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

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[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 Â± 11.0 (SE) x 1()"â€¢'.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 Â±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 Â¿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 4Â°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 37Â°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 0Â°C.The immunoprecipitates were collected by of pagecharges.Thisarticlemustthereforebeherebymarkedadvertisementin centrifugation at 12,000 x g at 4Â°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 4Â°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 37Â°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 37Â°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 immunoprecipitates 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 â€¢ [4,5-3H]leucine (100 /Â¿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. As proposed by Block et al. (15), a control was included in which the rats were given both radioactive Fig. 1. Coomassie blue stained immunoprecipitates of hepatoma 3924A syn labels at the same time and sacrificed 1 h later. The 1H/'4C ratio thase II. Lane A, partially purified anti-synthase II immunoglobin plus second obtained provided a correction factor for the isotopie ratio expected antibody, anti-rabbit IgG (32 *tg); Lane B, hepatoma 3924A synthase II immu- noprecipitate (34 /jg); Lane C, homogeneous hepatoma 3924A synthase II (17 without significant degradation. dg): Lane D, molecular weight standards (29 jig): myosin (M, 200,000), ÃŸ- galactosidase (M, 116,250). phosphorylase B (M, 92,500), bovine serum albumin (M, 66,200), and ovalbumin (M, 45,000). RESULTS The steady state activity of synthase II in hepatoma 3924A Synthetase 11 Immunoprecipitates was 8-fold higher than that of normal liver. Immunotitration studies by Reardon and Weber (6) had demonstrated previously that synthase II protein amount was 8.2-fold greater in the rapidly proliferating hepatoma 3924A than in normal liver. To investigate the mechanism(s) responsible for the increased syn thase II activity and amount in hepatoma 3924A, the in vivo rates of synthesis and degradation were determined. Antiserum Characterization. To properly apply the Â¡mimmo- chemical techniques required for this study, it was first neces sary to establish the antiserum specificity for synthase II from both liver and hepatoma 3924A. Analysis of the antiserum by immunodiffusion yielded a single precipitili line of identity, indicating that the antiserum produced against purified hepa toma 3924A antigen recognized and reacted with synthase II Gel Slice Number (4 mm) Fig. 2. Gel slice profile of tritiated liver and hepatoma 3924A synthase II from both liver and hepatoma (6). Denaturing polyacrylamide immunoprecipitates. Synthase II was quantitatively immunoprecipitated from gel electrophoresis of synthase II from liver or hepatoma 3924A liver (O) and hepatoma 3924A (â€¢)ofrats given injections of 100 >iCi[3H]leucine. tissue labeled previously with [3H]leucine, followed by quanti Sodium dodecyl sulfate polyacryÃamideelectrophoresis of the labeled immuno precipitates was conducted, and the gel lanes were cut into 4-mm slices and tative immunoprecipitation, showed three major protein bands counted for radioactivity. corresponding to synthase II (Mr 210,000), light (M, 25,000), and heavy (A/r 55,000 and 62,000) rabbit and goat immunoglo- labeled [3H]leucine into the enzyme precipitated by specific bin chains (Fig. 1). By cutting a corresponding gel lane into 4 antibody. Synthetic rates were expressed by the ratio of the mm slices and counting for radioactivity, virtually all of the enzyme to total soluble protein radioactivity per g of tissue. leucine label was located in a single peak, which coincided with These incorporation studies revealed that the isotopie leucine the synthase II protein visualized by Coomassie blue staining. ratio for liver synthase II was 17.4 Â±4.0 (SE) x 10~3, and the By this criterion, the polyclonal synthase II antiserum was ratio for the hepatoma 3924A enzyme was 168.2 Â±11.0 x 10~3. considered highly specific for both liver and hepatoma 3924A This synthetic rate for tumor synthase II was 9.7 times greater synthase II antigen and thus appropriate for these in vivo studies than that obtained for the liver enzyme (Table 1). (Fig. 2). In Vivo Degradation Rate. The apparent degradation rates of In Vivo Synthetic Rate. The relative rates of synthesis of liver synthase II for both liver and hepatoma 3924A were determined and hepatoma 3924A synthase II were determined from tumor- using the double isotope method in which [14C]leucine was bearing ACI/N rats by measuring the incorporation of pulse- injected i.p. and allowed to decay for a specified time, followed 3674

