2007.Full.Pdf

Home , Phosphoenolpyruvic acid, Triokinase

Cancer Research

VOLUME32 OCTOBER 1972 NUMBER10

[CANCER RESEARCH 32, 2007-2016, October 1972] Glycolysis, Respiration, and Anomalous Gene Expression in Experimental Hepatomas: G. H. A. Clowes Memorial Lecture1

Sidney Weinhouse The Fels Research Institute, Temple University School of Medicine, Philadelphia, Pennsylvania 19140

consider how limited has been our progress in the control or understanding of this disease. Nevertheless, I am truly pleased to share this honor with my many students and coworkers who are the real awardees. 1 owe particular thanks to my associate Jennie Shatton and her assistant Albert Williams; to Dr. D. Meranze, who has taken care of our histological work; to Dr. Harold Morris, who has been in every respect a true collaborator in our work of recent years; and to my colleagues in the Fels Institute for their constant help and intellectual stimulation. I express my sincere thanks to the Committee for choosing me as their awardee and to the Eli Lilly Company for their support of this Lectureship. It is a special pleasure to feel thus identified with the man in whose memory this award was established. I felt very close to Dr. Clowes during his later years. These were my earlier years, when some 22 years ago I was stepping gingerly into the controversial field of carbohydrate metabolism and energy transduction in tumors. I owe much to Dr. Clowes for many insights, gained not only from his published work in these fields, but also from many pleasant and profitable discussions generally held during the meetings of this Association. In those days, as many of you remember, Dr. Clowes, with his white hair and his impressive bushy eyebrows, was a familiar sight. It is a truism in science that we never really answer the truly important questions, but with succeeding generations and advancing knowledge we frame the questions in ever greater depth. For many years the biochemist has been asking the question of whether there is some unique biochemical feature that characterizes the neoplastic cell: a key to its disorderly growth, its lack of regulatability, its invasiveness. It is

1This paper is based on the twelfth G. H. A. Clowes Memorial Lecture given at the 63rd Annual Meeting of the American Association I am deeply appreciative of the honor of being awarded the for Cancer Research, Inc., Boston, Mass., May 5, 1972. The 1972 Clowes Lectureship. I accept this award with mixed experimental work of the author has been supported for many years by grants from the National Cancer Institute and from the American feelings: first, because I find it difficult to measure up to my Cancer Society. Current grants are Grants CA-12227, CA-10916, and distinguished predecessors; and second, because awards for BC-74M. cancer research seem somehow inappropriate when we

