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Isozyme Studies of Several Enzymes of Carbohydrate Metabolism in Human Adult and Fetal Tissues, Tumor Tissues, and Cell Cultures1

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Isozyme Studies of Several Enzymes of Carbohydrate Metabolism in Human Adult and Fetal Tissues, Tumor Tissues, and Cell Cultures1 [CANCER RESEARCH 38. 1323-1328, May 1978] Isozyme Studies of Several Enzymes of Carbohydrate Metabolism in Human Adult and Fetal Tissues, Tumor Tissues, and Cell Cultures1 Kathryn D. Hammond2 and Doris Balinsky3 Enzyme Research Unit, The South African Institute for Medical Research. Johannesburg, South Africa ABSTRACT role in liver metabolism are frequently replaced in hepatoma by forms characteristic of other adult tissues or of undiffer- The starch gel electrophoretic isozyme patterns of phosphoglucomutase. glucose-6-phosphate dehydrogen- entiated fetal or neonatal liver (10, 28). An earlier study (3) from our own laboratory showed that ase, hexokinase (HK), pyruvate kinase (PK), and lactate the HK4-5 and PK isozyme patterns typical of normal liver dehydrogenase in the cancerous and in the apparently uninvolved regions of livers from human primary hepa- are also altered in human primary hepatomas; occasional differences were observed for LDH. In the present study we toma patients were compared. The HK, PK, and lactate considered it worthwhile to extend our investigation of HK, dehydrogenase isozyme patterns in hepatoma were also PK, and LDH, with improved resolution of HK and PK compared with those in normal adult tissues (liver, mus isozymes, and to examine the isozyme patterns of PGM and cle, dura) and fetal tissues (liver, muscle, heart, brain). In addition, other human tumors (esophageal cancer, men- G6PD in human hepatoma. Also, it was of interest to compare the isozyme patterns in hepatoma and other hu ingioma, mesothelioma) and cell cultures of tumors (hep man tumors with those of fetal tissues. In addition, the atoma, esophageal cancer, HeLa) and fibroblasts were availability of tumor cell lines of hepatoma, esophageal examined. The phosphoglucomutase and glucose-6-phosphate de cancer, and HeLa permitted comparison with these sys tems. hydrogenase patterns in hepatoma were similar to those in host livers, except in one case in which the glucose-6- phosphate dehydrogenase pattern was altered in the MATERIALS AND METHODS tumor. Hepatoma patterns differed from those of normal Substrates, Cofactors, and Enzymes. Monosodium liver in that HK II was present, and the proportion of HK III phosphoenolpyruvate, the disodium salts of ATP, ADP, was reduced; the proportion of another form of HK (II,), NADP , NADH, and AMP, tetracyclohexylammonium fruc- observed in most of the tissues, was increased in hepa tose-1,6-bisphosphate, G6PD, and HK were obtained from toma. In cell lines of hepatoma and esophageal cancer, Boehringer Mannheim, Mannheim, Germany. NAD was the proportion of HK II was increased, compared with the from Sigma Chemical Co., St. Louis, Mo., and lactic acid respective tissues of origin. The PK patterns in host livers and disodium glucose 6-phosphate were from British Drug and hepatomas, although often different from normal Houses, Poole, England. liver, were variable, and there were no consistent trends. Other Chemicals. Dithiothreitol was obtained from Cal- Several host livers and hepatomas had additional bands biochem, Los Angeles, Calif. Phenazine methosulfate (possibly hybrids) between PK L and PK M . All other was from Sigma, and 2-(p-iodophenyl)-3-(p-nitrophenyl)-5- tumor tissues and cell lines had PK M . The lactate phenyl tetrazolium salt was from National Biochemical dehydrogenase patterns in hepatoma differed from the Corp., Cleveland, Ohio. Connaught hydrolyzed starch was liver patterns in only a few cases, when there appeared to from Connaught Medical Research Laboratory, Toronto, be a slight increase in the proportion of H subunits. The Ontario, Canada, and Bacto-Agar was from Difco Labora proportion of H subunits was also increased in hepatoma tories, Detroit, Mich. Other standard chemicals were ob cells. tained from British Drug Houses, E. Merck (Darmstadt, INTRODUCTION 4 The abbreviations used are: HK, hexokinase (ATP:r>hexose 6-phospho- transferase) (EC 2.7.1.1) |HK IV (or GK), the high-Km hexokinase, is generally Studies of experimental rat hepatomas have shown that known as glucokinase (ATP:D-glucose-6-phosphotransferase) (EC 2.7.1.2)]; metabolic alterations occurring in the tumors are associ PK, pyruvate kinase (ATP:pyruvate 2-O-phosphotransferase) (EC 2.7.1.40); LDH, lactate dehydrogenase (L-lactate:NAD- oxidoreductase) (EC 1.1.1.27); ated not only with changes in activities but also with PGM. phosphoglucomutase («-D-glucose-1,6-bisphosphate:(<-o-glucose-1- changes in isozyme patterns of many enzymes of interme phosphate phosphotransferase) (EC 2.7.5.1); G6PD, glucose-6-phosphate diary metabolism (10, 28). Isozymes that play a functional dehydrogenase (D-glucose-6-phosphate:NADPJ 1-oxidoreductase) (EC 1.1.1.49). 