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[CANCER RESEARCH 29, 1437-1446, July 1969] Hexokinase Isozyme Patterns of Experimental Hepatomas of Rats1 ••'<-*,V*:te¿-v*:.'J '. Shigeaki Sato, Taijiro Matsushima, and Takashi Sugimura Biochemistry Division, National Cancer Center Research Institute, Tsukiji, Chuo-ku, Tokyo, Japan SUMMARY Hexokinase isozymes in rat tissues were electrophoretically separated on cellulose acetate membrane. The method was very quick and gave reproducible results. By using this method, hexokinase isozyme patterns were studied on normal rat liver and experimental hepatomas with differing growth rates and degrees of differentiation. In normal rat liver, the hexokinase pattern obtained on cellu lose acetate membrane was identical with that obtained on starch gel by previous workers. There were four types of hexokinases, which corresponded to Types I, II, III, and IV hexokinases according to Katzen and Schimke, in order of increas ing mobility from the origin to the anode. Type IV hexokin ase, which was stained most intensely, was glucokinase, and duplication of Type IV hexokinase into two bands (IVS, slow form, and IVf, fast form) was occasionally observed. Morris hepatomas, as a slowly growing and less deviated hepatoma group, showed relatively predominant Type II hexo kinase in addition to Types I and III hexokinases, and they also had a faint Type IV hexokinase band. A rapidly growing and highly deviated hepatoma group, Yoshida ascites hepa tomas were characterized by the presence of only Type I hexo kinase and marked Type II hexokinase and by the loss of Type IV and Type III hexokinases. Yoshida sarcoma showed a simi lar hexokinase pattern to that of Yoshida ascites hepatomas. A slowly growing substrain of Yoshida sarcoma, LY 5, had also Type III and Type IV hexokinases, resembling Morris hepa tomas. These observations suggested that the genes for Types III and IV hexokinase isozymes were not deleted, but their expressions were blocked in the original Yoshida sarcoma. The regenerating liver was analogous to Morris hepatomas in hexokinase pattern. In fetal liver, Type II hexokinase was dominant, in addition to Types I, III, and weak IV hexo kinases. tive factor for the rate of glycolysis in various tumor strains (29). Meanwhile Gonzalez et al. (6) in 1964 separated rat liver hexokinase into four types with diethylaminoethyl cellulose column chromatography. Katzen and Schimke (12) in 1965 succeeded in separating four types of hexokinase in rat tissues with starch gel electrophoresis and mentioned the presence of the specific isozyme pattern in the specific tissue. Following these pioneering works, the hexokinase isozyme patterns of many tissues, including human materials, have been elucidated, and the enzymatic properties of each type of isozyme have been studied (3, 7, 10, 23). While many papers on isozyme patterns of hexokinase in the normal tissues have been pub lished, there have been only recent reports by Gumaa and Greenslade (8) and Shatton et al. (25) on the hexokinase iso zyme patterns of experimental hepatomas. From the studies using various concentrations of glucose, Sharma et al. (24) also reported relatively increased "low-iCm hexokinase" activities and decreased "highJCm -specific glucokinase" activity during experimental hepatocarcinogenesis in rats. We suggested the presence of two types of hexokinases in Yoshida ascites hepa toma cells by using different concentrations of glucose (27). In this paper we describe a new method for electrophoretic separation of hexokinase isozymes on cellulose acetate mem brane. By using this method, the hexokinase isozyme patterns were investigated on two groups of experimental hepatomas of rats, Morris hepatomas as a less deviated and slowly growing group, and Yoshida ascites hepatomas as a highly deviated and rapidly growing one. Yoshida sarcoma and its subs trains, in cluding a strain with slower growth rate than the