Enzyme Patterns in a Group of Transplantable Mouse Hepatomas of Different Growth Rates'

Home , Uridine kinase

[CANCER RESEARCH 31, 743â€”751,June 1971] Enzyme Patterns in a Group of Transplantable Mouse Hepatomas of Different Growth Rates'

E. Bresnick,2 E. D. Mayfield, Jr., A. G. Liebelt, and A. A. Liebelt

Departments of Pharmacology fE. B.J , Pathology fE. D. M.J , and Anatomy fA. G. L., R. A. L.J , Baylor College of Medicine, Houston, Texas 77025

SUMMARY Accordingly, the activities of host liver, normal liver, and hepatoma enzymes in the metabolism of pyrimidines and A series of transplantable hepatomas which arose amino acids were determined. The data are somewhat spontaneously in mice or in mice treated with goldthioglucose, suggestive of a correlation between growth rate and the urethan, or 3-methylcholanthrene has been tested for the activity of ATC and TdR kinase in the mouse hepatoma extent of deviation of enzyme activity from that of normal system. The results also clearly depict the individuality of each liver. The growth rates of these hepatomas varied from 2 1 to of the enzymic patterns of the hepatomas. 211 days. Deoxythymidine kinase, uridine kinase, carbamylphosphate synthetase , aspartate transcarbamylase, ornithine transcarbamylase, orotidylic acid pyrophosphorylase MATERIALS AND METhODS and decarboxylase, uracil reductase, histidase, tyrosine a-ketoglutarate transaminase , threonine-serine dehydrase , and Radiochemicals. 4 C (30 pCi/j.zmole), @ tryptophan pyrrolase activities were examined in control liver 4 C (28 pCi/jzmole), sodium C (5 and in the hepatomas. Deoxythymidine kinase and aspartate jiCi4tmole), orotic 6@ C (5 iCi/jimole), DL-aspartic transcarbamylase activities correlated to some extent with the acid-3-' 4C (23 j.zCi/pmole), uracil-2-' 4C (l0pCi/pmole),and growth rate of these hepatomas. Threonine-serine dehydrase, potassium cyanate-' 4C (5 pCi/@zmole) were purchased from histidase, and carbamylphosphate synthetase activities were New England Nuclear, Boston, Mass. Carbamylphosphate-' 4C @ almost undetectable in all the hepatomas, while uracil was prepared from C by the method of Spector et reductase was present in detectable amounts in only two of al. (40). The final product had a specific activity of 0.125 the hepatomas. jiCi/jimole; the product was dissolved in water immediately before use. Animals and Tumors. The mice used in these experiments INTRODUCTION were from inbred strains (or F, hybrids) maintained by brother X sister mating in the Kirschbaum Memorial In a previous study (6), the activities of a group of enzymes Laboratory4 at Baylor College of Medicine. The animals were that function in the synthesis of pyrimidines and of TdR3 housed 5 to a cage and were given Purina laboratory chow and kinase were determined in rat kidney adenocarcinomas of water ad libitum. varying growth rates, i.e., 80 to 400 days. In this series, the The GTG-induced hepatomas were found in C3Hf and CBA activity of TdR kinase appeared to correlate with the growth mice that had received at 2 months of age 1 i.p. injection of rate, while no such relationship was observed between the GTG in 0.9% NaCl solution, at 0.8 mg/g and 0.4 mg/g body rapidity in growth and the activities of ATC, dihydroorotase weight, respectively. and dihydroorotate dehydrogenase, and OMP Hepatomas were also induced in the inbred strains (a) by pyrophosphorylase and decarboxylase. A series of feeding 3-methylcholanthrene at a level of 1 mg in 0.2 ml olive transplantable mouse hepatomas with varying growth rates has oil once a week for 9 weeks beginning at 6 to 10 weeks of age, been developed by Dr. A. G. Liebelt. It was of interest to and (b) by 1 i.p. injection of urethan, 0.8 mg/g body weight, ascertain whether the growth of these mouse hepatomas might into newborn CBA mice. be directly related to the extent of deviation of enzyme Transplantation was done by s.c. implantation into the right activity from normal liver and to compare the results in this side of isogeneic mice with a trocar containing the 1- to 2-mm system with those obtained in the Morris hepatomas (32). fragments of viable hepatoma tissue. Specific information about the mouse strains and tumor lines is given in Table 1. I Supported by NIH Grant CA-l0893, National Science Foundation The mice were observed, the tumor growth was recorded, Grant GB 8010, and Robert A. WelchGrant Q-198. The Kirschbaum Memorial Laboratory was supported by Grants CA 45 17 and CA 6240. and when transplants were 1 to 1.5 cm in size the animals were Manuscript 16 of the Cancer Research Center at Baylor College of killed by cervical dislocation. A representative piece, free of Medicine. necrotic tissue, was placed in 10% neutral buffered formalin 2To whom reprint requests should be directed. 3The abbreviations used are: TdR, deoxythymidine; ATC, aspartate 4 A description of the laboratory and of the mouse strains which are transcarbamylase; OMP, orotidine 5'-phosphate; GTG, goldthioglucose; maintained in this facility is offered in Inbred Strains of Mice, No. 6, OTC, omithine transcarbamylase. companion issue to Mouse Newsletter No. 41, 1969, pp. 104â€”107. J. Received November 18, 1970; accepted January 27, 1971. Staats (ed.), Jackson Laboratory, Bar Harbor, Maine 04609.

