[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 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 JULY 1969 1437 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 37°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 30°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.