The behavior of the activity of the degradative pathway Hyamine hydroxide (hydroxide of Hyamine 10-X, Packard contrasts with that of the synthetic one. The catabolism of Instrument Co., Downers Grove, 111.) through the rubber thymidine to C02 is active in the newborn rat liver, and in stopper onto the glass fiber filter strip and then injection of 1 subsequent development the activity increases to the high ml of 50% TCA3 into the bottom of the flask. The flasks were levels observed in adult liver (Table 1). As a consequence of shaken an additional 30 min at 37°,after which time the the opposing behavior of the activity of the synthetic and rubber stoppers were removed and the filter strips were placed catabolic pathways, the ratios of the activity of the opposing in scintillation vials. Immediately, 10 ml of scintillator fluid pathways are of interest. The ratios are near 1 in the newborn (0.03% POPOP and 0.5% PPO in toluene from New England rat liver, and subsequently they gradually decrease, reaching a Nuclear) were added to each vial. The vials were counted for very small fraction in the adult liver. Thus, in the adult liver radioactive C02 on a Packard Tri-Carb liquid scintillation the activity of the degradative pathway is overwhelmingly spectrometer (Model 314 EX).
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