The Comparative Enzymology and Cell Origin of Rat Hepatomas II. Glutamate Dehydrogenase, Choline Oxidase, and Glucose-6-phosphatase* HENRY C. PITOT~ (McArdle Memorial Laboratory, The Medical School, University of Wisconsin, Madison, Wis.) SUMMARY The activities of glucose-6-phosphatase, glutamate dehydrogcnase, and choline ox[- dase were determined in some or all of ten rat hepatomas, including the Novikoff, Dunning L-C18, McCoy MDAB, and the Morris 3683, 39524A, and 51~3 hepatomas, together with primary hepatomas produced by feeding ethionine or 3%nethyl-4- dimethylaminoazobenzene, and transplanted hepatomas derived from the primary tumors induced with ethionine. Of these neoplasms, only the Morris hepatoma 51~3, the primary and transplanted ethionine-induced hepatomas, and one of the 3'-methyl-4-dimethylaminoazobenzene- induced tumors possessed significant glucose-6-phosphatase activity. These same tu- mors in addition to the Dunning L-C18 hepatoma had demonstrable glutamate dehydro- genase activity, whereas the other neoplasms tested failed to show significant activity of this enzyme. With the exception of the primary dye-induced neoplasm, which was not tested, only those neoplasms having significant glucose-6-phosphatase activities showed any choline oxidase activity. Of those neoplasms tested for tryptophan peroxidase activity only the Morris hepa- toma 51~3, the primary ethionine-induced hepatoma, and some of the Dunning L-C18 hepatomas had any demonstrable activity of this enzyme. In contrast to most of the enzymatic activities reported here, the threonine dehydrase activity of the Morris hepatoma 51r was of the order of 40 times the level of this enzyme in the livers of animals bearing this tumor. The significance of these results in relation to the biochemical and morphologic classification of experimental hepatic carcinomas, as well as the importance of study- ing the biochemistry of slowly growing, highly differentiated experimental neoplasms such as the Morris hepatoma 51~3, is discussed. In the first paper of this series (~4) the deoxy- the transplantable Dunning L-C18 hepatoma (7) cytidylie acid (dCMP) * deaminase activity and and the transplantable Morris hepatoma 51~23 the thymine degradative eapaeity of a number of (16), had demonstrable dCMP deaminase activ- primary and transplanted rat hepatomas were de- ity, but only one of the ten, the Morris hepatoma termined. Of ten hepatomas studied, all but two, 51523, eould degrade thymine. Normal rat liver had * This work was supported in part by a grant (No. C-646) 1The following abbreviations are used in the text: dCMP, to Prof. Van R. Potter from the National Cancer Institute, 5'-deoxyeytidylic acid; G-6-P, glucose-6-phosphate; G-6-Pase, National Institutes of Health, U.S. Public Health Service. glucose-6-phosphatase; GDH, glutamate dehydrogenase; CO, t Lillian Israel Memorial Fellow in Cancer Research of the choline oxidase; TPO, tryptophan peroxidase; TD, threonine dehydrase; DPN, diphosphopyridine nucleotide; TCA, tri- American Cancer Society, 1957-1960. chloroacetic acid; 3'-?r 8'-methyl-4-dimethylaminoazo- Received for publication April 4, 1960. benzene. 12652 Downloaded from cancerres.aacrjournals.org on September 29, 2021. © 1960 American Association for Cancer Research. PIToT--Enzymology and Origin of Rat Hepatomas. II 1~263 previously been shown to have no dCMP deami- Lowry and Lopez (13). Tim difference in the hi- nase activity (15) but did possess the necessary organic phosphate content of the control tube with enzyme system for the conversion of thymine 5~- no substrate and that containing G-6-P was a C t4 to C140_~ (5). In view of these findings it was measure of the G-6-P dephosphorylated by the postulated that both the Dunning and the 51~3 enzyme. hepatomas probably arose from cells that closely Glutamate dehydrogenase.--This enzyme was as- resembled a hepatic parenchymal cell. On the sayed in the forward reaction with the use of DPN other hand, those neoplasms having dCMP deami- and L-glutamic acid by a slight modification of the nase activity and no thymine degradative capacity procedure of Hogeboom and Schneider (10). The may have been more closely related to biliary final volume of the components was 3.0 ml., and epithelium (~1). the pH of the assay was 7.5. The change in optical To gain further biochemical evidence in support density at 340 mg was read every ~ minutes with of these postulates as well as to look for biochemi- the use of a Beckman model DU spectrophotome- cal differences between the highly differentiated ter and quartz cuvettes. The rate was followed Morris hepatoma 51~3 and rat liver, the activities until maximal rate was reached. This maximal rate of several other enzymes were determined in many was used in all calculations. of these hepatomas (~24). The results of this survey Choline oxidase.