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and processed for histological examination. The latter included containing 10 ml Bray's scintillation mixture (3), and the hematoxylin and eosin stain, periodic acid-Schiff-Alcian Blue radioactivity was ascertained in a Packard spectrometer. with and without diastase treatment for the presence of Uracil reductase was assayed by a modification of the glycogen, and occasionally an additional piece was fixed with method of Potter et al. (36). The assay mixture included ATP, Oil Red 0 to stain neutral fat. A second piece was used for the 14 jimoles; NADP, 1 pmole; glucose 6-phosphate, 6 j.zmoles; next transplantation passage while enzyme activity was MgCl2, 30 j.tmoles; uracil-2-' 4C, 0.5 pmole (0.1 jiCi); determined in the rest of the tumor tissue (generally, the supernatant preparation as described above, 0.1 ml; and tumor mass from about 4 to 6 mice was combined). buffered sucrose, pH 8.0 (0.20 M sucrose-0.04 M Enzyme Assays. All assays were conducted under conditions nicotinamide-0.012 M Tris-6 mM KC1) to 2.0 ml. The in which the activity was directly proportional to the amount incubation was conducted for 15 mm at 37Â°in 50-mi flasks of enzyme and the time of incubation. TdR kinase was assayed containing 0. 1 ml 5 N KOH in the center well, and radioactive as described previously (7). The assay system consisted of 14 CO2 was assayed as described above. ATP, 5 mM;MgCl2, 5 mM;TdR-2-'4C,0.l jiCi(3.3 mj.zmoles); Carbamylphosphate synthetase was determined by supernatant fraction, 0.01 to 0.05 ml; 0.05 M Tris buffer, pH modifications of the methods of Brown and Cohen (9) and 8, to a total volume of 0.25 ml. The enzyme consisted of a Marshall and Cohen (28). The assay system consisted of supernatant fraction obtained after the centrifugation of a NH4C1, S j.imoles; sodium bicarbonate-' 4C, 10 j.imoles (1 pCi); 20% homogenate of the tumor or liver in 0.25 M sucrose at ATP, 5 pmoles; enzyme; L-omithine, 5 pmoles; MgCl2, 10 100,000 X g for 60 mm. The incubations were conducted in pmoles, N-acetylglutamate, S pmoles; and 0.05 M test tubes at 37Â°for I 5 mm after which time the enzymatic glycylglycine buffer, pH 8.0, to a total volume of 1.0 ml. The reaction was stopped by immersing the tubes in a boiling water enzyme consisted of a supernatant fraction obtained after the bath for 3 mm. centrifugation at 4000 X g for 15 mm of a 10% homogenate of The specific activity of ATC was determined by a liver or tumor in 0. 1% cetyltrimethylammonium bromide. modification of existing methods (5, 14). The assay system After a 15-mm incubation at 37Â°, the mixture was included DL-aspartic acid-3-'4C, 0.1 pCi (2 pmoles); deproteinized with 0.2 ml 4 N hydrochloric acid, an aliquot of carbamylphosphate, S pmoles; supernatant preparation, 0.2 the deproteinized material was placed on a planchet, and the ml; and 0.2 M Tris buffer, pH 9.2, to 1.5 ml. The incubation planchet was heated at 100Â°for 30 mm in a ventilated hood. was conducted at 37Â°for 10 mm, after which time 0.2 ml 4 N The radioactivity on the planchet that represented the @ perchloric acid was added. The deproteinized extract was acid-stable 4C, i.e., citrulline, was then determined. passed through a column of Dowex 50-H@, 200 to 400 mesh The specific activity of OTC was determined by a (0.6 x S cm). Carbamyl aspartic acid-' 4C passed through the modification of the method of Brown and Cohen (9). The ion exchange column whereas aspartic acid was retained. The assay system consisted of L-ornithine, 20 pmoles; column was washed twice with 1 ml water. The radioactivity 4 C, 2.5 pmoles (0.07 pCi); supernatant of the combined eluates was estimated in a Packard Tri-Carb preparation; and 0.045 M glycylglycmne buffer, pH 8.0, to 2.0 liquid scintillation spectrometer, with the use of Bray's solvent ml. The enzyme source was the same as described in the system (1 8). The efficiency of counting was 75%. carbamylphosphate synthetase assay. The incubation was Uridine kinase activity was assayed in a system similar to conducted for 15 mm at 37Â°,and the reaction was terminated @ the one reported by Skold (39). The assay mixture included with 0.2 ml 4 N hydrochloric acid. C was assayed n4 C, 0.5 jzmole (0.1 pCi); AlP, 2.5 pmoles; MgCl2, as described above. 10 jimoles; 3-phosphoglycerate, 7 pmoles; enzyme; and 0.1 M The protein in the homogenates and extracts was estimated Tris buffer, pH 7.5, to 0.25 ml. The incubation was conducted by the colorimetric procedure of Lowry et al. (27), with at 37Â°for 15 mm. The radioactive product was assayed in the bovine serum albumin as the reference standard. same manner as in the TdR kinase assay. The specific activity of threonine-serine dehydrase was OMP pyrophosphorylase and decarboxylase activities were assayed by the method of Sayre et a!. (38) as modified by assayed by the method of Rubin et aL (37). The assay mixture Bottomley et a!. (2). The assay system consisted of included phosphoribosylpyrophosphate, 0.5 pmole; orotic L-threonine or L-serine, 50 pmoles; pyridoxal phosphate, 10 4 C, 0.1 jimole (0.1 pCi); ATP, 1 jimole; mpmoles; phosphate buffer, pH 8.0, 100 pmoles; enzyme 3-phosphoglycerate, 2 @imoles;MgCl2 1.5 @zmoles;potassium preparation; and distilled water to 2.0 ml. The enzyme phosphate, 1 j.zmole; ribose 5-phosphate, 0.5 jimole; consisted of a 20% homogenate in 0.25 M sucrose. The supernataht preparation, 0.1 ml; and 0.05 M Tris buffer, pH reaction was carried out in test tubes for 30 mm at 37Â°and 7.4, to 1.5 ml. The incubation was conducted for 15 mm in a was stopped with 2.0 ml 10% trichloracetic acid. Aliquots 50-ml Erlenmeyer flask containing a center well in which was were assayed for keto acid production by the method of placed 0.1 ml S N KOH and a fluted square ofpaper, 1 x 1 cm. Friedman and Haugen (16). The reaction medium was placed around the center well, and The procedure used for the assay of tryptophan pyrrolase the flasks were capped with rubber serum caps. At the end of was a modification (35) of the method of de Castro et aL (1 5), the incubation, 0.2 ml 4 N perchloric acid was added through with the use of the coupling procedure of Brown and Price the caps to the reaction medium by means of a syringe and (10). The assay system consisted of L-tryptophan, 9 pmoles; needle. The flasks were then shaken for 30 mm to ensure Tris-HC1 buffer, pH 7.5, 100 moles; 0.025% hematin, 0.02 ml; complete release of â€˜â€œCO2. At the end of this time, the fluted enzyme preparation (see above); and water to 2.0 ml. The filter paper was removed and placed in a scintillation vial reaction was run in test tubes at 37Â°for 30 mm and stopped