OCTOBER 1972 2007

Downloaded from cancerres.aacrjournals.org on September 30, 2021. © 1972 American Association for Cancer Research. Sidney Weinhouse becoming ever more clear that this is a simplistic question mat admits of no useful answers at this time. If we phrase our question to ask whether all cancers differ in some single, unique feature from all normal tissues, the answer is simply no. Paradoxically, however, if we ask whether a particular neoplasm differs from its particular tissue of origin, the answer NORMAL is that there are so many differences that causal relationships are thoroughly obscured. The spectacular advances of recent years in the molecular biology of the cell, instead of providing NEOPIASTIC an answer to the secret of cancer, have only made all the more i i evident the complexity of this baffling problem. There are two LACTICACIDFORMATION aspects of cancer biochemistry that I wish to cover in my Chart 1. Diagrammatic representation of aerobic and anaerobic lecture this evening. I do so not because these provide new glycolysis in normal and tumor slices, illustrating the Pasteur effect and dazzling insights or breakthroughs, as they are described in the the Warburg hypothesis. Sunday supplements, but because perhaps they lift one tiny corner of the curtain that obscures our view of the molecular basis of cancer. although a Pasteur effect was observed, the presence of oxygen The first of these deals with aberrations of genetic did not lead to elimination of glycolysis and aerobic glycolysis expression, as revealed by isozyme alterations in experimental remained high. Warburg regarded the persistence of aerobic hepatomas. It is becoming increasingly evident that a common glycolysis as a key to the neoplastic transformation. phenotypic feature of cancer is a misprogramming of genetic He proposed that the high aerobic glycolysis is the result of information leading to aberrant synthesis of protein. a defect in respiration, that cancer results when the cell Moreover, there is a recurrent pattern in cancer of the responds to an irreversible injury to its respiratory mechanism appearance of fetal proteins, suggesting that genes that were by adopting a fermentative metabolism. According to active in fetal tissue, but had been repressed during normal Warburg, such cells cannot maintain the differentiated state, embryonic development, become reactivated in cancer. and as undifferentiated cells they grow in uncontrolled The second deals with a peculiar metabolic property of fashion. This dictum, vigorously propounded by an illustrious cancer cells, namely, the high aerobic glycolysis. This subject figure in biology, captured the imagination of cancer has had a long history of controversy that has contributed researchers, and it is difficult to exaggerate the influence of more confusion than enlightenment; indeed, it has become this theory on the direction of cancer research for decades. tiresome in its constant reiteration. I bring it up again tonight Moreover the Pasteur effect, although still not well understood, has been a focal point for countless studies on for two reasons: first, to describe new information which metabolism and its regulation.2 allows us to put this phenomenon in a newer perspective; and secondly, as we shall see, to show that it is not entirely It was not until about 1950, however, when isotope tracer unrelated to the first topic, namely, the anomalous gene studies showed that tumors could oxidize glucose to C02 at expression of cancer cells. rates comparable with those of normal tissues, that the Warburg hypothesis was seriously questioned (90-92). High Aerobic Glycolysis in Tumors Although Warburg defended his theory vigorously (85, 86), many efforts to discover defects, or even substantive About a half-century ago Otto Warburg, an illustrious and alterations of respiration in cancer, were unsuccessful. A colorful, indeed a legendary figure in cancer research, variety of experimental techniques demonstrated that tumors pioneered in what may be called modern cell biology by utilize oxygen at low to moderate rates, they contain a full applying what were then modern techniques and concepts of complement of respiratory enzymes and coenzymes, they have physical chemistry to studies of the cancer cell (84). He was mitochondria, which appear normal morphologically and the first to note that slices of the most diverse tumors had one functionally (although they may be low in number in some common property: they produced large amounts of lactic acid tumors), the citric acid cycle is operative, and they couple from glucose. The essence of his experimental observations, oxidation with ATP production. In the light of our more somewhat oversimplified, is shown in Chart 1. When normal advanced knowledge of respiration, mitochondria! function, tissue slices are incubated in a nutrient medium containing and the interrelationships of the mitochondria with glucose, but in the absence of oxygen, there is a rapid and carbohydrate metabolism, the Warburg hypothesis is now continuous production of lactic acid, a process which Warburg regarded at best as a gross oversimplification of exceedingly termed glycolysis. However, when the tissues were incubated complex regulatory mechanisms (4,41, 57,90). in oxygen, glycolysis virtually ceased. This decrease in glycolysis brought about by oxygen was named by Warburg the Pasteur effect, after an observation made some 100 years 2The Pasteur effect and its associated phenomenon, the Crabtree ago by Pasteur that, when yeast is grown in air, it loses the effect, are subjects of a vast literature to which only the most cursory treatment can be given here. For further information on these effects as capability to ferment glucose to ethanol. well as on other control factors in glycolysis, the reader may refer to The behavior of tumor slices Warburg found, however, was reviews by Racker (57), Aisenbcrg (4), Chance (15), Newsholme and quite different. Anaerobic glycolysis was very high; and, Gevers (49), Scrutton and Utter (66), and Atkinson (8).

