Hexokinase Isozyme Patterns of Experimental Hepatomas of Rats1

[CANCER RESEARCH 29, 1437-1446, July 1969]

Hexokinase Isozyme Patterns of Experimental Hepatomas of Rats1 â€¢â€¢'<-*,V*:teÂ¿-v*:.'J'.

Shigeaki Sato, Taijiro Matsushima, and Takashi Sugimura Biochemistry Division, National Cancer Center Research Institute, Tsukiji, Chuo-ku, Tokyo, Japan

SUMMARY tive factor for the rate of glycolysis in various tumor strains (29). Meanwhile Gonzalez et al. (6) in 1964 separated rat liver Hexokinase isozymes in rat tissues were electrophoretically hexokinase into four types with diethylaminoethyl cellulose separated on cellulose acetate membrane. The method was column chromatography. Katzen and Schimke (12) in 1965 very quick and gave reproducible results. By using this succeeded in separating four types of hexokinase in rat tissues method, hexokinase isozyme patterns were studied on normal with starch gel electrophoresis and mentioned the presence of rat liver and experimental hepatomas with differing growth the specific isozyme pattern in the specific tissue. Following rates and degrees of differentiation. these pioneering works, the hexokinase isozyme patterns of In normal rat liver, the hexokinase pattern obtained on cellu many tissues, including human materials, have been elucidated, lose acetate membrane was identical with that obtained on and the enzymatic properties of each type of isozyme have starch gel by previous workers. There were four types of hexo- been studied (3, 7, 10, 23). While many papers on isozyme kinases, which corresponded to Types I, II, III, and IV hexo- patterns of hexokinase in the normal tissues have been pub kinases according to Katzen and Schimke, in order of increas lished, there have been only recent reports by Gumaa and ing mobility from the origin to the anode. Type IV hexokin Greenslade (8) and Shatton et al. (25) on the hexokinase iso ase, which was stained most intensely, was glucokinase, and zyme patterns of experimental hepatomas. From the studies duplication of Type IV hexokinase into two bands (IVS, slow using various concentrations of glucose, Sharma et al. (24) also form, and IVf, fast form) was occasionally observed. reported relatively increased "low-iCm hexokinase" activities Morris hepatomas, as a slowly growing and less deviated hep- and decreased "highJCm -specific glucokinase" activity during atoma group, showed relatively predominant Type II hexo experimental hepatocarcinogenesis in rats. We suggested the kinase in addition to Types I and III hexokinases, and they presence of two types of hexokinases in Yoshida ascites hepa also had a faint Type IV hexokinase band. A rapidly growing toma cells by using different concentrations of glucose (27). and highly deviated hepatoma group, Yoshida ascites hepa In this paper we describe a new method for electrophoretic tomas were characterized by the presence of only Type I hexo separation of hexokinase isozymes on cellulose acetate mem kinase and marked Type II hexokinase and by the loss of Type brane. By using this method, the hexokinase isozyme patterns IV and Type III hexokinases. Yoshida sarcoma showed a simi were investigated on two groups of experimental hepatomas of lar hexokinase pattern to that of Yoshida ascites hepatomas. A rats, Morris hepatomas as a less deviated and slowly growing slowly growing substrain of Yoshida sarcoma, LY 5, had also group, and Yoshida ascites hepatomas as a highly deviated and Type III and Type IV hexokinases, resembling Morris hepa rapidly growing one. Yoshida sarcoma and its subs trains, in tomas. These observations suggested that the genes for Types cluding a strain with slower growth rate than the original III and IV hexokinase isozymes were not deleted, but their strain, were also investigated. The hexokinase isozyme patterns expressions were blocked in the original Yoshida sarcoma. thus obtained on hepatomas were compared with those in the The regenerating liver was analogous to Morris hepatomas in normal liver, the regenerating liver, and fetal liver. The prob hexokinase pattern. In fetal liver, Type II hexokinase was lem of cellular disdifferentiation on carcinogenesis based on dominant, in addition to Types I, III, and weak IV hexo the experimental results will be discussed. kinases. MATERIALS AND METHODS INTRODUCTION Tumors. Morris hepatomas, Nos. 7316A, 7793, 7794A, and An increased rate of anaerobic glycolysis had been regarded 7795, were transplanted intramuscularly in both hind legs of for long to be one of the characteristics of tumor tissues. In about 9- to 10-week-old Buffalo strain rats. They were main 1966, however, Weinhouse (29) pointed out that increased tained on diet CE-2 (diet from the Central Laboratory of Ex glycolysis was not always observed in Morris hepatomas. He perimental Animal, Japan). Three to four weeks after trans described also that the level of hexokinase [ATP:D-hexose plantation, rats were killed by decapitation, and tumors were 6-phosphotransferase (EC.2.7.1.1)] activity was a determina- removed; necrotic, hemorrhagic, and nontumorous materials were carefully trimmed off. About one million cells of Yoshida ascites hepatomas or Supported in part by a grant from the Ministry of Education and sarcomas were intraperitoneally transplanted into Donryu Ministry of Health, Japan. strain male rats 9 to 10 weeks old. Five to ten days later, the Received October 7, 1968; accepted March 26, 1969. rats were killed by decapitation. The ascitic fluid was aspirated