5 The isozyme nomenclature used is as follows: G6PD isozymes are ' This work was supported in part by a grant from the Medical Research designated G6PD A', the faster-moving form corresponding to the A' phenotype, and G6PD B', the slower-moving form corresponding to the B' Council of South Africa. 1 This work forms part of a thesis submitted for a Ph.D. degree to the phenotype (5); HK isozymes are designated HK I, HK II. HK III. and HK IV, in University of the Witwatersrand, Johannesburg. South Africa. Present ad order of increasing electrophoretic mobility toward the anode (16); PK dress: Department of Chemical Pathology, St. Mary's Hospital Medical isozymes are designated PK L (the major liver form). PK M, (the muscle School, London W. 2 1PG, England. form), and PK M2 (the minor liver form) (15); LDH isozymes are designated 3 Present address: Department of Biochemistry and Biophysics. Iowa LDH,, LDH,, LDH,, LDH, and LDHS, in order of decreasing electrophoretic State University, Ames, Iowa 50011. To whom requests for reprints should be mobility toward the anode. The LDH isozymes are tetramers. made up of HM addressed. subunits. These 5 isozymes are equivalent to H4 (LDH,), H:,M (LDH,), H,M, Received May 6, 1977; accepted February 10, 1978. (LDH,), HM:, (LDH4), and M, (LDHS). MAY 1978 1323 Downloaded from cancerres.aacrjournals.org on September 25, 2021. © 1978 American Association for Cancer Research. K. D. Hammond and D. Balinsky Germany), and Hopkin and Williams (Chadwell Heath, Eng activation by Mg2+.The reaction was initiated by addition of glucose 1-phosphate. Km's and their S.E.'s were calculated land). Source of Material. Human fetal and adult liver and with the computer program of Cleland (8) for a 1-substrate hepatomas were obtained and prepared as was described reaction. in a previous article (13). Normal dura and meningioma were obtained during surgery from patients of Caucasian RESULTS AND DISCUSSION origin by Dr. K. Lewer Allen. Esophageal cancer tissues Characterization of Cell Lines. Initially, the isozyme pat obtained during surgery and mesothelioma obtained at terns in the cell lines were compared with those of human autopsy were provided by Dr. J. A. Hunt; the patients were tissues and with those of other cell lines growing in the Bantu-speaking Negro males of South African origin. Au same laboratory to confirm that there had been no contam topsy tissues were obtained within 6 hr of death, and fetal ination during culture. The mobilities of the isozymes of material was obtained as soon as possible after abortion, G6PD, HK, PK, and LDH corresponded with those of human usually within 2 to 6 hr. Tissues were kept on ice if tissues and could be distinguished from those of MDBK electrophoresis was to be carried out immediately; other wise, they were stored at -20°. cells (of bovine kidney origin) and of rat hepatoma cells. Human erythrocyte G6PD occurs in many genetic variant Hepatoma cells lines P and A, established from the forms, of which the commonest is the B* variant; a relatively tumors of patients in whom primary hepatocellular carci common variant occurring in Negroes is the A* variant (5). noma had been confirmed histologically, were provided by It was only possible to distinguish electrophoretically be Dr. O. W. Prozesky (27) and Dr. J. J. Alexander (22), tween human cells and VERO (vervet monkey kidney) cells respectively. A human esophageal cancer cell line was also if the human cells had the G6PD B* isozyme. As shown in provided by Dr. J. J. Alexander. Human fibroblasts and Chart 1 and Fig. 1, the G6PD isozyme present in hepatoma HeLa cells were supplied by Dr. J. M. Whitcutt. HeLa cells cells P and esophageal cancer cells corresponded to the B+ were also supplied by Dr. 0. W. Prozesky. Cells obtained in phenotype, thus distinguishing these cells from HeLa cells, serum-free medium after trypsinization were centrifuged at which are frequent tissue culture contaminants and have 50 x g for 5 min at room temperature and washed twice the G6PD A* isozyme. In hepatoma cells A, G6PD A* was with 0.9% NaCI solution. Extracts were prepared immedi present, as in the original tumor from which they were ately. derived. These cells could, however, be distinguished from Preparation of Extracts. Tissue extracts were prepared HeLa cells by other criteria (22). G6PD A* was also the form at 0°either in the homogenizing medium of Shonk and present in the fibroblasts, which were obtained from the Boxer (30), containing 1 mM dithiothreitol, or in 10 mM Tris- same patient as hepatoma cells A. HCI buffer (pH 7.4) containing 1 mM dithiothreitol, as PGM. The reduced activity of PGM in the fast-growing described previously for pyruvate carboxylase (13). Cell hepatoma, observed previously (3), did not appear to be extracts were prepared as described previously (13). The associated with a change in isozyme pattern. No apparent supernatants were kept on ice if electrophoresis was to be differences were observed between the patterns in host carried out immediately; otherwise, they were stored at -20°. livers and the corresponding hepatomas derived from 6 different individuals. Examples are shown in Chart 1: Pa- Horizontal Starch Gel Electrophoresis.
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