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]

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-phosphotransferase) (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 (Â«-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

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 -20Â°. 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 0Â°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 -20Â°. livers and the corresponding hepatomas derived from 6 different individuals. Examples are shown in Chart 1: Pa- Horizontal Starch Gel Electrophoresis. For PGM and G6PD, electrophoresis and staining were carried out as described by Shaw and Koen (29). Electrophoresis and staining of HK isozymes were essentially the same as those described by Katzen and Schimke (16); resolution of the isozymes was, however, considerably improved by increas ing the current to 1.6 to 1.8 ma/cm of gel width. The gel and electrode buffer used for the separation of PK isozymes was 0.02 M barbital (pH 8.6) containing 2.5 mM EDTA, 0.1 mM dithiothreitol, and 0.1 mM fructose 1,6-bisphosphate. HL, H, H12 H2 HL3 H3 Electrophoresis was carried out for 17 hr at 1.5 to 1.8 ma/ cm of gel width. The PK isozymes were stained as described by Balinsky ef al. (3); 20 mM AMP was added to inhibit adenylate kinase; blanks in the absence of phosphoenolpyruvate showed no bands of activity. Separation of LDH isozymes was carried out with the discontinuous buffer system of Poulik (26); the stain was the same as that described by Shaw and Koen (29). Determination of Apparent K,,,'s for PGM. PGM was assayed as described by Shonk and Boxer (30), except that 0.15 mM NADH was used, and glucose 1-phosphate concen HL, H HC OC He tration was varied between 0.33 and 3 mM. The crude Chart 1. Diagrammatic representation of the starch gel electrophoretic enzyme extracts, prepared in the homogenizing medium of isozyme patterns of PGM (top) and G6PD (bottom) in human host livers (HL) Shonk and Boxer (30), were preincubated at 30Â°for 10 min and the corresponding hepatomas (H), hepatoma cells P and A (HCP and HCA),esophageal cancer cells (OC), HeLa cells (He), and fibroblast cells (F). in the absence of glucose 1-phosphate to allow enzyme Subscripts 1. 2, and 3 indicate different patients. Arrow, origin.