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Downloaded from cancerres.aacrjournals.org on September 26, 2021. © 1969 American Association for Cancer Research. S. Sato, T. Matsushima, and T. Sugimura and was rapidly diluted about five times with cold saline, and mM 2-mercaptoethanol, and 10 mM glucose for two hours at tumor cells were harvested by centrifuging at 100 X g for 3 25 volts/cm. After electrophoresis, bands of hexokinase iso minutes. After three to four repetitions of washing in this way, zymes were stained by placing the membrane en a 1% agar gel the tumor cells became almost free from contaminating red plate which had been laid on a glass and contained 0.1 M or blood cells, leukocytes, or peritoneal cells, as determined by 0.5 mM glucose, 5 mM ATP, 5 mM MgCl2, 0.4 lU/ml G6PD, 1 examination under a light microscope. Tumor cells were final mM NADP, 2 mM KCN, 25 Mg/ml phenazine methosulfate, 0.4 ly well packed by centrifuging at 1,400 X g for 5 minutes. mg/ml nitroblue tetrazolium, and 0.1 M Tris-HCl buffer (pH Normal Adult Regenerating and Fetal Livers. For the nor 7.4). Staining was carried out for 40 minutes in the dark at 37Â°C.Densitometry of hexokinase isozyme bands on a mem mal control, Donryu strain rats 9 to 10 weeks old were mainly used. Buffalo and Wistar strain rats were also checked, but not brane was performed with a Densicord, Model 542 of Photo- much difference was detected among these three strains. The volt with a 525 m/Z filter. As a blank, glucose and ATP were regenerating liver was obtained 24 hours after the removal of omitted from the agar gel plate for staining. about two thirds of the liver of a male Donryu strain rat 9 to 10 weeks old. The fetal liver was collected from fetuses from a RESULTS pregnant rat of the same strain 2 to 5 days prior to the ex pected parturition. The normal and the regenerating livers Hexokinase Activity. The sum of activities of hexokinase were thoroughly perfused with cold saline, and the fetal liver and glucokinase assayed with 0.1 M glucose on the enzyme was carefully rinsed with cold saline. extracts of the normal rat liver, Morris hepatomas, Yoshida Preparation of Enzyme Extracts. The livers or the tumor ascites hepatomas, Yoshida sarcomas, the regenerating liver, tissues of Morris hepatomas were homogenized in an equal and the fetal liver are shown in Table 1. The value was ex volume of cold 0.1 M Tris-HCI buffer (pH 7.4) containing 5 pressed as the specific activity. Table 1 also includes the data ntiM EDTA2, 5 mM 2-mercaptoethanol, and 10 mM glucose on the survival time of the rat bearing each tumor. The specific with a Potter-Elvehjem type homogenizer with a Teflon pestle. activities of Morris hepatomas were much lower than that of The cells of Yoshida ascites hepatomas and sarcomas were the normal liver, in accordance with the results obtained by homogenized in three volumes of the same buffer with a Vir- Weinhouse (29), while the activities of all strains of Yoshida Tis 45 homogenizer operating at 25,000 rpm for 4 minutes. hepatomas and Yoshida sarcomas were markedly higher than These homogenates were centrifuged at 105,000 X g for 60 that of the normal liver. The activity of the regenerating liver minutes or at 164,000 X g for 40 minutes. The supernatant was almost the same as that of the normal liver, and