original strain, were also investigated. The hexokinase isozyme patterns thus obtained on hepatomas were compared with those in the normal liver, the regenerating liver, and fetal liver. The prob lem of cellular disdifferentiation on carcinogenesis based on the experimental results will be discussed. MATERIALS AND METHODS INTRODUCTION An increased rate of anaerobic glycolysis had been regarded for long to be one of the characteristics of tumor tissues. In 1966, however, Weinhouse (29) pointed out that increased glycolysis was not always observed in Morris hepatomas. He described also that the level of hexokinase [ATP:D-hexose 6-phosphotransferase (EC.2.7.1.1)] activity was a determina- Supported in part by a grant from the Ministry of Education and Ministry of Health, Japan. Received October 7, 1968; accepted March 26, 1969. JULY 1969 Tumors. Morris hepatomas, Nos. 7316A, 7793, 7794A, and 7795, were transplanted intramuscularly in both hind legs of about 9- to 10-week-old Buffalo strain rats. They were main tained on diet CE-2 (diet from the Central Laboratory of Ex perimental Animal, Japan). Three to four weeks after trans plantation, rats were killed by decapitation, and tumors were removed; necrotic, hemorrhagic, and nontumorous materials were carefully trimmed off. About one million cells of Yoshida ascites hepatomas or sarcomas were intraperitoneally transplanted into Donryu strain male rats 9 to 10 weeks old. Five to ten days later, the rats were killed by decapitation. The ascitic fluid was aspirated 1437 Downloaded from cancerres.aacrjournals.org on June 17, 2017. © 1969 American Association for Cancer Research. S. Sato, T. Matsushima, and T. Sugimura and was rapidly diluted about five times with cold saline, and tumor cells were harvested by centrifuging at 100 X g for 3 minutes. After three to four repetitions of washing in this way, the tumor cells became almost free from contaminating red blood cells, leukocytes, or peritoneal cells, as determined by examination under a light microscope. Tumor cells were final ly well packed by centrifuging at 1,400 X g for 5 minutes. Normal Adult Regenerating and Fetal Livers. For the nor mal control, Donryu strain rats 9 to 10 weeks old were mainly used. Buffalo and Wistar strain rats were also checked, but not much difference was detected among these three strains. The regenerating liver was obtained 24 hours after the removal of 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 pregnant rat of the same strain 2 to 5 days prior to the ex pected parturition. The normal and the regenerating livers were thoroughly perfused with cold saline, and the fetal liver was carefully rinsed with cold saline. Preparation of Enzyme Extracts. The livers or the tumor tissues of Morris hepatomas were homogenized in an equal volume of cold 0.1 M Tris-HCI buffer (pH 7.4) containing 5 ntiM EDTA2, 5 mM 2-mercaptoethanol, and 10 mM glucose with a Potter-Elvehjem type homogenizer with a Teflon pestle. The cells of Yoshida ascites hepatomas and sarcomas were homogenized in three volumes of the same buffer with a VirTis 45 homogenizer operating at 25,000 rpm for 4 minutes. These homogenates were centrifuged at 105,000 X g for 60 minutes or at 164,000 X g for 40 minutes. The supernatant thus obtained was used as an enzyme extract for the assay of hexokinase activity and for the electrophoresis of hexokinase isozymes. Enzyme Assay. The sum of hexokinase and glucokinase ac tivities was assayed spectrophotometrically at 30°Cby measur ing the rate of NADPH2 formation at 340 m/i. One ml of the reaction mixture contained the following at the final concen trations indicated: 0.1 M glucose, 5 mM ATP sodium salt, 5 mM MgCl2, 0.4 international unit/ml G6PD, 5 mM NADP tetrasodium salt, 5 mM 2-mercaptoethanol, and 0.3 M TrisHCl buffer (pH 7.4). The reaction mixture included also 0.1 ml of the appropriately diluted enzyme extract. Continuous recording of the optical density was performed