The Comparative Enzymology and Cell Origin of Rat Hepatomas II

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 tumors in addition to the Dunning L-C18 hepatoma had demonstrable glutamate dehydrogenase 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 hepatoma 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 procedure 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 activity in the Novikoff hepatoma has been previously 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 hepatoma 51~3, the ethionine-induced primary and o 1200 t2_ transplanted hepatomas, and one hepatoma induced by 3'-Me-DAB consistently showed signifi- 800 cant amounts of the enzyme. In addition, the Dun- ning hepatoma also has been found to possess 400 GDH activity (s The single 3'-M:e-DAB-in- 2:2 duced hepatoma with a high GDH activity was I!?iIil: ...... the same neoplasm that possessed a significant L 5 E E M D 5 H 3 M N t I T TDU 9 I 6C 0 level of G-6-Pase. Two dye-induced hepatomas of V 2 P I AN 2 80 V the "small-cell" type (~5) showed little if any 5 B 4A 5 P GDH activity. There was no significant loss of TISSUE CHART l.--Glueose-6-phosphatase activity of ten rat hepatomas. Each vertical line or point represents one animal. ASSAY: Key to Symbols LIV = Liver GLUTAMATE + DPN ---- 51~3 = Morris hepatoma 51~3 -r" 200 o~-KETOGLUTARATE + DPNH ETP = Primary hepatomas induced by dietary ethionine ETI = First- and second-generation transplants of ETP hA_z MDABP = Primary hepatomas induced by dietary 8r-Me- t-- DAB rr"O DUN = Dunning L-C18 hepatoma O_ 39~4A = Morris hepatoma 89~4A ILl = H-1 hepatoma (described in text) ,o0 3688 = Morris hepatoma 8683 ::I:: MCC = ~cCoy hepatoma a_Z NOV = Novikoff hepatoma _J O other hand, the other 8'-Me-DAB-induced tumor =X with little or no G-6-Pase activity was morphologically compatible with a cholangio-carcinoma. Al- T U 6 9 C 0 I though in the case of primary neoplasms it would V 2 P I N 8 2 C V 5 _B 3 4A be argued that any activity found might be due, at P TISSUE least in part, to contaminating liver, histologic sec- CHANT ~.--Glutamate dehydrogenase activity of ten rat tions of the tumors did not justify such a conclu- hepatomas. For key to symbols see legend to Chart 1. sion in these experiments. It is evident from these data that, upon trans- GDH in the first-generation transplants of the plantation in animals, the ethionine-induced neo- ethionine-induced hepatomas, but the H-1 tumor plasm lost some of its G-6-Pase activity. Simul- possessed very little of the enzyme. taneously, there appeared to be some gain in de- The variability found in the case of this enzyme oxycytidylic acid deaminase activity upon trans- may be due to the difficulties encountered in re- plantation of these neoplasms (e4). This change was leasing all the activity from the cells of the tissues most marked after the tumor had been explanted used. Previous work (10) has indicated that special to tissue culture, grown in vitro for several weeks, and then inoculated into a new host. The resulting H. E. Swim and H. C. Pitot, unpublished.

Downloaded from cancerres.aacrjournals.org on September 29, 2021. © 1960 American Association for Cancer Research. P~ToT--Enzymology and Origin of Rat Hepatornas. II 1~65 steps must be taken to make all the GDH of the reported by Auerbach and Waisman (~). Pitot et cell available for assay. In view of this, the very al. (~0) have also shown that primary hepatomas, low or negligible GDH activities of six of the possessing a low TPO activity, as well as the Dun- hepatomas probably represent low activities of the ning hepatoma, do not have the capacity to in- enzyme. In particular, Novikoff (17) has reported crease their TPO in response to tryptophan ad- the presence of GDH in acetone powder extracts of ministered in vivo or in slices incubated with tryp- the Novikoff hepatoma; however, Allard et al. (1) were unable to show GDH activity in homogenates of the Novikoff hepatoma. I0 ASSAY: Choline oxidase.--Chart 3 shows the CO activ- :E TRYPTOPHAN ity of seven of this series of hepatomas. Only the z KYNURENINE ,~ 8 CO activity of the Morris hepatoma 51~8 was of I-- o the same order as that found in liver. The primary rY and transplanted ethionine hepatomas exhibited n 6 about one-fifth the activity of liver. The presence x 4 of the enzyme in the Dunning hepatoma as well as O3 U.I its virtual absence in the Novikoff hepatoma has __1 been previously reported (~0). As with the other ~ 2 o I I,.