744 CANCER RESEARCH VOL.31

Downloaded from cancerres.aacrjournals.org on September 28, 2021. © 1971 American Association for Cancer Research. Hepatoma Growth Rate and Enzymatic Activity with 1.0 ml 10% trichloracetic acid. The kynurenine was days. On the other hand, hepatoma CBA 29 had an initial assayed colorimetrically by the method described by Pitot et growth period of 162 days and stabilized at I 10 days after 9 a!. (35). transplant generations. The transplantation generation for the Histidase activity was assayed by the method of Tabor and various tumors used in this particular study ranged from T-2 to Mehier (41) as modified by Pitot et al. (35). The assay was run T-20 and the growth period for these same tumors at this time in quartz cuvets containing 2-mercaptoethanol, 5 pmoles; was 21 1 to 21 days, respectively. reduced glutathione, 10 pmoles; L-histidine, 30 pmoles; The architecture of the primary tumors was similar to supematant fraction; and 15 mM sodium pyrophosphate to normal liver with parenchymal-type cells arranged in cords 3.0 ml. The reaction was followed in a Beckman DU around a central blood vessel but lacking a true lobular pattern spectrophotometer at 277 nm at 2- to 3-mm intervals. A blank (Fig. 1). This morphological pattern in general remained stable consisted of a cuvet containing all constituents except in the majority of the transplanted tumors used (Fig. 2), with enzyme. The enzyme consisted of a supernatant fraction the exception of 2 hepatomas, C3Hf l7l9A and CBA 8379. obtained after the centrifugation at 100,000 X g for 60 mm of These 2 hepatomas changed from a liver-like architecture to a a 20% homogenate in 0.25 M sucrose. tumor composed of sheets of squamous-type cells (Fig. 5). Tyrosine a-ketoglutarate transaminase was assayed by the The distribution of glycogen was similar to that of normal method described by Pitot and Morris (34). The assay medium liver (Fig. 3) in approximately one-half of the tumor lines in of 3 ml consisted of pyridoxal phosphate, 10 mpmoles; the first transplant generation (Fig. 4). However, 5 of these sodium diethyldithiocarbamate, 10 pmoles; a-ketoglutarate, tumors were free of histochemically demonstrable glycogen pH 7.0, 50 pmoles; 0.2 M K2HPO4 , pH 8.0; L-tyrosine, 6 following subsequent transplantation as well as at the time pmoles; supernatant preparation (see above). The incubation when used for enzyme analyses. Several of the transplanted was carried out in test tubes at 37Â°for 15 mm and stopped tumor lines showed evidence of an accumulation of neutral with 1.0 ml 6 N perchioric acid. The enol-borate complex was lipid droplets as determined by histochemical stains. In routine determined by the method of Lin et al. (26). histological techniques, the accumulation of lipid droplets was manifested by highly vacuolated cells. Several of the tumor lines showed an increase in nuclear size as compared RESULTS with normal liver, but this observation was not followed up with precise measurements. One tumor (CBA/S 16) had, upon Description of Tumors. The origin, the growth examination by electron microscopy, numerous cytoplasmic characteristics, and the morphological features of the infoldings into the nucleus proper which appeared to be transplanted hepatomas used in this study are listed in Table 1. nucleoli when observed with light microscopy. In general, the The original hepatomas arose in mice of the CBA/S, CBA/Ki, number and size of the nucleoli in all tumors were similar to C3Hf, and DBA/2 inbred strains; all transplantation lines were those of normal liver. maintained by serial transplantation in mice of the same strain Cytoplasmic inclusion bodies were present in all of the of origin except for hepatoma C3Hf 1719A which was passed C3Hf primary tumors (Fig. 6) but were not present in tumors in C3HfX CE F, hybrids from the 12th transplant generation following subsequent transplantation. These inclusion bodies (T-12) to the 18th transplant generation. A separate transplant are protein-rich structures found in hepatomas arising in only line designated C3Hf l7l9B was derived from the same certain strains of mice (22, 24). hepatoma (C3Hf 1719) after T-4 and maintained exclusively in Detailed mitotic counts were not carried out, but if 4 to 6 C3Hf hosts up to and beyond T-l6. mitotic figures were seen in an area covered by approximately The original hepatomas arose either spontaneously in 100 parenchymal cells, the tumor was classified as a untreated mice or in mice treated with GTG (25), urethan mitotically active tumor. This classification correlated well (24), or 