with a Gilford multiple sample absorbance recorder, model 2000. One unit of hexokinase activity was expressed as one micromole of glucose-6-phosphate formed per minute. This assay method is a slight modification of that described by Salas et al. (21). Pro tein was measured by the method of Lowry et al. with crystal line bovine serum albumin as a standard (14). Electrophoresis. Three or six jul of the enzyme extract was placed at the center of a cellulose acetate membrane (Gelman, 1 x 6-f inches). Electrophoresis was carried out at 0°C,using veronal buffer (pH 8.6, I = 0.05) containing 5 mM EDTA, 1 2The abbreviations used are: NADP, nicotinamide adenine dinucleotide phosphate; NADPH2, reduced nicotinamide adenine dinucleotide phosphate; G6PD, gJucose-6-phosphate dehydrogenase; EDTA, ethylenediaminetetraacetic acid. 1438 mM 2-mercaptoethanol, and 10 mM glucose for two hours at 25 volts/cm. After electrophoresis, bands of hexokinase iso zymes were stained by placing the membrane en a 1% agar gel plate which had been laid on a glass and contained 0.1 M or 0.5 mM glucose, 5 mM ATP, 5 mM MgCl2, 0.4 lU/ml G6PD, 1 mM NADP, 2 mM KCN, 25 Mg/ml phenazine methosulfate, 0.4 mg/ml nitroblue tetrazolium, and 0.1 M Tris-HCl buffer (pH 7.4). Staining was carried out for 40 minutes in the dark at 37°C.Densitometry of hexokinase isozyme bands on a mem brane was performed with a Densicord, Model 542 of Photovolt with a 525 m/Z filter. As a blank, glucose and ATP were omitted from the agar gel plate for staining. RESULTS Hexokinase Activity. The sum of activities of hexokinase and glucokinase assayed with 0.1 M glucose on the enzyme extracts of the normal rat liver, Morris hepatomas, Yoshida ascites hepatomas, Yoshida sarcomas, the regenerating liver, and the fetal liver are shown in Table 1. The value was ex pressed as the specific activity. Table 1 also includes the data on the survival time of the rat bearing each tumor. The specific activities of Morris hepatomas were much lower than that of the normal liver, in accordance with the results obtained by Weinhouse (29), while the activities of all strains of Yoshida hepatomas and Yoshida sarcomas were markedly higher than that of the normal liver. The activity of the regenerating liver was almost the same as that of the normal liver, and the fetal liver had less hexokinase activity than the normal liver. Patterns of Hexokinase Isozymes on Electrophoresis. In Chart 1, schematic patterns of hexokinase isozymes on the normal liver, all strains of Morris hepatomas, Yoshida ascites hepatomas, and Yoshida sarcomas, the regenerating liver, and the fetal liver stained with 0.1 M glucose are given. Photographs of the typical hexokinase isozyme patterns of the normal liver, Morris hepatomas, Yoshida ascites hepa tomas, Yoshida sarcomas, the regenerating liver, and the fetal liver are given in Figs. 1—3,and their densitometoric tracings on membrane are illustrated in Charts 2—4.The normal rat liver showed four bands migrating to the anode and one faint band on the cathodic side. Since the cathodic band was stained even in the absence of glucose and ATP in the staining gel, this band was not regarded as an isozyme of hexokinase. This band may correspond to that described as alcohol dehydrogenase by Kaplan and Beutler (2, 10). The four anodic bands were Types I, II, HI, and IV hexokinases in order of increasing mobility according to Katzen and Schimke (12). Types I, II, and III hexokinases were stained throughout the whole experiment at glucose concentration of either 0.1 M or 0.5 mM. The color intensities of Types I and II hexokinases 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 that Types I, II, and III bands are lowiCm hexokinases, while Type IV band is a hexokinase with a high Km value for glu cose, i.e., glucokinase as described by Katzen and Schimke (12). CANCER RESEARCH VOL. 29 Downloaded from cancerres.aacrjournals.org on June 17, 2017. © 1969 American Association