the fetal thus obtained was used as an enzyme extract for the assay of liver had less hexokinase activity than the normal liver. hexokinase activity and for the electrophoresis of hexokinase Patterns of Hexokinase Isozymes on Electrophoresis. In isozymes. Chart 1, schematic patterns of hexokinase isozymes on the Enzyme Assay. The sum of hexokinase and glucokinase ac normal liver, all strains of Morris hepatomas, Yoshida ascites tivities was assayed spectrophotometrically at 30Â°Cby measur hepatomas, and Yoshida sarcomas, the regenerating liver, and ing the rate of NADPH2 formation at 340 m/i. One ml of the the fetal liver stained with 0.1 M glucose are given. reaction mixture contained the following at the final concen Photographs of the typical hexokinase isozyme patterns of trations indicated: 0.1 M glucose, 5 mM ATP sodium salt, 5 the normal liver, Morris hepatomas, Yoshida ascites hepa mM MgCl2, 0.4 international unit/ml G6PD, 5 mM NADP tomas, Yoshida sarcomas, the regenerating liver, and the fetal tetrasodium salt, 5 mM 2-mercaptoethanol, and 0.3 M Tris- liver are given in Figs. 1â€”3,and their densitometoric tracings HCl buffer (pH 7.4). The reaction mixture included also 0.1 on membrane are illustrated in Charts 2â€”4.The normal rat ml of the appropriately diluted enzyme extract. Continuous liver showed four bands migrating to the anode and one faint recording of the optical density was performed with a Gilford band on the cathodic side. Since the cathodic band was stained multiple sample absorbance recorder, model 2000. One unit of even in the absence of glucose and ATP in the staining gel, this hexokinase activity was expressed as one micromole of glu- band was not regarded as an isozyme of hexokinase. This band cose-6-phosphate formed per minute. This assay method is a may correspond to that described as alcohol dehydrogenase by slight modification of that described by Salas et al. (21). Pro Kaplan and Beutler (2, 10). The four anodic bands were Types tein was measured by the method of Lowry et al. with crystal I, II, HI, and IV hexokinases in order of increasing mobility line bovine serum albumin as a standard (14). according to Katzen and Schimke (12). Electrophoresis. Three or six jul of the enzyme extract was Types I, II, and III hexokinases were stained throughout the placed at the center of a cellulose acetate membrane (Gelman, whole experiment at glucose concentration of either 0.1 M or 1 x 6-f inches). Electrophoresis was carried out at 0Â°C,using 0.5 mM. The color intensities of Types I and II hexokinases veronal buffer (pH 8.6, I = 0.05) containing 5 mM EDTA, 1 did not change with both concentrations of glucose. Type III hexokinase was stained slightly more intensely at 0.5 mM glu cose than at 0.1 M glucose. The band of Type IV hexokinase was very weak at 0.5 mM glucose, but markedly stained at 0.1 M glucose concentration. From these observations it is evident 2The abbreviations used are: NADP, nicotinamide adenine that Types I, II, and III bands are lowiCm hexokinases, while dinucleotide phosphate; NADPH2, reduced nicotinamide adenine Type IV band is a hexokinase with a high Km value for glu dinucleotide phosphate; G6PD, gJucose-6-phosphate dehydrogenase; cose, i.e., glucokinase as described by Katzen and Schimke EDTA, ethylenediaminetetraacetic acid. (12).