3-methylcholanthrene (20) and all but 1 tumor with the rapidly growing tumors with growth periods between developed in male mice. Two of the hepatomas studied, CBA 21 and 47 days. 2192 and DBA/2 5199, have been discussed previously with Several of these transplanted tumors were followed beyond respect to â€œmorphological transformationâ€• as well as the transplantation generation used for enzyme analysis. physiological changes during the course of serial Hepatoma CBA/S 16 has remained morphologically similar transplantation (21 , 23). from T-2 to T-7 and hepatoma CBA 2192, except for an No relationship was found between the type of increase in lipid droplets, has remained essentially the same in experimental treatment or age of host at time of tumor appearance between T-1 S and T-24. Two tumors, C3Hf 2275 diagnosis and morphological appearance of the original and CBA 29, have undergone a morphological transformation hepatoma. The age of the hosts in which the primary since they were used in the present study similar to that hepatomas were found ranged from 42 1 to 840 days. The described earlier (21). growth period, that is, the time from trocar inoculation until Enzymes of Pyrimidine Metabolism. The activities of the the tumor reached approximately I cm in diameter, for the enzymes involved in pyrimidine metabolism are compared in first transplant generation ranged from 162 to 403 days. Table 2. ATC and OTC represent competitive pathways for the However, with subsequent transplantation generations, the utilization of carbamylphosphate in pyrimidine and urea growth period for each tumor became progressively less. CBA syntheses, respectively. ATC is elevated in all hepatomas 8379 had an initial growth period in T-l of 307 days, but after except one, Tumor 16, and is maximum in 17l9B hepatoma. Stransplantgenerationsthegrowthperiodwasreducedto21 On the other hand, the activity of the catabolic enzyme, OTC,

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Table 1 Characteristicsofhepatomas usedfor enzyme studies

studiesGrowthStrainOriginaltumor and firsttransplant generationMorphological for enzyme

of mouse primaryGrowthofand sexAge with characteristicsTransplantedtumorused tumor-bear of tumor of first trans changes ing mouseTreatmentahepatoma (days)period T-l (days)of plantation generation1'Transplant generations'period (days)@Morphologicalas compared with T-1

CBA/S 16d GTG 840 266 Hepatic-like cells arranged T-2 211 Decreasedglycogen. in cords. Increased vascu larity. Increased glycogen. Increased vacuolization (lipid). Increased nuclear size. C3Hf 3375d Spontaneous 472 209 Hepatic-like cells arranged T-2 168 Decreased glycogen and in cords. Increased vascu absence of cytoplasmic larity. Increased glycogen. inclusions. Increased vacuolization (lipid). Cytoplasmic inclusions. CBA/S lSOo Spontaneous 675 246 Hepatic-like cells arranged in T-3 140 No change. cords. Increased vascularity. Increasedglycogen.Increased nuclear size. CBA 21929 MC 723 224 Hepatic-like cells arranged in T-15 113 Increased vascularity, cords. Vascularity, glycogen, glycogen, and vacuoli vacuolization similar to normal zation (lipid). liver, normal nuclear size. CBA 29d UR 535 162 Squamous type cells arranged T-9 110 No change. in sheets. Increased vascularity. Increased vacuolization (lipid). Normal nuclear size. CBA 7727d Spontaneous 612 377 Hepatic-like cells arranged in T-3 64 Increased nuclear size. cords. Increased vascularity. Increased glycogen. Increased vacuolization. Normalnuclear size. C3Hf 1719B GTG 421 403 Hepatic-like cells arranged in T-16 47 Cellular transformation.c cords. Increased vascularity. Decreased vascularity and Normal glycogen. Increased glycogen. Cytoplasmic in vacuolization. Cytoplasmic clusions absent and in inclusions.Normal nuclear creased mitosis. size. DBA/2 5199d Spontaneous 685 268 Squamous-type cells arranged T-39 40 Increased vascularity and in sheets. Normal vascularity. mitosis. Increased glycogen. Increased vacuolization (lipid). Normal nuclear size. C3Hf l719A GTG 421 403 Hepatic-like cells arranged in T-18 24 Cellular transformation.@' cords. Increased vascularity. Decreased glycogen with Normal glycogen. Increased normal vascularity. Cyto vacuolization (lipid). Cyto plasmic inclusions absent plasmic inclusions. Normal and increased mitosis. nuclear size. CBA 8379d Spontaneous 610 307 Hepatic-like cells arranged in T-5 2 1 Cellular transformation.C cords. Significant increased Normal vascularity and vascularity. Decreased glycogen. increased mitosis. Normal vacuolization (lipid). Normal nuclear size.