Table 1 In vivodeterminationof the rates of synthesisand degradationfor rat hostliverand hepatoma3924A II activity incorporation ratio rateK,(h-')0.0100(100) (nmol/h/mg (synthase Il/per cyto- solicIO"3)17.4 protein x TissueHost protein)9.2Â±0.1*(100)c (/<')"30.0 liver Â±0.4 (100) Â±4.0(100) (100)

Hepatoma 3924ASynthase 72.9 Â±3(792)''Antiserum 246 Â±5(820)''"H 168.2 Â±11.0(967)''Degradation 0.0106(106)Mh)69.3 65.5 (95) " From Ref. 6. * Mean Â±SE of 4 determinations for each tissue. c Numbers in parentheses, percentage of host liver. J Significantly different from values of host liver (P < 0.05). by a second, pulse i.p. administration of [3H]leucine (13), as arginine, determined that the half-life of liver cytosolic protein described in "Materials and Methods." Simultaneously, the was 122.4 h. incorporation of the two isotopie forms of leucine into total cytosolic protein was measured. The apparent degradation rate, DISCUSSION K,,, was determined from the slope of a normalized linear least squares semilogarithmic plot of the 3H/14C ratios obtained from the immunoprecipitates versus time of decay (7*= 0, 24, 48, 72 It was proposed that an ordered, integrated imbalance in the metabolism and enzymology of tumor cells was due to a repro- h). The apparent Kamino acid cannot be readily reutilized for protein syn and this rise correlated positively with tumor proliferative rates thesis because the guanidino carbon is hydrolyzed by arginase (4). The molecular correlation concept classified the behavior to urea. These investigations, using the labeled guanidino-L- of synthase II activity as transformation- and progression- linked (4, 5, 16). Imnui noi hral ion experiments conducted by Reardon and Weber (6) revealed that the elevations of synthase II in a spectrum of hepatomas of slow (20), intermediate (7787), and rapid (3924A) growth rates were due to corresponding elevations in the concentration of immunotitratable enzyme protein amounts. These neutralization studies also indicated 0.8- that the immunological properties of synthase II were similar in liver and hepatoma 3924A and that the neoplastic transfor

0. 7 - mation in the tumor did not cause major structural alterations in this enzyme that would have interfered with antibody-antigen recognition and/or interaction (6).

0.6- The results demonstrated that the synthetic rate for hepatoma synthase II was 9.7-fold higher compared to host liver. Since the degradation rate for liver synthase II (tVl= 69.3 h) was similar to the rate for hepatoma 3924A enzyme (t>,,= 65.5 h), o 0.5- the increase in the hepatoma synthase II activity and amount te.Â« was probably due primarily to an increase in enzyme synthesis in the presence of an unaltered rate of catabolism. This is in accord with the enzyme assay and immunotitration studies 0.14- which had shown previously that synthase II activity and o.o- amount in the hepatoma 3924A were 8- to 10-fold higher than 24 Â«8 72 in liver. Novel aspects of this study include the following: (a) Time (h) use of specific antiserum for immunoprecipitation of the rate- Fig. 3. Determination of the apparent degradation rates of control liver and limiting enzyme of the pyrimidine de novo synthetic pathway, hepatoma synthase II. AC1/N tumor-bearing rats were administered [uCjleucine synthase II, in liver and rapidly proliferating hepatoma 3924A; (25 /iC'/lOO g rat) at T= 0 and injected with [3H]leucine (100 nCi/100 g rat) at (Â¿>)determination in liver and hepatoma of the in vivo synthetic T = 0, 23, 47, and 71 h and killed I h after this second injection. After centrifugation at 105,000 x g O'C for 30 min, I ml of the supernatant fluid was and degradative rates of synthase II by pulse-labeling and iso precipitated with excess antibody. The immunoprecipitates were collected by topie decay; and (c) demonstration that increased synthase II centrifugation through 2 ml of a I M sucrose cushion. The pellet was then dissolved in 300 ;