1438 CANCER RESEARCH VOL. 29

Table 1

Activities Survival times (units/gm protein) (days) Normal liver 16.2" (11.3,12.7,16.4, 18.1, 22.4*) Morris hepatoma 7316A (2.8, 3.9, 4.9, 5.9, 6.1) Morris hepatoma 7793 6.8 (2.3, 4.6, 8.1, 12.2) 28-35 Morris hepatoma 7794A 4.8 (1.7,7.8) 90-180 Morris7795Yoshida hepatoma 2.8(2.6,3.0)20.131.338.2 287821 ascites hepatoma AH13AH 44AH62F (22.9, 25.0, 32.2, 54.4, 56.3) AH65CAH 13.410.638.038.424.3 101171018 66AH66FAH84AAH

127 (10.6,22.2,40.1) AH 130 38.6 (13.6,63.5) 11 AH 150A 63.2 (65.4,61.0) 13 AH225A 18.4 (15.6,21.2) 125-7127-830 AH272AH 30.327.3 7974Yoshida (11.0,12.3,29.0,55.8)39.143.7 sarcomaLY5LY52LY54LY80Regenerating (33.7,40.2,57.3) 52.744.122.320.7 7-107-107-10

liver (18.3,20.9,22.8) Fetal liver4.7 4.7 (4.2,5.2)21 Total hexokinase activities of normal liver, tumors, regenerating and fetal livers, and survival times of tumor-bearing rats. "Average value. *Value of individual determination.

Hexokinase Patterns of Hepatomas and Sarcomas. All strains Hexokinase Patterns of Regenerating Liver and Fetal Liver. of Morris hepatomas examined (Nos. 7316A, 7793, 7794A, Fig. 3 represents the hexokinase isozyme patterns of the regen and 7795) had Types I, II, III, and IV hexokinases, among erating and the fetal livers. In both cases, four types of hexo which Type II hexokinase was predominant. Type IV band in kinases were present, but Type IV was weak and Type II was Morris hepatomas was faint and slightly shifted to the anode relatively intense. from the position of Type IV in the normal liver. This band Duplication of Type IV Hexokinase. The duplication of was weaker with 0.5 mM glucose than with 0.1 M glucose. Type IV band was often observed on the cellulose acetate Four strains of Morris hepatomas had almost the same patterns membrane, and slow and fast forms were designated IVS and of hexokinase isozymes. A faint cathodic band which was re IVf respectively. Hansen et al. (9, 18) reported that the band garded nonspecific was also detected in all cases. of Type IV hexokinase duplicated on starch gel electrophoresis In all strains of Yoshida ascites hepatomas, Types I and II when it was carried out in the absence of EDTA in veronal hexokinases were recognized, and some strains showed a weak buffer. In our experiments, the duplication of Type IV hexo band of Type III hexokinase. No strain had Type IV hexo kinase was found either with or without EDTA in veronal kinase. Type II hexokinase was remarkable in all strains. It buffer in some normal livers, in pregnant rat livers, and in may be noticed that rats bearing Yoshida ascites hepatomas some livers of hepatoma-bearing rats, but in some normal livers with Type III hexokinase exhibited longer survival times than the duplication was not observed under any condition, even in those without it. the absence of EDTA in the extracting buffer. Yoshida sarcoma and the derived strains were subjected to analyses of hexokinase isozyme patterns. Yoshida sarcomas, DISCUSSION the original strain and the derived strains, LY 52, LY 54, and LY 80, showed almost the same hexokinase isozyme patterns A method for the rapid, distinct, and reproducible demon as Yoshida hepatomas. The rats bearing these tumors survived stration of hexokinase isozymes in animal tissues by electro about a week after transplantation. Exceptionally, a strain, LY phoresis on cellulose acetate membrane was established. In 5, which allows the rats bearing this tumor to survive for about contrast to starch gel electrophoresis for separation of hexo a month, showed relatively intense Type III hexokinase as well kinase isozymes, cellulose acetate membranes are readily avail as weak Type IV hexokinase. able, and much time required for electrophoresis and staining