a MC, 3-methylcholanthrene; UR, urethan. b As compared with normal liver morphology. C Adenocarcinoma-like cell type. is decreased in all hepatomas. The reduction in enzymatic hepatoma except hepatoma 7727, which contained 10% of activity was least in hepatoma 29 (CBA host) and maximal in that present in normal liver. hepatoma 1719A (C3Hf host). The initial enzyme for The ability of the tumors to degrade pyrimidines was carbamylphosphate synthesis, carbamylphosphate synthetase, assessed by a measurement of the activity of the rate-limiting was also assayed, but no activity was detected in any enzyme in the catabolic pathway, uracil reductase. The

746 CANCER RESEARCH VOL. 31

Table 2 Enzyme activity in mouseliver and hepatoma

protein/hr)Carbamyl Activity(m@moles/mg

ofphosphateUridineTdRorotic Conversion toUracilTissueATCsynthetaseOTCkinasekinaseUMPreductaseControl acid

(22)CBA/Sliver'@41.0 Â±3.9 (l7)@'36.0 Â±3.7 (18)139 Â±21 (17)6.7 Â±1.6 (17)11.1 Â±0.1 (17)85.1 Â±10.6 (17)716 Â±100 (4)C3Hftumor 1641.0; 42.00; 025.6; 43.63.6; 4.415.2; 16.0n.d.'@1 1.0 Â±2 846CBA/Stumor 3375n.d.n.d.17.6; 28.815.1; 10.645.6; 46.476.2; 76.8734; (4)n.d.n.d.CBAtumor 15076.8 Â±2.3(4)0 (3)21.2 Â±3.2 (3)4.4 Â±0.3 (3)1 16.4 Â±2.2 (3)CBAtumor 2192115.8 Â±11.6(4)0 (3)61.2 Â±9.2(3)10.8 Â±1.6 (3)142.8 Â±37.1 (3)77.4 Â±1.8 (8)184 Â±62 (4)CBAtumor 2972.6 Â±18.0(4)0 (3)72.0 Â±10.6 (3)5.2 Â±0.6(3)27.1 Â±7.3 (3)140.2 Â±13.2 (4)43 Â±10 (6)C3Hftumor 772764.8 Â±7.5 (10)3.6 Â±0.4 (10)42.0 Â±5.1 (10)12.4 Â±2.0 (10)47.8 Â±5.2 (10)n.d.19 Â±5 (4)n.d.n.d.C3Hftumor 1719B315.6 Â±34.7 (4)0 (4)34.4 Â±8.5 (4)30.4 Â±2.6 (4)744 Â±110 (13)DBA/2tumor 1719A207.6 Â±54.0(4)0 (4)8.4 Â±1.9 (4)15.2 Â±4.3(4)425 Â±114 (4)142 Â±10(13)14 Â±4 (4)CBA tumor 5199131.4 Â±41(3)0 (3)29.2 Â±1.9 (3)6.8 Â±1.5 (4)104.4; 73.3n.d.11 Â±2 8379209 Â±50(3)0 (3)10.8; 1 1.239.6 Â±8.3 (3)788 Â±140 (3)n.d.n.d.

a The enzyme activities in livers of DBA, CBA/S, CBA, and C3Hf mice did not differ significantly and have been grouped together in this category. Unless the values for host liver are given, it can be assumed that these did not significantly vary from control liver presented in the table. The table is arranged according to increasing growth rate of the hepatomas. b Mean Â±S.E. No. in parentheses, no. of groups. Each group consisted of the combined tumor mass from 4 to 6 mice. C n.d., not determined.