ASSAY: P 2 N V .. GHOLI NE--r 02--.. BETAINE 3 _z 400]-t TISSUE L~ I-- CHART 4.--Tryptophan peroxidase activity of liver and rro 300, four rat hepatomas. For key to symbols see legend to Chart 1.

o 200 6O c~ ASSAY: _] THREONINE " o I00 rP 5O -i'- a KETOBUTYRATE

z L 5 E D E 3 N 4O t-- I I T U T 90 0 V 2 P N I 2 V rY 5 4A o_ 30 TISSUE (_9 CHART 8.--Choline oxidase activity of six rat hepatomas. ~ 2O 1.1_1 For key to symbols see legend to Chart 1. ..J o I0 mitochondrial enzyme in this study, GDH, the CO activity did not appear to differ in the first III 1 generation transplanted compared with the pri- L 5 I I mary hepatomas produced by ethionine feeding. V 2 Tryptophan peroxidase and threonine dehydrase. TISSUE 3 --In Charts 4 and 5 are, respectively, the specific CHART 5.--Threonine dehydrase activity of Morris hepa- activities of the TPO activity of rat liver and four toma 5123 and of liver in rats bearing hepatoma 5123. hepatomas and of the TD activity of the Morris hepatoma 51~3 as compared with the activity of tophan in vitro. This failure of the hepatoma to re- the livers in animals bearing hepatoma 51~3. TPO spond is not due to the presence or absence of an activity was low in primary ethionine-induced inhibitor or to the absence of formylase in the tu- hepatomas and in hepatoma 51~3, and low as well mor cell (19). The capacity of the enzyme-forming as variable in the Dunning hepatoma. The pres- system in the Morris hepatoma 51~3 to synthesize ence of the enzyme in primary tumors induced by new TPO in response to tryptophan in vivo and in ethionine, as well as in the Dunning hepatoma, has vitro is presently under investigation in this labora- been previously reported (18, ~0). The absence of tory. TPO activity in the Novikoff hepatoma has been In contrast to almost all the other enzymes