1324 CANCER RESEARCH VOL. 38

Downloaded from cancerres.aacrjournals.org on September 25, 2021. © 1978 American Association for Cancer Research. Isozymes in Human Tissues and Cells tients 1 and 2 had the PGM-2 phenotype, whereas Patient 3 had the PGM-1 phenotype. The apparent Km's of host liver and hepatoma PGM for glucose 1-phosphate were also similar [1.46 Â± 0.04 rriM (S.E.) and 1.38 Â± 0.03 HIM, respectively]; Weberei al. (38) had reported that PGM from a fast-growing rat hepatoma had a higher Km for this substrate than had PGM from normal liver. FH FB FM FL HC. O OC NO Mer G6PD. The G6PD isozyme patterns in host livers and hepatomas obtained from 5 different individuals were simi lar and in each case corresponded to either the A" or the B" phenotype. In 1 case, in the surgical host liver and hepa toma, a different G6PD variant was detected and, as shown in Fig. 1, 2 bands of activity were apparent in both tissues. In the host liver, however, the intensity of the band with slower mobility was greater than that of the faster-moving band, whereas in hepatoma the pattern was reversed. Several possible explanations can be envisaged. The sim HCp HCA O OC NO Men Mes He F plest is that human liver G6PD shows analogous heteroge neity, as found in rat liver G6PD in which the bands are interconvertible and simply represent different degrees of sulfhydryl oxidation (33) or aggregation (14), with more alteration in the enzyme from the hepatoma than from the host liver, as occurs with rat hepatoma AH 30 G6PD (33). Since the patient was a female, she could be heterozygous for 2 forms of G6PD, with a greater proportion of cells in the hepatoma producing the faster-moving G6PD band than FH FB FM FL O OC NO Mei in host liver. By the Lyon hypothesis (21), only 1 X chromo Chart 2. Diagrammatic representation of the starch gel electrophoretic some in each cell is activated. Hence, on the assumption isozyme patterns of HK (A), PK (B). and LDH (C) in human fetal heart (FH), that the hepatoma arose from a single cell, one would fetal brain (FB), fetal muscle (FM), fetal liver (FL), normal muscle (NM), normal liver (A//.), host liver (Hi.), hepatoma (H), hepatoma cells P and A expect to see only a single band of G6PD in the hepatoma. (HC,. and HC,), esophageal cancer (O). esophageal cancer cells (OC), nor However, since in hepatoma cells many genes that are mal dura (ND), meningioma (Men), mesothelioma (Mes), HeLa cells (He), inactive in the parent tissue are switched on, the inactive X and fibroblast cells (F). Arrow, origin. chromosome could be switched on in some hepatoma cells and produce the other G6PD variant. Further experiments hepatoma and in fetal and host liver than in normal liver; would be required to distinguish between these various HK II was the predominant isozyme of hepatoma cells in possibilities. culture. HK III was observed in fetal, normal, and host liver HK. In Fig. 1B, the 2 halves of the gel were stained with and occurred in hepatoma, although the proportion of HK III low (0.5 mw) and high (0.1 M) glucose concentrations, in the hepatoma was lower than in the noncancerous livers. respectively. As can be seen, at 0.5 mM glucose, HK I, II, The patterns in fetal tissues showed no age-related differ and III are stained; at 0.1 M glucose, the intensities of HK I ences. Tissue-specific differences were, however, appar and II are unchanged, HK III is inhibited, and HK IV becomes ent, although the patterns differed somewhat from those of visible. In addition, another band, intermediate in mobility adult tissues. Changes to adult patterns must presumably between HK II and HK III, becomes visible, which we have occur at a later stage, perhaps at birth or after. HK IA was designated HK llr. HK llf was not observed in blanks from observed only in fetal liver. which either glucose or ATP had been omitted, but it was The patterns in all host livers and hepatomas differed detected in adult and fetal tissues whether or not EDTA was from the normal liver pattern in that HK II was present and included in the electrophoresis buffers, although in the the proportion of HK II, was increased. As in normal liver, absence of EDTA all of the isozymes stained much more HK I was usually the major isozyme in host livers and hepa faintly. HK II, was first described by Benson and de Jong tomas. In one case, however, HK II was the major form in (4), although it was observed only in fetal liver. They both host liver and hepatoma, and in another it was the showed that it differed from tryptic digestion products of major form in the hepatoma although not in the corre HK I and HK II. HK II can appear as 2 bands when subjected sponding host liver. In all cases there was a greater propor to electrophoresis in the absence of thiols, but in the work tion of HK II compared to HK III in hepatomas than in host described here dithiothreitol was present throughout. At livers; the proportion of HK II, also appeared to be increased present, it is not certain whether HK II, is a separate isozyme in the tumor. These changes are suggestive of a trend or whether it represents a degradation modification of 1 of toward the fetal liver pattern, consistent with the view that the other isozymes. Its kinetic properties have been studied the fetal state tends to be reproduced in cancer (25, 28). (7). The replacement of HK III by HK II in the hepatoma confirms As can be seen in Chart 2, HK I was the predominant iso our earlier observations (3) and is consistent with findings zyme in all specimens except the hepatoma cell cultures. reported for some rat hepatomas (10, 28). Increases in HK II The proportions of HK II and HK II, were higher in human have also been observed in human cervical and corpus