Table 3 Amino acid metabolismin mouseliver and hepatoma

TyrosineTissueHistidaseaa-ketoglutarate

dehydraseControl transaminaseTryptophan pyrrolaseSerine-threonine

(25)CBA/Sliver1.0 Â±0.1 (l9)@'220 Â±61 (6)512 Â±78 (21)7120 Â±560 (4)n.d.CBAtumor 160 (4)n.d.C280 Â±35 (4)CBAtumor 33750 (2)568; 968nd.0 (6)CBAtumor 21920 (3)133 Â±28(6)412 Â±73 (4)98 Â±30 (8)CBAtumor 290 (4)112 Â±33(4)400 Â±90(3)10 Â±3 (6)DBA/2tumor 77270.1 Â±0.1(6)n.d.80 Â±30 (6)700 Â±140 (4)C3Hftumorl7l9An.d.136;1561140Â±tumor 5199n.d.n.d.200 Â±15(4)0 160(3)590Â±50(6)CBA tumor 83970 (9)n.d.n.d.nd.

a Histidase activity is expressed as the change in absorbance at 270 nm/mg protein/hr. All others are expressed as mMmoles product produced/mg protein/hr. b Mean Â±S.E. No. in parentheses, no. of groups. C n.d., not determined.

activity of this enzyme was reduced in almost all hepatomas all of the hepatomas (Table 2). The enzymatic activities ranged (Table 2). The activity was less than 10% of the corresponding from 1.5- to 75-fold higher than normal liver activity. The control liver value except in hepatomas 2 192 and 3375, which slowest growing tumors, hepatomas 16 and 3375, were contained approximately 25 and 100% of the normal liver elevated only slightly, while the fast-growing hepatomas, value , respectively. 17l9A, 1719B, and 8379, exhibited the highest activities. The relative conversion of orotic acid to UMP by normal Enzymes of Amino Acid Metabolism. The activities of liver and by the hepatomas is also shown in Table 2 . The tryptophan pyrrolase and tyrosine a-ketoglutarate combined activities of OMP pyrophosphorylase and transaminase are shown in Table 3 . It is evident that a wide decarboxylase showed no reduction from control liver in any range of tryptophan pyrrolase activity was present in the of the hepatomas which were assayed; an increase was hepatomas. The enzyme level varied from 15 to 200% of the apparent in hepatomas 29 and 1719A. corresponding normal liver value. The fast-growing hepatomas, Uridine kinase, a â€œsalvageâ€•enzyme involved in UMP 7727 and S199, possessed the least activity, while the formation, showed a wide variation in activity (Table 2). Three fast-growing hepatoma, 1719A, exhibited the highest activity. hepatomas showed decreased activity while 6 others were 2- to Histidase activity was undetectable in S hepatomas and barely 6-fold higher than the corresponding host liver value. The most detectable in the 7727 tumor. Serine-threonine dehydrase active in this regard was hepatoma 8379, a fast-growing tumor. activity was also markedly reduced in all the tumors assayed; The specific activity of TdR kinase is markedly elevated in no activity was demonstrable in hepatomas 3375 and 5199.