JULY 1969 1439

- Origin experiments, some livers showed the duplication of Type IV *I1' M IV hexokinase even in the presence of EDTA in veronal buffer. This was also observed by Shatton et al. (25). The band of Normal liver il Type IV hexokinase was detected also in Morris hepatomas, a strain of Yoshida sarcoma and the fetal liver. In these cases the Morrishepatoma 7316A\A ID band shifted a little further to the anode as represented in Morrishepatoma 7793 1 i a Chart 1, but it remains to be investigated that these bands Morrishepatoma 7794A IKD1 1 e o Morris hepatoma 7795 o i B D Yoshidaascites hepatoma AH 13 AH 44 AH 62F AH 65C AH 66 Liver AH 66F A H 84 A AH 127 AH 130 AH I50A A H 225 A A H 272 A H 7974 Yoshidasarcoma LY5 Morris Hepatoma LY52 D LY54 73I6A LY80 Regenerating liver I Fetal liver Chart 1. Schematic representation of hexokinase isozyme patterns of normal liver, Morris hepatomas, Yoshida ascites hepatomas, Yoshida sarcomas, and regenerating and fetal livers. Biack, hatched, and open bars indicate intensely, moderately, and weakly stained bands at 0.1 M glucose respectively.

Yoshida Hepatoma is saved. A number of samples can be applied at the same time in a single chamber, and much smaller amounts of the reagents AH 127 were required for staining the isozyme bands. Moreover, the pattern of hexokinase isozymes of the normal liver obtained with this method is reproducible and coincides well to that on starch gel electrophoresis reported by Katzen and Schimke

The duplication of Type IV hexokinase was often detected in the livers of normal, pregnant, and some hepatoma-bearing rats. Pilkis and Hansen (18) described an immunologie differ Chart 2. Densitometric tracings of hexokinase isozyme bands on cel ence between Types IVf and IVS hexokinases, and they noted lulose acetate membranes of normal liver, Morris hepatoma 7316A, and that Type IVf disappeared in fasting and diabetes. Type IVS Yoshida ascites hepatoma AH 127. Hexokinase isozyme bands were hexokinase was also found in the kidney and testis. In our stained at 0.1 M glucose.

1440 CANCER RESEARCH VOL. 29

The hexokinase isozyme patterns of Morris hepatomas used in the present experiment are not so deviated from that of the normal liver except for the relative predominance of Type II hexokinase and the decrease of Type IV hexokinase. Yoshida ascites hepatomas are derived from hepatomas in duced by azo dyes, dimethylaminoazobenzene, or 3'-methyl-di- Liver methylaminoazobenzene (30). Their morphologic and bio chemical properties are fairly deviated from those of the nor-

Liver Yoshida Sarcoma

Regenerating Liver

Yoshida Sarcoma LY5

Chart 3. Densitometric tracings of hexokinase isozyme bands of nor mal liver, Yoshida sarcoma, original strain, and Yoshida sarcoma LY 5. Hexokinase isozyme bands were stained at 0.1 M glucose. Fetal Liver

correspond to Type IVf hexokinase. In any case, these findings indicate the existence of glucokinase also in some tumor tissues. Shatton et al. (25) also detected Type IV hexokinase in Morris hepatomas in addition to Types I, II, and III. The dupli cations of Type I and II hexokinase bands have been also Chart 4. Densitometric tracings of hexokinase isozyme bands of nor observed with starch gel electrophoresis (10â€”12), but these mal, regenerating, and fetal livers. Hexokinase isozyme bands were were not noticed in our experiment. stained at 0.1 M glucose.