for Cancer Research. Rat Hepatoma Isozyme Patterns Table 1 Activities (units/gm protein) Normal liver 16.2" (11.3,12.7,16.4, Morris hepatoma 7316A Morris hepatoma 7793 Morris hepatoma 7794A Morris 7795Yoshida hepatoma (2.8, 3.9, 4.9, 5.9, 6.1) 6.8 (2.3, 4.6, 8.1, 12.2) 4.8 (1.7,7.8) (2.6,3.0)20.131.338.2 2.8 Survival times (days) 18.1, 22.4*) 28-35 90-180 287821 ascites hepatoma 13AH AH 44AH62F AH65CAH 66AH66FAH84AAH 127 AH 130 AH 150A AH225A 272AH AH 7974Yoshida (22.9, 25.0, 32.2, 54.4, 56.3) 13.410.638.038.424.3 (10.6,22.2,40.1) 38.6 (13.6,63.5) 63.2 (65.4,61.0) 18.4 (15.6,21.2) 30.327.3 (11.0,12.3,29.0,55.8)39.143.7 101171018 11 13 125-7127-830 sarcomaLY5LY52LY54LY80Regenerating (33.7,40.2,57.3) 52.744.122.320.7 liver Fetal liver4.7 7-107-107-10 (18.3,20.9,22.8) 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 of Morris hepatomas examined (Nos. 7316A, 7793, 7794A, and 7795) had Types I, II, III, and IV hexokinases, among which Type II hexokinase was predominant. Type IV band in Morris hepatomas was faint and slightly shifted to the anode from the position of Type IV in the normal liver. This band was weaker with 0.5 mM glucose than with 0.1 M glucose. Four strains of Morris hepatomas had almost the same patterns of hexokinase isozymes. A faint cathodic band which was re garded nonspecific was also detected in all cases. In all strains of Yoshida ascites hepatomas, Types I and II hexokinases were recognized, and some strains showed a weak band of Type III hexokinase. No strain had Type IV hexo kinase. Type II hexokinase was remarkable in all strains. It may be noticed that rats bearing Yoshida ascites hepatomas with Type III hexokinase exhibited longer survival times than those without it. Yoshida sarcoma and the derived strains were subjected to analyses of hexokinase isozyme patterns. Yoshida sarcomas, the original strain and the derived strains, LY 52, LY 54, and LY 80, showed almost the same hexokinase isozyme patterns as Yoshida hepatomas. The rats bearing these tumors survived about a week after transplantation. Exceptionally, a strain, LY 5, which allows the rats bearing this tumor to survive for about a month, showed relatively intense Type III hexokinase as well as weak Type IV hexokinase. JULY 1969 Hexokinase Patterns of Regenerating Liver and Fetal Liver. Fig. 3 represents the hexokinase isozyme patterns of the regen erating and the fetal livers. In both cases, four types of hexo kinases were present, but Type IV was weak and Type II was relatively intense. Duplication of Type IV Hexokinase. The duplication of Type IV band was often observed on the cellulose acetate membrane, and slow and fast forms were designated IVS and IVf respectively. Hansen et al. (9, 18) reported that the band of Type IV hexokinase duplicated on starch gel electrophoresis when it was carried out in the absence of EDTA in veronal buffer. In our experiments, the duplication of Type IV hexo kinase was found either with or without EDTA in veronal buffer in some normal livers, in pregnant rat livers, and in some livers of hepatoma-bearing rats, but in some normal livers the duplication was not observed under any condition, even in the absence of EDTA in the extracting buffer. DISCUSSION A method for the rapid, distinct, and reproducible demon stration of hexokinase isozymes in animal tissues by electro phoresis on cellulose acetate membrane was established. In contrast to starch gel electrophoresis for separation of hexo kinase isozymes, cellulose acetate membranes are readily avail able, and much time required for electrophoresis and staining 1439 Downloaded from cancerres.aacrjournals.org on June 17, 2017. © 1969 American Association for Cancer Research. S. Sato, T. Matsushima, and T. Sugimura - Origin I1' * M IV il Normal liver \A Morrishepatoma 7316A Morris hepatoma 7793 Morris hepatoma 7794A Morris hepatoma 7795 I D i a eo o i BD 1 IKD1 1 Yoshidaascites hepatoma AH 13 AH 44 AH 62F AH 65C AH 66 AH 66F A H 