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DISCUSSION exhibited a similar close correlation in the Morris hepatomas (reviewed in Ref. 42). Thus, it had previously been shown (18, Up to 1960, the Novikoff tumor was the most commonly 44) that incorporation of thymidine into tumor DNA closely used and most extensively studied hepatoma. The latter, correlated with the growth rate of the hepatomas. A however, differs markedly from normal liver both in terms of comparable finding was reported for a group of kidney morphology and in biochemistry (reviewed in Ref. 19). It adenocarcinomas (17). We had previously reported on the appeared desirable to develop a series of hepatomas which activities of TdR kinase in the Morris hepatomas (8) and in the would deviate only minimally from normal tissue. kidney adenocarcinomas (7). The quantitative aspects bore a Accordingly , a number of tumors of varying growth rates were direct relationship to growth rate in these cases. In other obtained in inbred strains of rats, largely through the rapidly proliferating tissues TdR kinase activity was markedly administration of N-(2-fluorenyl)phthalamic acid and elevated (7, 11, 29). N-(2-fluorenyl)diacetamide by Morris et aL (3 1, 32). The activities of two of the enzymes involved in the de novo The tumors were divided according to growth rate into 3 synthesis of pyrimidines, carbamylphosphate synthetase and series, the slow-growing, the medium-growing, and the rapidly ATC, were also influenced by the growth rate of the growing hepatomas. The slow-growing tumors were those that hepatomas, but in different directions. The former was required transplantation every 1.5 to 6 months, were progressively reduced with increasing growth (33); the latter histologically well-differentiated trabecular hepatocarcinomas, was increased (4). ATC activity was also markedly elevated in and had few mitotic figures. The medium-growing tumors other rapidly proliferating tissues (5, 7, 12, 13). required transplantation every 4 to 6 weeks, were less We have reported here the characteristics of a group of well-differentiated, and showed more mitotic figures. The mouse hepatomas that arose either spontaneously or as a result rapidly growing hepatomas had an anaplastic histology, of administration of urethan, GTG, or 3-methylcholanthrene. required less than 3 weeks for transplantation, and were The hepatomas were observed in mice that were between 421 mitotically quite active. Some of the hepatomas showed and 840 days old. The first transplantation time (T-l) varied increasing growth rates with increasing numbers of transfers; between 162 and 403 days. The second transplantation period others showed little change. Each tumor line, apparently, (T-2) of Tumors 16 and 3375 was little changed. A substantial possessed its own growth characteristics. The histology of decrease in subsequent transplantation time was noted in these Morris hepatomas has been extensively studied (30). many of the other hepatomas. The morphology of the Although the well-differentiated hepatomas are hepatomas resembled that of normal liver in T-l , although characterized by many biological and biochemical similarities several demonstrated an increased nuclear size and increased to normal liver, the less well-differentiated and more glycogen deposition; cytoplasmic inclusion bodies were also rapid-growing tumors possess more extensive differences in noted in several. Upon subsequent transplantations, glycogen this regard. For example, bile secretion was observed in a was decreased in hepatomas 16, 3375, 1719B, and l7l9A. number of the former. Glycogen was also seen, although, in Increased mitosis was observed in 1719A and 1719B, in 8379, general, the content decreased from transplant generation to andin5199. generation. In this mouse hepatoma series, uracil reductase activity In a recent review by Weber and Lea (42), the enzyme proved a sensitive marker. With the exception of hepatomas content of Morris hepatomas has been classified according to 3375 and 2192, enzyme activity was markedly reduced, the correlation of activity to the growth rate of the tumor. almost to the point of nonexistence in the other tumors. In The activities of certain key enzymes involved in the latter 2 tumors, uracil reductase was present at carbohydrate, lipid, protein, and nucleic acid metabolism approximately 100 and 25%, respectively, of control liver exhibited a remarkable relationship in this regard. activity. A decrease in gluconeogenesis and a concomitant rise in With a singular exception of hepatoma 7727, glycolysis were noted in the rapidly growing hepatomas when carbamylphosphate synthetase activity was not present in any compared with either normal liver or the slow-growing tumors of the tumors. In this tumor, enzyme activity was only 10% of (42). The activities of the key gluconeogenic enzymes, i.e., control liver level. Thus, in contrast to the Morris hepatoma pyruvate carboxylase , phosphoenolpyruvate carboxykinase, series (see above), no direct correlation to growth rate was fructose 1,6-diphosphatase, and glucose 6-phosphatase, were seen with the mouse tumors. depressed in parallel with the increase in growth rate. On the ATC and TdR kinase were markedly elevated in most of the other hand, the key glycolytic enzymes, hexokinase, mouse hepatomas, with a suggestion of correlation to growth phosphofructokinase, and pyruvate kinase, increased in rate. Hepatoma 7727 seemed somewhat unique in that the activity in direct relation to increasing growth rate. magnitude of the increase was only 1.5- and 4-fold, A close inverse relationship was also observed between the respectively. The other transcarbamylase, OTC, was markedly increasing growth rate of the Morris hepatomas and the depressed in activity in the mouse hepatomas, with no activities of certain enzymes that functioned in the catabolism apparent correlation with growth rate. of amino acids (1, 29, 42). Thus, tryptophan pyrrolase, Amino acid metabolism was profoundly altered in the serine-threonine dehydrase, glutamate dehydrogenase , and mouse hepatomas. Thus, histidase activity was almost OTC were reduced in activity in proportion to the increasing nonexistent in 5 hepatomas and serine-threonine dehydrase growth capacity of the tumors. activity was depressed. No correlation of the magnitude of the Certain of the enzymes involved in nucleic acid metabolism reduction of the latter enzyme was observed with growth rate.