2008 CANCER RESEARCH VOL. 32

The Morris Hepatomas Table 1 Properties of Morris hepatomas It was not until the development of the Morris hepatomas (46-48, 51) that the significance of high aerobic glycolysis Degree of differentiation to neoplasia was seriously questioned. Some of these tumors High Well Poor grow in uncontrolled fashion, albeit slowly, metastasize, and ultimately kill their hosts and yet have a low rate of glycolysis. GrowthCytologyGlycolysisRespirationSlowNearnormalLowHighModerateSomeabnormalitiesLowModerateRapidAbnormalRapidModeratelylow Induced originally by feeding of carcinogenic chemicals, they are transplantable in the host inbred strain. Some of these resemble morphologically, and also retain at least some metabolic features of, the adult differentiated tissue. Potter (55) named these minimal-deviation hepatomas, but it is important to recognize that they represent a wide spectrum of marked differences in their glycolytic capability and, second, deviation from the parent cell. As shown in Table 1, they may to attempt to determine to what extent the morphological and conveniently be divided into three classes on the basis of their metabolic differences could be related to differences in degree of differentiation as judged by usual histolÃ³gica! activities of specific enzymes. criteria.3 The highly differentiated tumors, of which there are At about this time Markert and Moller (43) discovered that only a few examples, grow very slowly. The growth rate, lactate dehydrogenase of diverse systems exists in which can be assessed roughly from the average time between multimolecular forms, each of which has its unique kinetic transplantations, ranges from 3 to 4 months up to well over 1 properties; and it subsequently became evident that each of year. The well-differentiated tumors grow somewhat more these individual molecular structures has its specific functional rapidly, taking from 2 to 6 months for a transplant generation, role (80, 94). A host of similar examples of multimolecular and the poorly differentiated tumors have growth rates of less forms was soon discovered. Markert named these isozymes, than 1 month down to about 10 days. The highly and the subspecialty of isozymology has burgeoned into differentiated tumors have the normal liver chromosome prominence, with the recognition that physiological function number and karyotype, the well-differentiated differ slightly, may be greatly influenced by the relative proportions of and the poorly differentiated deviate markedly in chromosome isozymes in tissues (38, 70, 81).4 Originally, our interest was karyotype and number from those of rat liver (51). directed to the molecular forms of the enzyme hexokinase, Respiration decreases moderately with loss of differentiation, which catalyzes the initial step in glucose utilization, its but the most striking biochemical feature of these tumors is conversion to glucose 6-phosphate. the low or negligible glycolysis in the well-differentiated hepatomas, a behavior which is in decided contrast with the Glucose-ATP Phosphotransferases usual high level of glycolysis in the poorly differentiated tumors. In studies of the role of these hexokinase isozymes in The low aerobic glycolysis of these tumors, originally regulation of normal carbohydrate metabolism in rat liver, we observed by Weber (88) and Aisenberg and Morris (5) and discovered about 10 years ago a new isozyme, subsequently their colleagues, was confirmed in our laboratory (20), and termed glucokinase (18, 67). This isozyme in Chart 2 is shown about 10 years ago we began a study of their biochemistry and in the shaded bars. Liver also contains three other hexokinases, metabolism. Our objectives at that time were, first, to explore shown collectively in Chart 2, clear bars. Glucokinase has a set possible enzymatic alterations that would account for the of distinctive kinetic properties that endow it with unique significance to hepatic function (26, 33, 67, 82). Moreover, it 3In gross appearance, the highly and well-differentiated hepatomas is under tight regulatory control by the animal through its resemble liver in color and consistency. The cells are large and contain responsiveness to hormones such as insulin and to nutritional abundant pale-staining eosinophilic cytoplasm, and they contain conditions. Glucokinase activity is low in fasted or variable quantities of glycogen. The nuclei are round, with large carbohydrate-deprived rat liver, and the hexokinase isozyme nucleoli, and there are occasional double nucleated cells. The cells are activities are also low. On carbohydrate feeding, there is about arranged in sheets, often in a lobular pattern; there are canaliculi, sometimes with bile pigment; and sinusoids are conspicuous with lining a 5-fold increase of glucokinase. Glucokinase is also extremely cells resembling Kupffer cells. There may be bands of connective tissue, low in the diabetic rat, and after insulin treatment it also with macrophages containing hemosiderin, ceroid, lipid, and bile increases greatly. Hexokinase activity remains essentially pigment. There are many transition stages between different constant, however. During liver regeneration the hexokinases hepatocellular carcinomas, based on architecture, cytology, and staining characteristics, often within a particular hepatoma. The choice between sharply increased without marked change in glucokinase (68). well differentiated and highly differentiated is a matter of judgment I call your attention particularly to the fetal pattern, in which based on the above-mentioned criteria. hexokinase activity is relatively high and glucokinase is absent The poorly differentiated hepatomas are grossly firm and gray or (10,83). white. The cells are smaller, with great variations in size and pattern, and are laid down in crowded sheets. There is complete loss of We explored possible alterations of this key enzyme in the hepatocellular and lining cell pattern. Mitoses are frequent, with only rare canalicular formation and pigment deposition. A more detailed 4For more detailed treatment of the subject of isozymes in cancer, description of the gross and microscopic characteristics may be found readers may refer to several monographs (34, 52, 81) and a recent in earlier reviews (47, 48, 51). review (17).