MAY 1978 1325

carcinomas (18). The regulatory isozyme HK IV, which ferentiated rodent liver, PK L occurs only in parenchymal disappears in rat hepatomas (10, 30), was detected in the cells, and PK M, occurs only in nonparenchymal tissue (9, fresh surgical host liver although not in the corresponding 35). PK M.,, however, occurs in fetal liver hepatocytes and hepatoma. This was the only time that this isozyme was can be switched on in regenerating liver parenchyma (34, observed in any human material, and it was no longer 37). The cellular localization in humans has not been exam detected after the tissue had been frozen. ined. Neither PK M, nor the intermediate bands between PK The esophageal cancer tissue examined in this work had L and PK M, have previously been observed in hepatomas. only HK I, whereas meningioma and mesothelioma (derived However, the gene for PK M, occurs in all cells; hence, from either the visceral or parietal pleura) had HK II and HK there is no reason why it, along with other proteins that do Mrin addition to HK I. not normally occur in liver (e.g., aldolase C), cannot be The patterns in the hepatoma cell lines differed from the expressed in these abnormal cells. Increasing growth rate hepatoma tissue patterns in that HK II was the predominant and loss of differentiation in rat hepatomas is associated isozyme, the proportion of HK llr was decreased, and HK III with progressive replacement of PK L by PK M2 (10, 28). was absent. The original tumor from which hepatoma cells Since PK is under hormonal and nutritional control (19, A were derived had HK I, HK II, HK II,, and HK III, with HK I 34), its variable content in host livers could be due to the the predominant isozyme. HK II was present in esophageal poor nutritional states of these patients. The altered pattern cancer cells in addition to HK I. The apparent increase in in the host liver could also be due to other factors, (a) The proportion of HK II in hepatoma cells and the appearance liver has been exposed to the same carcinogenic stimuli of this isozyme in esophageal cancer cells may be related that caused the hepatoma. (b) Humoral factors put out by to the glucose content of the culture medium, since Katzen transplantable rat hepatomas have been shown to cause ef al. (17) have shown that, in human liver cells, HK II alterations in host liver PK isozyme patterns (32); a similar increases in the presence of high concentrations of glu phenomenon could occur in the case of human primary cose. hepatomas. The altered pattern in hepatomas is not en PK. The PK isozymes M, and rVLwere separated under tirely due to reduction of PK L since the overall PK activity the conditions used. In normal muscle only PK M, was increases (3); this is evidently due to an increase of PK M2. detected. The major isozyme of normal liver was PK L, and Human hepatoma PK M is not subject to most of the there was a small amount of PK M..,.Age-related differences allosteric controls regulating PK L and has a lower Kmfor in the patterns in fetal tissues were again not apparent. The phosphoenolpyruvate (1, 2); hence, its increase would lead patterns in fetal muscle and liver were similar to the adult to efficient glycolysis even at low concentrations of phos patterns, whereas in rat the adult patterns are acquired only phoenolpyruvate. In normal liver under these conditions, after birth (15). An additional band of mobility slightly less phosphoenolpyruvate would be diverted into gluconeogen- than that of PK L was occasionally detected in fetal liver. esis; this is further prevented in hepatoma by low levels of This isozyme appears to have similar mobility to that re the gluconeogenic enzymes pyruvate carboxylase and ported for erythrocyte PK (23), and it presumably corre phosphoenolpyruvate carboxykinase (13). sponds to the erythrocyte isozyme of hemopoietic tissue. All other tumor tissues examined had PK M.,. An isozyme The patterns in host livers and hepatomas were extremely with slower mobility than the PK M of liver has been variable; a diagrammatic representation is given in Chart 3. reported in lung, stomach, and jejunum carcinomas and There were no consistent differences between host liver also in placenta (31). and hepatoma, and the patterns of both were usually Both hepatoma cell lines had only PK M.,, whereas the different from the normal liver pattern. In one case, PK L original tumor from which hepatoma cells A were derived was the major form in both host liver and hepatoma, and in appeared to have the M, isozyme. Esophageal cancer cells another both tissues had only the M., isozyme. One of the also had only PK M,. Alterations in PK Â¡sozymepatterns hepatomas appeared to have only the M, isozyme, and have previously been observed in tissue culture. Walkerei another appeared to have a mixture of the M, and M2forms. al. (36) found that dividing normal rat liver cells had PK M Several host livers and hepatomas showed 2 additional instead of PK L. strongly staining bands between PK L and PK M.,. These LDH. The patterns in normal muscle and liver were could be hybrids, as in the case with bovine kidney (6). similar, with LDH4 and LDH:, as the major forms. No age- This observation is of interest since it shows that PK L and related differences were observed in the isozyme patterns PK M.,can occur in the same cell in human hepatoma. In dif- in fetal tissues, and tissue-specific differences were not apparent. In all cases, LDH..,was the major form, LDH2 and LDH.,were present, and only traces of LDH, and LDH:,were found; these findings are consistent with those of Pfleiderer and Wachsmuth (24). The patterns in host livers and hepatomas usually resem bled those of normal liver, although occasionally the pro portion of H subunits in hepatoma appeared to be slightly greater than they did in the corresponding host liver or in