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Downloaded from cancerres.aacrjournals.org on September 26, 2021. © 1969 American Association for Cancer Research. S. Sato, T. Matsushima, and T. Sugimura mal liver. They grow very fast, and the survival times of rats the paniculate hexokinase in Yoshida ascites hepatomas. The bearing them are four days to two weeks. Among seventy molecular species of hexokinase, released by Triton X-100, strains of Yoshida ascites hepatomas now available, we ex were the same as those of the soluble fraction. amined the hexokinase isozyme patterns in 13 strains, each of The regenerating and the fetal livers also possessed hexoki which showed different characteristics in survival times of rats nase isozyme patterns similar to those of hepatomas. Since bearing them, in the form of cell aggregation (free or island they are also rapidly proliferating, the possibility still remains forming), in chromosome number, or in sensitivity to chemo- that the predominance of Type II hexokinase might be merely therapeutic agents. All strains investigated had high hexokinase a reflection of the rapid growth of cells. activities, and they showed Type I and predominant Type II hexokinase isozymes. The appearance of Type III hexokinase was noticed in some strains of Yoshida ascites hepatomas, and ACKNOWLEDGMENTS the rats bearing these tumors survived a fairly longer period. No correlation was observed between the form of cell aggrega The authors are indebted to Dr. Hiroshi Sato, Sasaki Institute, Tokyo, tion or the chromosome number and the presence of Type III and to Dr. Harold P. Morris, NIH, Bethesda, Md., for giving us the strains of transplantable hepatomas of rats. The authors acknowledge hexokinase. the valuable discussion held with Dr. Waro Nakahara, Director of this Yoshida sarcoma was originally regarded as a reticulum cell Institute, and with Dr. Takashi Kawachi of this department. They wish sarcoma, but recently it is assumed to be a hepatoma. Hexo to express sincere thanks to Dr. Sidney Weinhouse, Fels Research Insti kinase isozyme patterns of Yoshida sarcomas were quite simi tute, Philadelphia, Pa., for reading this manuscript. lar to those of Yoshida ascites hepatomas. However, in the cells of LY 5, which grow slowly and kill animals about a month after the transplantation, Type III hexokinase was de REFERENCES tected in addition to Types I and II. Furthermore, it was of interest to note the presence of Type IV hexokinase. This 1. Adelman, R. C., Morris, H. P., and Weinhouse, S. Fructokinase, pattern was really close to those of Morris hepatomas. From Triokinase, ana Aldolases in Liver Tumors of the Rat. Cancer Res., these observations it may be suggested that the absence of 27: 2408-2413.1967. hexokinases III and IV in the original Yoshida sarcoma cells 2. Beutler, E., Shaw, C. R., Koen, A. L., Cuatrecasas, P., and Segal, S. does not necessarily mean the deletion of genes for these hexo "Galactose Dehydrogenase", "Nothing Dehydrogenase", and Alco kinase isozymes, but rather indicates blockage of their expres hol Dehydiogenase : Interrelation. Science, 156: 1516â€”1518, sion. Results obtained in our experiments coincide well with 1967. recent observations of Gumaa and Greenslade (8), who also 3. Brown, J., Miller, D. M., HoUoway, M. T., and LevÃ©,G.D. Hexoki reported