OCTOBER 1972 2009

Hexofcinose CD ducokinose E2 isozymes in the Morris hepatomas. Aldolase B, shown in Chart 4, shaded bars, is the essentially sole form of this enzyme in adult liver. This enzyme is highly active toward fructose normal, fostea Mm3 1-phosphate, the sole product of fructose metabolism in liver

normal, fed glucose (2), and therefore plays a crucial role in fructose metabolism, an important hepatic function. Aldolase A is the sole or diabetic predominant form in essentially all other nonhepatic tissues

ciabetK and is essentially the only form in early fetal liver (59). With insulin-treated embryonic development, aldolase A is repressed and aldolase B 48* nr appears and becomes essentially the sole form shortly after birth. Again, as with glucokinase, aldolase B is virtually the fetal sole form in highly differentiated hepatomas (3). With 05 K> 1-5 2O 25 decreased differentiation aldolase A makes its appearance; and, as with the hexokinase isozymes, the poorly differentiated hepatomas have almost completely lost aldolase B and have Chart 2. Relative activities of hexokinase isozymes in rat liver under various conditions. Values in this and subsequent charts are given in replaced it by a high activity of isozyme A. units (/amÃ³lesof glucose converted per min) per g tissue under the assay conditions described in the appropriate references. Pyruvate Kinase

We now consider an enzyme, pyruvate kinase, which is HEXOKINASECDAND 6LUCOKINASEC3 ISOZYMESINRATLIVERANDHEPATOMAS especially significant as a determinant of several important

HEPATOMAS hepatic functions, including that of energy transduction, and Mjlilydifferentiated the study of which has been especially illuminating. As shown Â«elldifferentiated in Chart 5, this enzyme also exists in multiple forms (21, 75, 77). Type II, shown in Chart 5, shaded bars, is the 1 poorlydifferentiated preponderant form in liver. As Weber et al. (89), Krebs and LIVER

ADULT CDBE^mSftffftarm FETAL Fetal^^ , EarlyNornu|ljMr

1.0 U

DNITSPERGRAM lateFetalHtpatomasWell Chart 3. Relative activities of hexokinase isozymes in rat liver and hepatomas. '.'."/."'-l'.'."1A Differentiated HepatomjsPoorly Morris hepatomas, with results shown in Chart 3 (69). We Differentiated[Mf(iaTftmas2 noted that the slow-growing, highly differentiated hepatomas 3 4 i6UNITSÂ« exhibit the same pattern of low hexokinase and high LIVER glucokinase values as normal adult liver, whereas with Chart 4. Relative activities of aldolase isozymes in liver and decreased differentiation glucokinase is largely lost. Moreover, hepatomas. these low glucokinase activities cannot be increased by carbohydrate or insulin administration. Most striking was the CZI TYPE I CD TYPE n very marked increase in the hexokinase activity and the virtual disappearance of glucokinase activity in the fast-growing, poorly differentiated hepatomas. We see in these tumors an NORMAL LIVER essentially complete suppression of an isozyme that is high in normal adult liver, in which it has a key physiological 9618A HEPATOMA function, and a marked derepression of isozymes that are normally low in the adult tissue. The resemblance of this WELL-DIFFERENTIATED pattern to that of the fetal liver is called to your attention. POORLY-DIFFERENTIATED Aldolase I I I Another extraordinary alteration of isozyme activities in 50 100 150 200 250 300 hepatomas is shown in Chart 4. The Schapiras, Sugimura, and units per gram associates had observed in certain hepatomas the presence of Chart 5. Pyruvate kinase isozymes in rat liver and hepatomas. Assays muscle aldolase (50, 61, 62, 64, 74); and since liver aldolase were carried out by an unpublished method developed by F. Farina, had been the subject of physiological studies in our laboratory taking advantage of the differential adsorption of the liver and (71), we were prompted to study the nature of the aldolase nonhepatic forms on DEAE-cellulose.