84 A AH 127 AH 130 AH I50A A H 225 A A H 272 A H 7974 Liver Yoshidasarcoma LY5 LY52 LY54 LY80 Regenerating liver Fetal liver experiments, some livers showed the duplication of Type IV hexokinase even in the presence of EDTA in veronal buffer. This was also observed by Shatton et al. (25). The band of Type IV hexokinase was detected also in Morris hepatomas, a strain of Yoshida sarcoma and the fetal liver. In these cases the band shifted a little further to the anode as represented in Chart 1, but it remains to be investigated that these bands D Morris Hepatoma 73I6A I 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 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 ence between Types IVf and IVS hexokinases, and they noted that Type IVf disappeared in fasting and diabetes. Type IVS hexokinase was also found in the kidney and testis. In our 1440 AH 127 Chart 2. Densitometric tracings of hexokinase isozyme bands on cel lulose acetate membranes of normal liver, Morris hepatoma 7316A, and Yoshida ascites hepatoma AH 127. Hexokinase isozyme bands were stained at 0.1 M glucose. CANCER RESEARCH VOL. 29 Downloaded from cancerres.aacrjournals.org on June 17, 2017. © 1969 American Association for Cancer Research. Rat Hepatoma Isozyme Patterns 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- in 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. 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 observed with starch gel electrophoresis (10—12), but these were not noticed in our experiment. Fetal Liver Chart 4. Densitometric tracings of hexokinase isozyme bands of nor mal, regenerating, and fetal livers. Hexokinase isozyme bands were stained at 0.1 M glucose. JULY 1969 Downloaded from cancerres.aacrjournals.org on June 17, 2017. © 1969 American Association for Cancer Research. 1441 S. Sato, T. Matsushima, and T. Sugimura mal liver. They grow very fast, and the survival times of rats bearing them are four days to two weeks. Among seventy strains of Yoshida ascites hepatomas now available, we ex amined the hexokinase isozyme patterns in 13 strains, each of which showed different characteristics in survival times of rats bearing them, in the form of cell aggregation (free or island forming), in chromosome number, or in sensitivity to chemotherapeutic agents. All strains investigated had high hexokinase 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 the rats bearing these tumors survived a fairly longer period. No correlation was observed between the form of cell aggrega tion or the chromosome number and the presence of Type III hexokinase. Yoshida sarcoma was originally regarded as a reticulum cell sarcoma, but recently it is assumed to be a hepatoma. Hexo kinase isozyme patterns of Yoshida sarcomas were quite simi 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 tected in addition to Types I and II. Furthermore, it was of interest to note the presence of Type IV hexokinase. This pattern was really close to those of Morris hepatomas. From these observations it may be suggested that the absence of hexokinases III and IV in the original Yoshida sarcoma cells does not necessarily mean the deletion of genes for these hexo kinase isozymes, but rather indicates blockage of their expres sion. Results obtained in our experiments coincide well with recent observations of Gumaa and Greenslade (8), who also reported predominant Type II hexokinase in experimental solid hepatomas and ascites tumors, and also with Shatton et al. (25), who described hexokinase isozyme patterns in hepa tomas and other tumors. Nakamura and Hosoda (16) detected a very low or almost nondetectable concentration of glucose in ascitic fluid of Ehrlich ascites tumor. This may be also true for Yoshida ascites hepatomas and Yoshida sarcomas. In the intramuscular ly transplanted Morris hepatoma tissues, the vascularization is supposed to be insufficient compared with the normal livers, and the exogeneous supply of glucose