HL, H, HL2 H2 HLj Hj HLÂ¿H^ HL5 H5 HLg Hg HL? H? HLg Hg normal liver. In the latter cases it was apparent that the hepatoma pattern showed some resemblance to that of Chart 3. Diagrammatic representation of the starch gel electrophoretic isozyme patterns of PK in human host livers (HL) and the corresponding fetal liver. Increases in the proportion of H subunits have hepatomas (H) of 8 different patients. Arrow, origin. also been observed in rat hepatomas (10, 28).

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The major isozymes present in esophageal cancer, nor 11. Fondy, T. P.. and Kaplan, N 0. Structural and Functional Properties of H and M Subunits of LÃ¡clate Dehydrogenases. Ann N. Y. Acad. Sci.. mal dura, meningioma, and mesothelioma (visceral or 779: 888-904, 1965. parietal pleura) were LDH, and LDH.,. In nonhepatic human 12. German. J. L., Evans, V. J.. Conner, J. A., and Westfall. B. B Character tumors, increases in the proportion of the H form have ization of Three Human Cell Lines by Chromosomal Complement and by Certain Biochemical Parameters. Reversible Alteration of Isozyme Pat occasionally been reported; however, in the majority of terns by Different Media. J. Nat. Cancer Inst.. 32 681-695. 1964. cases, increases in the proportion of the M form were ob 13. Hammond. K. D., and Balinsky. D. Activities of Key Gluconeogenic Enzymes and Glycogen Synthetase in Rat and Human Livers, Hepato served (20). mas, and Hepatoma Cell Cultures. Cancer Res., 38. 1317-1322. 1978. Although LDH, was not detected in hepatoma cells P, the 14. Holten, D. Relationships among the Multiple Molecular Forms of Rat proportion of H subunits in both the hepatoma cell lines Liver Glucose-6-phosphate Dehydrogenase. Biochim. Biophys. Acta. 268. 4-12, 1972. was greater than that in the majority of hepatomas. This was 15. Imamura, K., and Tanaka, T. Multimolecular Forms of Pyruvate Kinase also the case when hepatoma cells A were compared with from Rat and Other Mammalian Tissues. I Electrophoretic Studies. J the hepatoma from which they had been derived (Fig. 1). Biochem. Tokyo. 71: 1043-1051, 1972 16. Katzen. H. M., and Schimke. R. T. Multiple Forms of Hexokinase in the Changes in the LDH isozyme pattern, depending on culture Rat: Tissue Distribution, Age Dependency, and Properties. Proc Nati. conditions, have been observed in cell cultures derived Acad. Sei. U. S., 54: 1218-1225, 1965. 17. Katzen, H. M., Soderman, D D., and Nitowsky. H. M. Kinetic and from a number of species (11, 12). Electrophoretic Evidence for Multiple Forms of Glucose-ATP Phospho- transferase Activity from Human Cell Cultures and Rat Liver. Biochem Biophys. Res Commun. 79. 377-382, 1965. CONCLUSION 18. Kikuchi. Y.. Sato, S.. and Sugimura, T. Hexokinase Isozyme Patterns of Human Uterine Tumors. Cancer, 30. 444-447, 1972. The present study of PGM, G6PD, HK, PK, and LDH 19. Krebs, H. A., and Eggleston, L. V. The Role of Pyruvate Kinase in the Regulation of Gluconeogenesis. Biochem. J., 94. 3C-4C, 1965. isozymes amplifies and extends earlier work carried out in 20. Langvad. E. Lactate Dehydrogenase Isoenzymes in Cancer, pp. 26-67. this laboratory (3). As a result of improved separation of the Copenhagen: Akademisk Forlag. 1972 isozymes, additional forms of HK (HK llf) and PK (interme 21. Lyon, M. F. Sex Chromatin and Gene Action in the Mammalian X- Chromosome. Am. J. Human Genet., 14: 135-148. 1962. diate bands, possibly hybrids, between PK L and PK M_,) 22. Macnab, G. M.. Alexander, J. J., Lecatsas. G.. Bey. E. M., and Urbanow- were detected, and further differences between liver and icz, J. M. Hepatitis-B Surface Antigen Produced by a Human Hepatoma hepatoma patterns were apparent. Loss of the liver-specific Cell Line. Brit. J. Cancer, 34. 509-515, 1976. 23. Nakashima, K. Further Evidence of Molecular Alteration and Aberration forms of the key glycolytic enzymes, HK and PK, in human of Human Erythrocyte Pyruvate Kinase. Clin Chim. Acta. 55. 245-254. hepatoma, as in rat hepatoma (10, 28), suggests a loss of 1974. the differentiated functions with which these isozymes are 24. Pfleiderer, G.. and Wachsmuth, E. D. Alters und FunktionsabhÃ¤ngige Differenzierung der LactatdehydrogenÃ¤se Menschlicher Organe Bio associated in normal tissue. Since many differentiated func chem. Z.,334. 