predominant Type II hexokinase in experimental nase Isozymes in Liver and Adipose Tissue of Man and Dog. solid hepatomas and ascites tumors, and also with Shatton et Science, 155: 205-207, 1967. al. (25), who described hexokinase isozyme patterns in hepa 4. Davidova, S. Y., Shapot, V. S., and Solowjewa, A. A. Hexokinase Activity and Glycolytic Capacity of Plasma Membranes of Hepato tomas and other tumors. mas. Biochim. Biophys. Acta, 158: 303-305, 1968. Nakamura and Hosoda (16) detected a very low or almost 5. Emmelot, P., and Bos, C. J. Differences in the Association of Two nondetectable concentration of glucose in ascitic fluid of Glycolytic Enzymes with Plasma Membranes Isolated from Rat Ehrlich ascites tumor. This may be also true for Yoshida Liver and Hepatoma. Biochim. Biophys. Acta, 121: 434â€”436, ascites hepatomas and Yoshida sarcomas. In the intramuscular 1966. ly transplanted Morris hepatoma tissues, the vascularization is 6. GonzÃ¡lez,C., Ureta, T., Sanchez, R., and Niemeyer, H. Multiple supposed to be insufficient compared with the normal livers, Molecular Forms of ATP: Hexose 6-Phosphotransferase from Rat and the exogeneous supply of glucose is also thought to be Liver. Biochem. Biophys. Res. Commun., 16: 347â€”352, 1964. small. Under such circumstances, it seems reasonable that low 7. Grossbard, L., Weksler, M., and Schimke, R. T. Electrophoretic Km hexokinases would become dominant in such deviated tu Properties and Tissue Distribution of Multiple Forms of Hexoki nase in Various Mammalian Species. Biochem. Biophys. Res. mors as Yoshida ascites and Morris hepatomas. Commun., 24: 32-38,1966. In hepatocarcinogenesis the gene for high Km hexokinase 8. Gumaa, K. A., and Greenslade, K. R. Molecular Species of Hexoki might be switched off, and, in turn, the expression of the nase in Hepatomas and Ascites-Tumour Cells. Biochem. J., 107: genes for low Km hexokinases, especially for Type II hexo 22p, 1968. kinase, might be exaggerated. Similar observations have been 9. Hansen, R., Pilkis, S. J., and Krahl, M. E. Properties of Adaptive reported on aldolase (1, 15, 27), pyruvate kinase (26, 28), and Hexokinase Isozymes of the Rat. Endocrinology, Si: 1397-1404, fructose 1,6-diphosphatase (22). Such a phenomenon has been 1967. referred to as "switch-off and -on mechanism of gene on car- 10. Kaplan, J. C., and Beutler, E. Hexokinase Isozymes in Human cinogenesis" or "disdifferentiation" (27). Potter recently ex Erythrocytes. Science, 159: 215-216, 1968. pressed this as "oncogeny as blocked ontogeny" (19). An in 11. Katzen, H. M. The Effect of Diabetes and Insulin in vivo and in vitro on a Low Km Form of Hexokinase from Various Rat Tissues. crease of hexokinase activity has been described also in Biochem. Biophys. Res. Commun., 24: 531â€”536, 1966. cultured mammalian cells transformed to a malignant type by 12. Katzen, H. M., and Schimke, R. T. Multiple Forms of Hexokinase polyoma virus (17). in the Rat: Tissue Distribution, Age Dependency, and Properties. The presence of the particle-bound hexokinase in ascites tu Proc. Nati. Acad. Sei. U. S., 54: 1218-1225,1965. mor cells and experimental hepatomas has been reported by 13. Kosow, D. P., and Rose, I. A. Ascites Tumor Mitochondrial Hexo many workers (4, 5, 13, 20). We also observed the presence of kinase. II. J. Biol. Chem., 243: 3623-3630,1968.