--This enzyme was assayed comprise the subject of this paper. manometrically by a slight modification of the pro- cedure described by Richert and Westerfeld (~6). MATERIALS AND METHODS The substrate was added directly to the reactio~ The neoplasms used in this study were the same chamber in ~Tarburg vessels having no side-arms. as those employed in the previous paper (~4). The After a 5-minute equilibration period, the first tumor-bearing animals were stunned by a blow on reading was taken. Readings were then taken at the head and their necks immediately dislocated to 10, 30, and 50 minutes. The first 10-minute reading obtain paralysis of the lower extremities. The tu- was not included in the calculations; 0.5 and 1.0 mor and the liver were quickly removed and placed ml. of homogenate were assayed with all the tu- in ice-cold 0.154 M KCl. The tumor tissue was rap- mors. idly dissected and any necrotic material removed Tryptophan peroxidase.--This enzyme was as- as completely as possible; 1:4 homogenates of the sayed by the procedure of Knox and Auerbac5 tissues in 0.154 M KC1 were prepared by homogeni- (11). zation for 3 minutes in an all-glass Potter-Elve- Threonine dehydrase.--The method described hjcm homogenizer. The homogenizer was kept in an by Sayre et al. (~7) was used for the assay of thbs ice bath during this procedure to dissipate any enzyme. frictional heat. Samples of each of the tissues to be All enzyme assays were carried out within homogenized were taken for histologic examina- hours of the death of the animal. In particular, tion. The tissue was fixed for 16 hours in Lesher's best results were obtained with the choline oxidase fixative (1~) and then placed in 70 per cent ethanol assay when the homogenate was less than 1 hour until embedded, sectioned, and stained with hema- old. Unless otherwise specified, all enzyme activi- toxylin and eosin. ties were expressed as ttmoles of product formed All enzyme assays described in this paper were per hour per gram protein. Protein was determined performed with the whole homogenate. by the method of Lowry et al. (14). Glucose-6-phosphatase.--The procedure for the Dipotassium G-6-P and DPN were obtained assay of this enzyme was essentially that used by from the Sigma Co. L-Threonine, L-tryptophan, Cori and Cori (6); 0.1 and 0.~ ml. of the homoge- and L-glutamic acid were obtained from the Cali- hate was added to a 13 X 100 mm. pyrex culture fornia Corporation. Choline chloride was a product tube containing 0.13 ml. of 0.1 M potassium citrate of Nutritional Biochemicals Corporation. buffer, pH 6.8, 0.04 ml. of 0.1 M potassium G-6-P, and sufficient water to make a final volume of 1.0 RESULTS ml. For each tube containing G-6-P a duplicate Glucose-6-phosphatase.--In Chart 1 are showI~ tube without substrate was prepared. All tubes the G-6-Pase activities of ten hepatomas ex- were incubated for 30 minutes at room tempera- pressed as /,moles of phosphorus liberated as in- ture with occasional shaking. At the end of this organic phosphate/gin protein/hr. Enzyme values time 1.0 ml. of 10 per cent TCA was added to each which were less than 50 gmoles/gm protein/hr tube. After the precipitated protein had been cen- were considered to be within the limits of accuracy trifuged off, 0.5 ml. of the supernatant was used of the method and of questionable significance~. for phosphorus determination by the method of Therefore, by these standards, only the Morris Downloaded from cancerres.aacrjournals.org on September 29, 2021. © 1960 American Association for Cancer Research. 1~64 Cancer Research Vol. ~0, September 1960 hepatoma 51~8 and the primary and transplanted neoplasm (H-l) showed almost no G-6-Pase activ- ethionine-induced hepatomas as well as one of the ity but exhibited a high level of deoxycytidylic 3'-Me-DAB-induced tumors contained significant acid deaminase (~4). Histologically, hepatoma H-1 amounts of the enzyme. The histology of the dye- showed a somewhat more uniform cytologic pat- induced neoplasm which showed G-6-Pase activity tern than its parent primary tumor. The morphol- resembled the primary ethionine-induced neo- ogy and biochemistry of this neoplasm in vivo and plasms and cytologically could be classified as a in vitro will be discussed more extensively in a hepatoma of the large-celled variety (~5). On the further publication. 2 The absence of G-6-Pase ac- tivity in the Novikoff hepatoma has been pre- viously reported (~9). 3- ASSAY: Glutamate dehydrogenase.--The activities of this GLUCOSE- 6- PO4--,- z~ 1600 enzyme in the same series of hepatomas are seen in l.-- GLUCOSE + PO4 Chart ~. As with the G-6-Pase, the Morris hepa- toma 51~3, the ethionine-induced primary and o 1200 t2_ transplanted hepatomas, and one hepatoma in- duced by 3'-Me-DAB consistently showed signifi- 800 cant amounts of the enzyme.