Downloaded from cancerres.aacrjournals.org on September 29, 2021. © 1960 American Association for Cancer Research. :1~66 Cancer Research Vol. '20, September 1960 studied, the TD activity of the Morris hepatoma 51~3. This neoplasm possesses demonstrable activ- 51~3 was about 40 times the activity of this en- ity of all hepatic enzymes studied and lacks sig- zyme in the livers of animals bearing this neo- nificant dCMP deaminase. Whether or not this plasm. The TD activity of livers of normal Holtz- classification will retain its validity with further man rats was in the neighborhood of 10-~0 gmoles investigations will be decided in the future. The a-keto butyrate/hr/gm protein. 3 Greenberg et al. D-T- and D-T + hepatomas may be classified as (9) have previously shown that the total TD activ- "large cell" hepatomas (~5). ity of the livers of rats bearing Walker carcino- There is a fourth biochemical type of hepatic sarcoma ~56 is essentially unchanged as compared carcinoma described in this paper. This is the with the livers of normal rats. ~u or not the ethionine-induced carcinoma of the liver which high level of TD activity in the 51s hepatoma is possesses both dCMP deaminase activity as well merely a unique feature of this tumor or reflects as CO, GDH, G-6-Pase, and TPO activities. The some basic mechanism of the neoplastic process is degradation of thymine was not studied in the pri- a matter which must await further study. mary neoplasms of this group; however, the transplanted tumors showed no thymine degradative DISCUSSION activity (~4) and a lowered G-6-Pase activity A comparison of the GDH, CO, and G-6-t)ase ac- (Chart 1). The simplest explanation of these find- tivities with the dCMP deaminase and thymine ings is that these neoplasms are mixtures of the degradative activities (~24) of these hepatomas D+T - and the D-T- type of hepatoma in our lends further support to the concept that experi- classification. Histologic studies of these tumors in- mental hepatic carcinomas in the rat can be di- dicate that they are indeed composed of at least vided into at least three distinct classes with re- two distinct cell types comparable to the small and gard to their enzymology. These classes have been large cell hepatomas of Richardson el al. (~5). designated D+T -, D-T-, and D-T + (s When one of these tumors was stimulated to rapid The first type, D+T -, as exemplified by the growth by explantation to tissue culture and in- Novikoff hepatoma and Morris hepatoma 39~4A, oculated back into the animal, the small-cell hepa- has readily demonstrable amounts of dCMP de- toma predominated 2 and the enzymology pro- aminase but little or no G-6-Pase, GDH, CO, and gressed to a D+T - hepatoma. From these findings the thymine degradative enzyme system. G-6- it would appear that, on continued transplanta- Pase, CO, and the capacity to degrade thymine are tion, a tumor of mixed-cell type will eventually se- enzymatic properties which are found only in the lect out the most rapidly growing cell, of which the liver and kidney of the adult rat and may thus be neoplasm will eventualb~ be almost entirely com- considered as markers of hepatic or renal origin. :In posed. Such a phenomenon is certainly one form of contrast, dCMP deaminase does not occur in nor- the much discussed "dedifferentiation" of neo- real adult rat liver but probably does occur in plasms. proliferating bile duct cells or in some related epi- However, when the primary neoplasm is of only thelial cell of the liver (~1). Thus, neoplasms of this one cell type, then subsequent transplants will re- first type are probably more closely related to sult in neoplasms homogeneous with respect to biliary epithelial cells than to hepatic parenchymal cell type unless genetic mutations or deletions cells. Histologically, these neoplasms are almost change the cell population. In the case of the Dun- always composed of small basophilic cells corre- ning hepatoma repeated transplantation since sponding to the "small cell" type of hepatoma 1947 has not resulted in any major morphologic (~5). change in the tumor. After explantation to tissue The second type, D-T-, of which the Dunning culture and subsequent inoculation into the ani- hepatoma is the only clear-cut example in this se- mal, the resulting tumor is biochemically and ries, has no dCMP deaminase or capacity to de- morphologically essentially the same as the origi- grade thymine and little or no G-6-Pase activity, nal explant3 but does have GDH and CO activity. This neo- The Morris hepatoma 51~3 is a very slowly plasm has at least one of the enzymes characteris- growing neoplasm requiring several months to kill tic of normal liver and lacks the deaminase but it the host, whereas the Dunning hepatoma grows also lacks several of the other enzymes of normal somewhat more rapidly, killing the host in about a liver. month when implanted subcutaneously. However, The third type of hepatic carcinoma, D-T +, is both tumors grow more slowly than the Novikoff hepatoma, which when implanted in an animal can uniquely demonstrated by the Morris hepatoma lead to the death of the host in 7-10 days. As em- H. C. Pitot, unpub]ished observations. phasized previously (~4), it is of interest that both

~he 51~3 and Dunning tumors can be carried only neoplastic process itself. In view of the biochemical in inbred strains (7, 16). A possible explanation of (~4), biologic, and morphologic (16, 17) character- this is that slowly growing, highly differentiated istics of the Morris hepatoma 51~t8, this neoplasm neoplasms will be able to maintain their growth offers perhaps the best system, at present, for the only when host resistance is at a minimum. Ana- elucidation of any alterations in enzymatic and plastic, rapidly growing neoplasms such as the controlling mechanisms between the normal and Novikoff hepatonm will tend to overcome host re- malignant liver cell. Whether or not any difference sistance by their rapid rate of growth. Since many, that is found between the two can be generalized if not the majority, of human neoplasms are rela- to other neoplasms must await careful experimen- tively slowly growing and highly differentiated, tal investigation. the more highly differentiated experimental neoplasms of animals may offer model systems for ACKNOWLEDGMENTS study and comparison with their human counter- The author is indebted to Dr. Van R. Potter for many parts. helpful suggestions and stimulating discussions during the preparation of this manuscript. Weinhouse has recently suggested that rapid growth, i.e., cell division, may actually be the pri- REFERENCES mary cause of deletion of many enzymes in cancer 1. ALLARD, C., DELAMIRANDE,G.,and CANTERO, A. Enzymes tissues (30), and it appears probable that growth and Cytological Studies in Rat Hepatoma Transplants, per se is neither a unique characteristic nor a neces- Primary Liver Tumors, and in Liver Following Azo Dye sary prerequisite of the neoplastic cell. 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Downloaded from cancerres.aacrjournals.org on September 29, 2021. © 1960 American Association for Cancer Research. The Comparative Enzymology and Cell Origin of Rat Hepatomas II. Glutamate Dehydrogenase, Choline Oxidase, and Glucose-6-phosphatase

Henry C. Pitot

Cancer Res 1960;20:1262-1268.

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