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Increased Synthesis of Carbamoyl-Phosphate Synthase II (EC 6.3.5.5) in Hepatoma 3924A1

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Increased Synthesis of Carbamoyl-Phosphate Synthase II (EC 6.3.5.5) in Hepatoma 3924A1 [CANCER RESEARCH 46, 3673-3676, July 1986] Increased Synthesis of Carbamoyl-Phosphate Synthase II (EC 6.3.5.5) in Hepatoma 3924A1 Melissa A. Reardon2 and George Weber3 Laboratory for Experimental Oncology, Indiana University School of Medicine, Indianapolis, Indiana 46223 ABSTRACT labeling in vivo studies, the antiserum to synthase II was char acterized with regard to its specificity. The relative rates of Carbamoyl-phosphate synthase II (glutamine-hydrolyzing) (EC enzyme synthesis were then determined in rats carrying bilateral 6.3.5.5) (synthase II) is the first and rate-limiting enzyme in the de novo s.c. hepatomas. The animals were given injections of tritiated UTP biosynthetic pathway. Leucine pulse-labeling in the rat demon leucine, followed by synthase II immunoprecipitation. The ap strated that in the rapidly proliferating hepatonta 3924A the ratio of parent degradation rate of synthase II was measured using the radioactivity of synthase II to that of total cytosolic protein was 168.2 ± 11.0 (SE) x 1()"•'.Thissynthetic rate for the tumor enzyme was 9.7-fold double isotope method in which [14C]leucine was injected i.p. higher than that for the liver synthase II, 17.4 ±4.0 x 10"'. Since the and allowed to decay for a specified time followed by a second, degradation rate for hepatoma 3924A enzyme (t., = 65.5 h) was similar i.p. pulse of [3H]leucine. to the rate for liver synthase II (r,, = 69.3 h), the increase in tumor synthase II activity and amount was due primarily to an elevation in MATERIALS AND METHODS enzyme synthesis in the presence of an unaltered catabolic rate. The results indicate that the reprogramming of gene expression in the hepa Materials. Sodium [14C]bicarbonate and OCS cocktail were pur toma entails an increased production rate of the rate-limiting enzyme of chased from Amersham, Arlington Heights, IL, and Ready-to-Use UTP synthesis. This increase in the activity, concentration, and synthesis scintillation fluid III was from Eastman Kodak Co., Rochester, NY. L- of tumor synthase II should provide a heightened capacity for the de now [4,5-3H]leucine and L-[t/-l4C]leucine were obtained from ICN Radi- pyrimidine biosynthetic pathway, thus conferring a selective advantage ochemicals, Irvine, CA, and the Sepharose 6B was from Pharmacia to the cancer cells. Fine Chemicals, Uppsala, Sweden. Hydroxylapatite (Bio-Gel HTP) and reagents for protein assay and electrophoresis were from Bio-Rad Laboratories, Richmond, CA, and Protosol was from New England INTRODUCTION Nuclear, Boston, MA. All other reagents, also of the highest available purity, were from Sigma, St. Louis, MO. Carbamoyl-phosphate synthase II, the first and rate-limiting Biological Systems. Chemically induced, transplantable hepatoma enzyme in the de novo uridylate biosynthetic pathway, exists in 3924A was maintained as bilateral s.c. implants in male ACI/N rats of the cytosol as a multi-enzyme complex (M, 210,000) with 180-200 g of weight. Proliferation rate was measured in weeks required aspartate carbamoyltransferase (E.G. 2.1.3.2) and dihydro-or- between inoculation and growth of the tumor to reach a diameter of otase (EC 3.5.2.3), the second and third enzymes in this path 1.5 to 2 cm. White, male New Zealand rabbits of 3 to 4 kg were used way (1-3). Previous investigations in this laboratory showed in the production of antiserum. that synthase II4 activity increased in all rat hepatomas studied Synthase II Assay. Synthase II activity was assayed with potassium [14C]bicarbonate as substrate following the production of L-[carbamoyl- compared to normal liver, and the rise correlated with the 14C]citrulline in the presence of excess L-ornithine and ornithine car increase in tumor proliferative rates. In slowly growing hepa bamoyltransferase from Streptococcus faecalis (7). tomas, the increases in synthase II activity were 1.3- to 2.9- Protein Determination. The protein concentration was determined fold, in tumors of medium growth rate, 2.1- to 4.9-fold, and in directly from enzyme preparations by the method of Bradford (8), using rapidly growing hepatomas, 5.7- to 9.5-fold, higher than in the commercially available Bio-Rad reagent dye. Bovine serum albumin normal control liver (4, 5). Reardon and Weber (6) showed that was used routinely as the standard. in hepatomas of slow (20), intermediate (7787), and fast Synthase II Purification and Antiserum Production. Synthase II was (3924A) proliferative rates, synthase II activity increased 1.5-, purified to apparent homogeneity, and polyclonal antiserum was pro 2.3-, and 7.9-fold, and the amount of antiserum required to duced as described previously (6). neutralize the enzyme activity was 1.6-, 2.3-, and 8.2-fold higher In Vivo