is also thought to be small. Under such circumstances, it seems reasonable that low Km hexokinases would become dominant in such deviated tu mors as Yoshida ascites and Morris hepatomas. In hepatocarcinogenesis the gene for high Km hexokinase might be switched off, and, in turn, the expression of the genes for low Km hexokinases, especially for Type II hexo kinase, might be exaggerated. Similar observations have been reported on aldolase (1, 15, 27), pyruvate kinase (26, 28), and fructose 1,6-diphosphatase (22). Such a phenomenon has been referred to as "switch-off and -on mechanism of gene on carcinogenesis" or "disdifferentiation" (27). Potter recently ex pressed this as "oncogeny as blocked ontogeny" (19). An in crease of hexokinase activity has been described also cultured mammalian cells transformed to a malignant type polyoma virus (17). The presence of the particle-bound hexokinase in ascites mor cells and experimental hepatomas has been reported many workers (4, 5, 13, 20). We also observed the presence 1442 in by tu by of the paniculate hexokinase in Yoshida ascites hepatomas. The molecular species of hexokinase, released by Triton X-100, were the same as those of the soluble fraction. The regenerating and the fetal livers also possessed hexoki nase isozyme patterns similar to those of hepatomas. Since they are also rapidly proliferating, the possibility still remains that the predominance of Type II hexokinase might be merely a reflection of the rapid growth of cells. ACKNOWLEDGMENTS The authors are indebted to Dr. Hiroshi Sato, Sasaki Institute, Tokyo, and to Dr. Harold P. Morris, NIH, Bethesda, Md., for giving us the strains of transplantable hepatomas of rats. The authors acknowledge the valuable discussion held with Dr. Waro Nakahara, Director of this Institute, and with Dr. Takashi Kawachi of this department. They wish to express sincere thanks to Dr. Sidney Weinhouse, Fels Research Insti tute, Philadelphia, Pa., for reading this manuscript. REFERENCES 1. Adelman, R. C., Morris, H. P., and Weinhouse, S. 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NCI Monograph, 16: 1964. JULY 1969 Downloaded from cancerres.aacrjournals.org on June 17, 2017. © 1969 American Association for Cancer Research. 1443 5. Sato, T. Matsushima, and T. Sugimura 3 2 l origin +"i 2 3 4 cm Liver Morris Hepatoma 73I6A Morris Hepatoma 7793 Yoshida Hepatoma AH7974 AH 62 F AH 127 i 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 Downloaded from cancerres.aacrjournals.org on June 17, 2017. © 1969 American Association for Cancer Research. Rat Hepatoma Isozyme Patterns 32 origin + l * i 2 3 4 cm Liver Yoshida Sarcoma Yoshida Sarcoma LY 52 LY54 LY5 2 Fig. 2. Hexokinase isozyme patterns of normal liver and Yoshida sarcomas. Hexokinase isozyme bands were stained at 0.1 M glucose. JULY 1969 1445 Downloaded from cancerres.aacrjournals.org on June 17, 2017. © 1969 American Association for Cancer Research. S. Sato, T. Matsushima, and T. Sugimura origin + 3•2i li 4i i 2 3 4 cm Liver Regenerating Liver 3 Fetal Liver 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 Downloaded from cancerres.aacrjournals.org on June 17, 2017. © 1969 American Association for Cancer Research. Hexokinase Isozyme Patterns of Experimental Hepatomas of Rats Shigeaki Sato, Taijiro Matsushima and Takashi Sugimura Cancer Res 1969;29:1437-1446. Updated version E-mail alerts Reprints and Subscriptions Permissions Access the most recent version of this article at: http://cancerres.aacrjournals.org/content/29/7/1437 Sign up to receive free email-alerts related to this article or journal. To order reprints of this article or to subscribe to the journal, contact the AACR Publications Department at [email protected]. To request permission to re-use all or part of this article, contact the AACR Publications Department at [email protected]. Downloaded from cancerres.aacrjournals.org on June 17, 2017. © 1969 American Association for Cancer Research.