185-198, 1961. tions may not be present in fetal tissues, it is not surprising 25. Potter. V. R. Recent Trends in Cancer Biochemistry: The Importance of Studies on Foetal Tissue. Can. Cancer Conf.. 8 9-30, 1969. to find some resemblance between fetal and tumor tissues. 26. Poulik, M. D. Starch Gel Electrophoresis in a Discontinuous System of The differences observed between the isozyme patterns in Buffers. Nature. 780. 1477-1479, 1957. the cell lines and those of the respective tissues of origin 27. Prozesky, 0. W., Brits, C., and Grabow, W. 0. K. In Vitro Culture of Cell Lines from Australia Antigen Positive and Negative Hepatoma Patients. presumably result from adaptation of the cells to culture In: S. J. Saunders and J. Terblanche (eds.), Liver: Proceedings of an conditions. International Liver Conference with Special Reference to Africa, p. 358. London: Pitman Medical Books, 1973 28. Schapira, F. Isozymes and Cancer. Advan. Cancer Res., 78. 77-153. REFERENCES 1973. 29. Shaw, C. R.. and Koen, A. L Starch Gel Electrophoresis of Enzmes. In: 1. Balinsky, D., Cayanis, E.. and Bersohn, I. Comparative Kinetic Study of I. Smith (ed.), Chromatographie and Electrophoretic Techniques, Vol 2, Human Pyruvate Kinases Isolated from Adult and Fetal Livers and from pp. 325-364. London: William Heinemann Ltd.. 1968 Hepatoma. Biochemistry. 12: 863-870, 1973. 30. Shonk, C E., and Boxer, G. E. Enzyme Patterns in Human Tissues I. 2. Balinsky. D.. Cayanis, E., and Bersohn, I. The Effects of Various Methods for the Determination of Glycolytic Enzymes. Cancer Res., 24. Modulators on the Activities of Human Pyruvate Kinases Isolated from 709-731, 1964 Normal Adult and Foetal Liver and Hepatoma Tissue. Intern. J. Bio- 31 Spellman. C. M., and Fottrell. P. F. Similarities between Pyruvate Kinase chem., 4. 489-501. 1973. from Human Placenta and Tumours. Federation European Biochem. 3. Balinsky. D.. Cayanis, E., Geddes, E. W., and Bersohn, I. Activities and Soc. Letters. 37. 281-284, 1973. Isoenzyme Patterns of Some Enzymes of Glucose Metabolism in Human 32. Suda, M., Tanaka, T., Yanagi. S.. Hayashi. S.. Imamura. K., and Primary Malignant Hepatoma. Cancer Res., 33. 249-255, 1973. Taniuchi, K. Dedifferentiation of Enzymes in the Liver of Tumor-bearing 4. Benson, P. F., and de Jong, M. Multiple Forms of Hexokinase in Animals. Gann Monograph, 73: 79-93, 1972. Embryonic and Adult Human Tissues. Biochem. J.. 706. 15P, 1968. 33. Taketa, K , and Watanabe, A. Interconvertible Microheterogeneity of 5. Boyer, S. H., Porter, l. H., and Weilbacher, R. G. Electrophoretic Glucose-6-phosphate Dehydrogenase in Rat Liver Biochim. Biophys. Heterogeneity of Glucose-6-phosphate Dehydrogenase and Its Relation Acta, 235: 19-26, 1971. ship to Enzyme Deficiency in Man. Proc. Nati. Acad Sei. U. S., 48: 1868- 34. Tanaka, T., Harano, Y., Sue, F., and Morimura, H. Crystallization, 1876, 1962. Characterization and Metabolic Regulation of Two Types of Pyruvate 6. Cardenas, J. M.. Dyson. R. D., and Strandholm, J. J. Bovine and Chicken Kinase Isolated from Rat Tissues. J. Biochem. Tokyo, 62: 71-91, 1967 Pyruvate Kinase Isozymes. Intraspecies and Interspecies Hybrids. In: C. 35. Van Berkel. J. C.. Koster. J. F., and Hulsman, C. W. Distribution of L and L. Marken (ed.), Isozymes, Vol. 1, pp. 523-541. New York: Academic M Type Pyruvate Kinase between Parenchymal and Kupffer Cells of Rat Press. Inc.. 1975. Liver. Biochim Biophys. Acta, 276 425-429. 1972. 7. Cayanis, E., and Balinsky. D. Comparative Kinetic Properties of Human 36. Walker, P. R., Bonney. R. J., Becker, J. E.. and Potter, V. R. Pyruvate Hexokinases. Intern. J. Biochem., 6. 741-749, 1975. Kinase, Hexokinase and Aldolase Isozymes in Rat Liver Cells in Culture 8. Cleland, W. W. Computer Programmes for Processing Enzyme Kinetic In Vitro. 8: 107-114, 1972 Data. Nature, 198: 463-465, 1963. 37 Walker, P. R., and Potter, V. R. Isozyme Studies on Adult. Regenerating, 9. Crisp, D. M., and Pogson, C. I. Glycolytic and Gluconeogenic Enzyme Precancerous and Developing Liver in Relation to Findings in Hepato Activities in Pc-renchymal and Non-parenchymal Cells from Mouse Liver. mas. Advan. Enzyme Regulation. 70: 339-364, 1972. Biochem. J., 726. 1009-1023, 1972. 38 Weber, G.. Henry, M. C., Wagle, S. R., and Wagle, D S Correlation of 10. Criss. W. E. A Review of Isozymes in Cancer. Cancer Res , 37. 1523- Enzyme Activities and Metabolic Pathways with Growth Rate of Hepato 1542. 1971. mas. Advan. Enzyme Regulation, 2: 335-346. 1964