Downloaded from cancerres.aacrjournals.org on September 28, 2021. © 1986 American Association for Cancer Research. INCREASED SYNTHESIS OF HEPATOMA SYNTHASE II glutamine-dependent carbamoyl-phosphate synthetase and effect of magne the de novo biosynthetic pathway and in this way confer a sium ion as an essential activator. J. Biochem. (Tokyo), 72: 537-547, 1972. selective advantage to the cancer cells. 8. Bradford, M. M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem., 72: 248-254, 1976. REFERENCES 9. Weber, K., and Osborn, M. The reliability of molecular weight determinations by dodecyl sulfate-polyacrylamide gel electrophoresis. J. Biol. Chem., 244: 1. Jones, M. E. Pyrimidine nucleotide biosynthesis in animals: genes, enzymes, 4406-4412, 1969. and regulation of UMP biosynthesis. Ann. Rev. Biochem., 49: 253-279, 10. Mans, R. J., and Novelli, G. D. Measurement of the incorporation of 1980. radioactive amino acids into protein by a filter paper disk method. Arch. 2. Coleman. P. F.. Suttle, D. P., and Stark, G. R. Purification from hamster Biochem. Biophys., 94: 48-53, 1961. cells of the multifunctional protein that initiates de novo synthesis of pyrim- 11. Dunaway, G. A., and Weber, G. Effects of hormonal and nutritional changes idine nucleotides. J. Biol. Chem., 252: 6379-6385, 1977. on rates of synthesis and degradation of hepatic phosphofructokinase iso- 3. Mori, M., and Tatibana, M. Purification of homogeneous glutamine-depend- zymes. Arch. Biochem. Biophys., 162:629-637, 1974. ent carbamoyl phosphate synthetase from ascites hepatoma cells as a complex 12. Hopgood, M. F., and Ballard, F. J. Synthesis and degradation of phospho- with aspartate transcarbamoylase and dihydroorotase. J. Biochem. (Tokyo), enolpyruvate carboxylase in rat liver and adipose tissue. Biochem. J., 134: 78: 239-242, 1975. 445-453, 1973. 4. Aoki, T., Morris, H. P., and Weber, G. Regulatory properties and behavior 13. Glass, R. D., and Doyle, D. On the measurement of protein turnover in of activity of carbamoyl-phosphate synthetase II (glutamine-utilizing) in animal cells. J. Biol. Chem., 247: 5234-5242, 1972. normal and proliferating tissues. J. Biol. Chem., 257:432-438. 1982. 14. Arias, I. V. Doyle, D., and Schimke, R. T. Studies on the synthesis and 5. Aoki, T., and Weber. G. Carbamoyl phosphate synthetase II (glutamine- degradation of the endoplasmi reticulum of rat liver. J. Biol. Chem.. 244: hydrolyzing): increased activity in cancer cells. Science (Wash.), 272: 463- 3303-3315, 1969. 465. 1981. 15. Block, K. W., Siekevitz, P.. and Palade, G. E. Localization and turnover 6. Reardon, M. A., and Weber, G. Increased carbamoyl-phosphate synthetase studies of membrane nicotinamide adenine dinucleotide glycohydrolase in II concentration in rat hepatomas: immunological evidence. Cancer Res., 45: rat liver. J. Biol. Chem., 246: 188-195, 1971. 4412-4415, 1985. 16. Weber, G. Biochemical strategy of cancer cells and the design of chemother 7. Tatibana, M., and Shigesada, K. Requirements of a quantitative assay of apy: G. H. A. Clowes Memorial Lecture. Cancer Res., 43:3466-3492,1983.

3676

Melissa A. Reardon and George Weber

Cancer Res 1986;46:3673-3676.

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