2010 CANCER RESEARCH VOL. 32

Eggleston (36), Tanaka (76), and others have shown, the type PROPOSED CONTROL SITES IN II isozyme, like glucokinase, is dietary and hormone GLYCOLYSIS AND RESPIRATION dependent and also has kinetic properties that endow it with TRANSPHOSPHORYLATION STEPS functional importance in such processes as glucose utilization Fructo$e-1,6-Dipho$phate and gluconeogenesis. Again we see the same pattern repeated. Atdolase \\ In the very-slow-growing, highly differentiated 9618A Triose-Phospha^te hepatoma, type II pyruvate kinase is high; it is quite low in the TriÃ³seâ€”P >T Competes with well-differentiated tumors and is virtually absent in the Dehydrogenasef^NADH respiration for Pi fast-growing, poorly differentiated hepatomas. However, in 1,3â€”DiphosphoglycericAcid this last category there is an extraordinarily high activity of ADP ~^\| Competes with the type I pyruvate kinase, the form that is very low in adult ATP â€¢¿Â«â€”\ respiration for ADP liver. 3â€”PhosphoglycericAcid The high type I pyruvate kinase activity in these poorly differentiated hepatomas has provided an important clue to Phosphoenolpyruvic Acid their high glycolytic activity and also provides a possible ADP â€”¿sj Competes with explanation for the Pasteur effect. In considering enzymatic ATP â€¢¿Â«â€”'t respiration for ADP sites for an inhibitory effect of respiration in glycolysis, it is logical to consider those enzymes that carry out transfers of Pyruvic Acid L'N/:VIADH JMAD"'-*+ phosphate to and from phosphorylated intermediates. In Chart kw 6 are shown the two sequential transphosphorylation steps leading from glucose to fructose 1,6-diphosphate, catalyzed Lactic Acid respectively by hexokinase and phosphofructokinase. Much Chart 7. Transphosphorylating enzymes competing with respiration attention has been focused in recent years on for ADP and PÂ¡. phosphofructokinase because of its remarkable allosteric properties. It is strongly inhibited by ATP and citrate and is considered from time to time, attention in recent years has deinhibited by AMP, ADP, and PÂ¡; numerous kinetic generally been diverted to phosphofructokinase (53). experiments point to feedback control on this enzyme by ATP Prompted by the extraordinarily high activities of pyruvate and citrate as the major determinant of glycolytic activity in kinase in the highly glycolyzing, poorly differentiated muscle and possibly also in brain (49,53,66). hepatomas, we reconsidered this possibility; and we Subsequent transphosphorylation reactions are diagrammed investigated the interactions between glycolytic and in Chart 7. These are: triÃ³se phosphate dehydrogenase, where respiratory systems of well- and poorly differentiated PI is taken up into 1,3-diphosphoglycerate; phosphoglycerate hepatomas in model systems, with the purpose of measuring kinase, where phosphate is transferred from the role of pyruvate kinase in determining the relative extents 1,3-diphosphoglycerate to ADP to yield 3-phosphoglycerate of glycolytic and respiratory ATP production. We were able in and ATP; and pyruvate kinase, where phosphate is transferred these experiments to establish that the transphosphorylating from phosphoenolpyruvate to ADP to yield pyruvate and steps of glycolysis beyond fructose diphosphate do compete ATP. These three reactions are especially significant since they with respiration for available ADP and to obtain evidence may compete directly for ADP and PÂ¡with the mitochondria! pointing to high pyruvate kinase activity as an important respiratory system, where ADP and PÂ¡are converted to ATP determinant of the high aerobic glycolysis of tumors (41). by oxidative phosphorylation coupled with electron transport. Further studies now under way in our laboratory on the They thus are obvious sites for the Pasteur effect, and indeed chemical and kinetic properties of the tumor pyruvate kinase this was suggested 30 years ago, independently by Johnson should help to resolve the long-standing question of the role of (31) and Lynen (42). Although the enzyme has been this enzyme in aerobic glycolysis.

PROPOSED CONTROL SITES IN GLYCOLYSIS Fetal Pyruvate Kinase in Poorly Differentiated Hepatomas AND RESPIRATION I would now like to discuss another feature of pyruvate Hexokinase and Phosphofructokinase Glucose kinase which is particularly noteworthy. In work carried out \f ATP Inhibited by about 5 years ago in our laboratory by Farina et al. (21), the Hexokinase [ Glucose-6-P and ADP properties of the pyruvate kinase of the Novikoff hepatoma suggested that it was a third isozyme different from either the Glucose-6-Phosphate liver or muscle forms. Later, Criss (16) in our laboratory, using isoelectric focusing, also observed a unique isozyme in poorly Fructose-6-P Inhibited by Deinhibitedby |^- ATP ATP ADP differentiated hepatomas; and a number of other investigators Phosphofructokinase i Citrate AMP (13, 35, 63, 76, 77) showed, by means of gel electrophoresis, JV..ADP Pi that an isozyme of pyruvate kinase was present in certain Fructose-1,6 -di phosphate hepatomas that migrated like a form in fetal rat liver. Chart 6. Phosphorylation sites of glucose and fructose 6-phosphate, Recently, Shatton (J. B. Shatton, H. P. Morris, and S. leading to formation of fructose 1,6-diphosphate. Weinhouse, unpublished work) has been able to separate the