Determination of the Relative Rates of Synthase II Synthesis. The incorporation of L-[4,5-3H]leucine (specific activity, 58 Ci/mmol) than in control liver (6). Therefore, the increase in synthase II into synthase II was determined by pulse labeling and specific immu activity in the hepatomas was due to an elevation in the amount noprecipitation. Each hepatoma 3924A-bearing rat was given an injec of enzyme protein. To investigate the mechanism of this in tion i.p. of L-[4,5-3H]leucine (100 ¿iCi/100g) in a final volume of 0.4 creased tumor synthase II activity, the contributions of the ml physiological saline. Four h later the rats were killed, and the livers synthetic and degradative rates of hepatoma 3924A synthase II and tumors were removed immediately and homogenized in 4 volumes were determined and compared to those in control normal liver. of extraction buffer. After centrifugation at 105,000 x g at 4°Cfor 30 To apply the immunochemical techniques used in these pulse- min, l ml of the supernatant fluids was precipitated with excess anti body and incubated for 30 min at 37°C,followed by a 4-h incubation Received1/10/86;revised3/19/86;accepted3/26/86. on ice. Goat anti-rabbit IgG was added, and the incubation was contin Thecostsof publicationofthisarticleweredefrayedinpartbythe payment ued for another 4 h at 0°C.The immunoprecipitates were collected by of pagecharges.Thisarticlemustthereforebeherebymarkedadvertisementin centrifugation at 12,000 x g at 4°Cfor20 min and then washed 3 times accordancewith18U.S.C.Section1734solelytoindicatethisfact. 1Supported by USPHS Grant Numbers CA-13526 and CA-05034 awarded by by centrifugation at 7,000 x g at 4°Cfor20 min through a 1 M sucrose the National Cancer Institute, Department of Health and Human Services. cushion (2 ml). The pellet was then dissolved in 300 n\ Protosol and 1Some of the data in this paper are from a thesis to be submitted by M. A. incubated for 12 h at 37°C,after which 10 ml of OSC fluid was added, Reardon in partial fulfillment of the requirement for the degree of Doctor of Philosophy in the Biochemistry Department, Indiana University School of Med and the radioactivity was counted. icine, Indianapolis, IN 46223. To establish the specificity of the immunoprecipitates, both liver and 3To whom requests for reprints should be addressed, at the Laboratory for hepatoma 3H monolabeled antigen-antibody complexes were prepared Experimental Oncology, Indiana University School of Medicine, 702 Barnhill and electrophoresed on a sodium dodecyl sulfate 5% polyacrylamide Drive, Indianapolis, IN 46223. 4The abbreviations and trivial names used are: synthase II, carbamoyl-phos- gel (9). To locate the radioactivity, the gel was partially frozen and cut phate synthase II; OCS, organic counting scintillant. into 4-mm slices. Each gel slice was placed in a glass scintillation vial 3673 Downloaded from cancerres.aacrjournals.org on September 28, 2021. © 1986 American Association for Cancer Research. INCREASED SYNTHESIS OF HEPATOMA SYNTHASE II and incubated at 37°Cfor 12 h with 500 M'of Protosol. After the vials were cooled, 10 ml of OCS fluid were added, and the radioactivity was ABCD counted. A second procedure involved visualization of the immunopre- cipitates by staining corresponding lanes with Coomassie blue. Radioactivity incorporated into total cytosolic protein was measured from the 105,000 x g supernatant fraction using the method of Mans and Novelli (10) as modified by Dunaway and Weber (11). The relative rates of synthesis were expressed as the ratios of [3H]leucine incorpo rated into s\n tliase II relative to that incorporated into total cytosolic protein (12). This method allowed for possible variations in the amount of label injected and also for changes in the precursor leucine pool. In Vivo Determination of the Apparent Rates of Synthase II Degra dation. The apparent degradation rate for synthase II was determined by the double isotope method of Glass and Doyle (13). Each tumor- bearing rat was given an injection i.p. of L-[f/-MC]leucine (25 ^Ci/100 g) (specific activity, 300 mCi/mmol) in a final concentration of 0.4 ml physiological saline at time 0 (T = 0 h). A second i.p. injection of i • [4,5-3H]leucine (100 /¿Ci/100g) (specific activity, 60 Ci/mmol) was given at T = 0, 23, 47, and 71 h. The animals were killed l h after the ('H)leucine administration. Therefore, the rats were exposed to the I4C label for 1, 24, 48, and 72 h and to the tritium label for 1 h. The apparent degradation rates were calculated from the estimated 3H/I4C ratios measured from the synthase II immunoprecipitates, where the 'II counts represented the initial time point on the decay curve and the "( ' counts represented the amount of radioactivity remaining in the synthase II protein after the specified time intervals as described (13, 14). The incorporation of [l4C]leucine and [3H]leucine in the cytosolic protein was measured simultaneously.
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