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Fig. 1. Isozyme patterns in human livers, hepatomas. and hepatoma cells Starch gel electrophoresis was carried out as was described in "Materials and Methods." (A) G6PD: left to right, hepatoma cells P, surgical host liver, surgical hepatoma, HeLa cells, erythrocytes (G6PD B). esophageal cancer cells, normal liver (G6PD A), hepatoma cells P, and HeLa cells. (B) HK: left to right, normal muscle, normal liver, hepatoma cells P, hepatoma (surgical), host liver (surgical), fetal liver, fetal liver, host liver (surgical), hepatoma (surgical), hepatoma cells P, normal liver, and normal muscle. The gel on the left was stained with 0.5 mM glucose; the gel on the right was stained with 0.1 M glucose. (C) PK:/eft to right, hepatoma cells P. normal muscle, normal liver, fetal liver, host liver, hepatoma. hepatoma cells P, normal muscle, and host liver. (D) LDH; left to right, hepatoma, normal liver, normal muscle, and fetal liver. (E) LDH; left to right, hepatoma cells P. hepatoma from which hepatoma cells A were derived, host liver, and hepatoma cells A.

1328 CANCER RESEARCH VOL. 38

Downloaded from cancerres.aacrjournals.org on September 25, 2021. © 1978 American Association for Cancer Research. Isozyme Studies of Several Enzymes of Carbohydrate Metabolism in Human Adult and Fetal Tissues, Tumor Tissues, and Cell Cultures

Kathryn D. Hammond and Doris Balinsky

Cancer Res 1978;38:1323-1328.

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