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Tryptophan pyrrolase activity, on the other hand, was elevated Measure of Hepatoma Growth Rate. Cancer Res., 26: 465â€”469, in the 1719A hepatoma. In the other tumors, this activity was 1964. reduced but again in no direct relationship to growth. 18. Lea, M. A., Morris, H. P. and Weber, G. DNA Metabolism in Liver In summary, a series of transplantable mouse hepatomas and Kidney Tumors of Different Growth Rates. Cancer Res., 28: have been developed, which exhibit many of the 71â€”74, 1968. 19. LePage, G. A., and Henderson, J. F. Progr. Exptl. Tumor Res., 1: morphological characteristics of normal liver. However, these 440â€”476,1960. tumors vary greatly in their enzymic complement and are 20. Liebelt, A. G., and Liebelt, R. A. Chemical Factors in Mammary quite deviant from the normal liver pattern. Each tumor Tumorigenesis. In: Carcinogenesis, a Broad Critique. University of exhibited its own individuality in this respect, which could be Texas M. D. Anderson Hospital and Tumor Institute at Houston, an expression of genetic origin or of the manner of induction pp. 315â€”340. Baltimore: The Williams & Wilkins, Co., 1966. of the neoplasia. 21. Liebelt, A. G., and Liebelt, R. A. Transplantation of Tumors. In: H. Busch (ed.), Methods in Cancer Research, Vol. 1, pp. 142â€”242. New York: Academic Press, Inc., 1967. REFERENCES 22. Liebelt, A. G., Liebelt, R. A., Rogers, T., Sykes, J., and Dmochowski, L. Eosinophilic Cytoplasmic Inclusions in Mouse Hepatomas. Proc. Am. Assoc. Cancer Res., 6: 40, 1965. 1. Auerbach, V. H., and Waisman, H. A. Amino Acid Metabolism of 23. Liebelt, R. R., Liebelt, A. G., Gulledge, A., and Calvert, J. Novikoff Hepatoma. Cancer Res., 18: 543â€”547, 1958. Autoregulation: Normal Organ and Tumor Homeostasis. In: 2. Bottomley, R. H., Pitot, H. C., and Morris, H. P. Metabolic Proliferation and Spread of Neoplastic Cells. University of Texas Adaptations in Rat Hepatomas. IV. Regulation of Threonine and M. D. Anderson Hospital and Tumor Institute at Houston, pp. Serine Dehydrase. Cancer Res., 23: 392â€”399,1963. 733â€”768. Baltimore: The Williams & Wilkins Co., 1967. 3. Bray, G. A. A Simple Efficient Liquid Scintillator for Counting 24. Liebelt, R. A., Liebelt, A. G., and Lane, M. Hormonal Influences Aqueous Solutions in a Liquid Scintillation Counter. Anal. on Urethan Carcinogenesis in C3H/f Mice. Cancer Res. , 24: Biochem., 1.@279â€”285,1960. 1869â€”1879,1964. 4. Bresnick, E. Feedback Inhibition of Aspartate Transcarbamylase in 25. Liebelt, R. A., and Perry, J. Action of Goldthioglucose on the Liver and in Hepatoma. Cancer Res., 22: 1246â€”1252, 1962. Central Nervous System. In: C. F. Code (ed.), Handbook of 5. Bresnick, E. Early Changesin Pyrimidine Biosynthesisafter Partial Physiology, Section 6, Vol. 1, pp. 271â€”286. Washington, D.C: Hepatectomy. J. Biol. Chem., 240: 2550â€”2556, 1965. American Physiological Society, 1967. 6. Bresnick, E., Mainigi, K. D., Mayfield, E. D., Jr., and Morris, H. P. 26. Lin, E. C. C., Pitt, B. M., Civen, M., and Knox, W. E. The Assay of Activities of Enzymes of Pyrimidine Nucleotide Synthesis in Aromatic Amino Acid Transaminations and Keto Acid Oxidation Slowly Growing Kidney Tumors. Cancer Res., 29: 1932â€”1936, by the Enol Borate-Tautomerase Method. J. Biol. Chem., 233: 1969. 668â€”673, 1958. 7. Bresnick, E., and Thompson, U. B. Properties of Deoxythymidine 27. Lowry, 0. H., Rosebrough, N. J., Farr, A. L., and Randall, R. J. Kinase Partially Purified from Animal Tumors. J. Biol. Chem., 240: Protein Measurement With the Folin Phenol Reagent. J. Biol. 3967â€”3974,1965. Chem., 193: 265â€”275,1951. 8. Bresnick, E., Thompson, U. B., Morris, H. P., and Liebelt, A. G. 28. Marshall, M., and Cohen, P. P. An Immunochemical Study of Inhibition of Thymidine Kinase Activity in Liver and Hepatomas Carbamyl Phosphate Synthetase. J. Biol. Chem., 236: 7 18â€”724, by UP and d-CTP. Biochem. Biophys. Res. 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Some Characteristics of Transplantable Rat Hepatoma 5 123 Induced by 12. Calva, E., and Cohen, P. P. Carbamylphosphate-Aspartate Transcarbamylase Activity in Regenerating Rat Liver.Cancer Res., Ingestion of N-(2-Fluorenyl)phthalamic Acid. Cancer Res., 20: 19: 679â€”683,1959. 1252â€”1254, 1960. 13. Calva, E., Lowenstein, J. M., and Cohen, P. P. 32. Morris, H. P. and Wagner, B. P. Induction and Transplantation of Carbamylphosphate-Aspartate Transcarbamylase in Tumors. Rat Hepatomas with Different Growth Rates (Including â€œMinimal CancerRes.,19:101â€”103,1959. Deviationâ€• Hepatomas). In: H. Busch (ed.), Methods in Cancer 14. Curci, M. R., and Donachie, W. D. An Attempt to Find Pyrimidine Research, Vol. 4, pp. 125â€”152. New York: Academic Press, Inc., Inhibitors of Mammalian Aspartate Carbamyltransferase. Biochim. 1968. Biophys. Acta, 85: 338â€”341,1964. 33. Ono, T., Blair, D. G. R., Potter, V. R., and Morris, H. P. The 15. de Castro, F. T., Brown, R. R., and Price, J. M. 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Fig. 1. A primary hepatoma from CBA mouse 2192. H & E, X 512. Fig. 2. Transplanted CBA 2192 hepatoma after the 15th transplant generation. H & E, x 500. Fig. 3. Distribution of glycogen as determined by the periodic acid-Schiff staining reaction in normal liver of CBA mouse. x 610. Fig. 4. Distribution of glycogen as determined by the periodic acid-Schiff staining reaction in transplanted hepatoma CBA 9214. x 628. Fig. 5. Transplanted C3Hf 1719A hepatoma which has undergone a morphological transformation from a typical hepatic parenchymal type of cell population as in Fig. 1 to a squamous-typecellular arrangement. H & E, X 518. Fig. 6. A primary hepatoma of a C3Hf mouse demonstrating the presence of cytoplasmic inclusion bodies. H & E, x 1320.

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E. Bresnick, E. D. Mayfield, Jr., A. G. Liebelt, et al.

Cancer Res 1971;31:743-751.

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