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14. Lowry, O. H., Rosebrough, N. J., Farr, A. L., and Randall, R. J. Liver and Skeletal Muscle of the Mouse. Biochim. Biophys. Acta, Protein Measurement with the Polin Phenol Reagent. J. Biol. 159: 130-140, 1968. Chem., 193: 265-275, 1951. 23. SchrÃ¶ter, W., and Tillmann, W. Hexokinase Isozymes in Human 15. Matsushima, T., Kawabe, S., Shibuya, M., and Sugimura, T. Aldo- Erythrocytes of Adults and New Borns. Biochem. Biophys. Res. lase Isozymes in Rat Tumor Cells. Biochem. Biophys. Res. Commun., 31: 92-97, 1968. Commun., 30: 565-570, 1968. 24. Sharma, R. M., Sharma, C., Donnelly, A. J., Morris, H. P., and 16. Nakamura, W., and Hosoda, S. The Absence of Glucose in Ehrlich Weinhouse, S. Glucose-ATP Phosphotransferases during Hepatocar- Ascites Tumor Cells and Fluid. Biochim. Biophys. Acta, 158: cinogenesis. Cancer Res., 25: 193â€”199,1965. 712-218,1968. 25. Shatton, J. B., Morris, H. P., and Weinhouse, S. Kinetic, Electro- 17. Paul, J. Metabolic Processes in Normal and Cancer Cells. E. J. phoretic and Chromatographie Studies on Glucose-ATP Phospho- Ambrose, and F. J. C. Roe, (eds.), The Biology of Cancer, pp. transferase in Rat Hepatomas. Cancer Res., 29: 1161-1172, 1969. 52â€”64.London: D. Van Nostrand Company, Inc., 1966. 26. Suda, M., Tanaka, T., Sue, F., Harano, Y., and Morimura, H. Dedif- 18. Pilkis, S. J., and Hansen, R. J. Resolution of Two High-Km ATP: ferentiation of Sugar Metabolism in the Liver of Tumor-bearing D-Hexose 6-Phosphotransferase Bands by Starch-gel Electrophore- Rat. Gann Monograph, Ã•:127-141, 1966. sis. Biochim. Biophys. Acta, 159: 189-191, 1968. 27. Sugimura, T., Matsushima, T., Kawachi, T., Hirata, Y., and 19. Potter, V. R. Recent Trends in Cancer Biochemistry. 8th Bienn. Kawabe, S. Molecular Species of Aldolases and Hexokinases in Ex Can. Cancer Conf., in press. perimental Hepatomas. Gann Monograph, Ã•:43â€”49,1966. 20. Rose, I. A., and Warms, J. V. B. Mitochondrial Hexokinase. Re 28. Tanaka, T., Harano, Y., Morimura, H., and Mori, R. Evidence for lease, Rebinding and Location. J. Biol. Chem., 242: 1635-1645, the Presence of Two Types of Pyruvate Kinase in Rat Liver. 1967. Biochem. Biophys. Res. Commun; 21: 55â€”60,1965. 21. Salas, M., ViÃ±uela,E., and Sols, A. Insulin-dependent Synthesis of 29. Weinhouse, S. Glycolysis, Respiration and Enzyme Deletions in Liver Glucokinase in the Rat. J. Biol. Chem., 238: 3535â€”3538, Slow-growing Hepatic Tumors. Gann Monograph, 1: 99â€”115, 1963. 1966. 22. Sato, K., and Tsuiki, S. Fructose 1,6-Diphosphatase of Mouse 30. Yoshida, T., and Sato, H. Ascites Tumor-Yoshida Sarcoma and Ehrlich Ascites Tumor and its Comparison with the Enzymes of Ascites Hepatoma(s). NCI Monograph, 16: 1964.

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3 2 lorigin +"i 2 3 4 cm

Liver

Morris Hepatoma 73I6A Morris Hepatoma 7793 Yoshida Hepatoma AH7974

AH 62 F i AH 127

Fig. 1. Hexokinase isozyme patterns of normal liver, Morris hepatomas, and Yoshida ascites hepatomas. Hexokinase isozyme bands were stained at 0.1 M glucose.

1444 CANCER RESEARCH VOL. 29

origin + 32 l * i 2 3 4 cm

Liver

Yoshida Sarcoma

Yoshida Sarcoma LY 52 LY54

LY5

Fig. 2. Hexokinase isozyme patterns of normal liver and Yoshida sarcomas. Hexokinase isozyme bands were stained at 0.1 M glucose.

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origin + 3â€¢ 2 i li 4 i i 2 3 4 cm

Liver

Regenerating Liver Fetal Liver 3

Fig. 3. Hexokinase isozyme patterns of normal, regenerating and fetal livers. Hexokinase isozyme bands were stained at 0.1 M glucose.

1446 CANCER RESEARCH VOL. 29

Shigeaki Sato, Taijiro Matsushima and Takashi Sugimura

Cancer Res 1969;29:1437-1446.

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