OCTOBER 1972 2011

Downloaded from cancerres.aacrjournals.org on September 30, 2021. © 1972 American Association for Cancer Research. Sidney Weinhouse pyruvate kinase isozymes by gradient elution from DEAE-cellulose. As shown in Chart 8, adult liver has a major form which is strongly held by the resin, whereas the skeletal GRADIENTCOITIONOF 50.0 muscle form is eluted earlier. However, a third form, present in FETALLIVERPK 40.0 ISOZYMES the Novikoff and other poorly differentiated hepatomas, is not 30.0 retained by the resin and comes off in the void volume. 200 Chart 9 shows the pyruvate kinase isozyme pattern of 20-day rat fetal liver. There are three peaks, one of which comes off like the adult liver isozyme, but the major peak is clearly like the tumor form. Thus, although further work will 10.0 be necessary to establish the identity of these isozymes, it appears that this is another illustration of the expression, in poorly differentiated tumors, of a fetal liver protein that has been repressed in the adult hepatocyte. 5.0 Dr. Sato from our laboratory reported at these meetings (60) that isozymes of the a and b forms of glycogen

0 100 200 300 400 500 600 700

ML. Chart 9. Elution pattern of pyruvate kinase (pk) isozymes of 20-day rat fetal liver. Procedure was the same as that described for Chart 8.

phosphorylase of poorly differentiated hepatomas are Kinetically and immunologically similar to those of fetal rat liver forms and are distinctly different from the corresponding a and b forms of adult liver or muscle phosphorylases. Similar findings of fetal liver isozymes in liver tumors have been made i by Katanuma et al. (32) for glutaminase and by Ichihara et al. I (30) for the branched-chain amino acid aminotransferases. i These few examples doubtlessly represent a general phenome non of reactivation in tumors of genes that have been repressed during embryonic development. Thus far the phenomenon has largely been restricted to liver tumors, and its exploration in other tumor systems should be very interesting.

Fatty Acids as Respiratory Fuel

The question arises as to what the well-differentiated, ZOO 300 400 6OO 6OO TOO slow-growing tumors, which utilize glucose poorly, use for metabolic fuel. The answer is that they use fatty acids. Table 2 shows data taken from work done by Leah Block-Frankenthal Chart 8. Gradient elution of pyruvate kinase isozymes from and John Langan from our laboratory on the oxidation of DEAE-cellulose columns. Columns of DEAE-cellulose, 40 x 1.1 cm, 14C-labeled butyric and palmitic acids (12). They demonstrated were equilibrated with a solution consisting of 0.25 M sucrose, 10 mM Tris-HCl, and 1 mM EDTA at pH 7.5. Tissue homogenates were that, whereas the well-differentiated hepatomas oxidize long- prepared in an equal volume of the same solution and were centrifugea and short-chain fatty acids about as well as liver, the poorly for 1 hr at 105,000 X g at 2Â°.After the supernatant was assayed for differentiated tumors are clearly unable to do so. It is total activity, 1 to 10 ml, depending on the activity, were placed on the particularly interesting to note also from this figure that the column and eluted with a linear KC1gradient, 0 to 0.5 M in the same well-differentiated hepatomas also retain the capability (a buffer solution, and 5-ml fractions were collected at a rate of 60 ml/hr. characteristic liver function) for converting long- and Elution curves: â€¢¿,liver;A, muscle; o, hepatomas. KC1concentration, determined by gravimetric Cl~ assay. Pyruvate kinase was assayed short-chain fatty acids to acetoacetate, whereas this capability is nearly completely lost in the poorly differentiated spectrophotometrically by coupling with lactate dehydrogenase and hepatomas. Thus, as the poorly differentiated hepatomas measuring of decrease in absorption at 340 nm, with: 0.1 ml of the eluate, 0.02 ml 3 mM NADH, 0.28 ml of a stock solution containing 5 express genes that increase their capability for glucose mM phosphoenolpyruvate, 2 mM ADP, 0.75 unit/ml lactate utilization, they suppress other genes that code for that part of dehydrogenase, 50 mM glycylglycine (pH 7.5), 100 mM KC1, 10 mM their cellular machinery responsible for oxidation of fatty MgCl,, and water to 0.4 ml total volume. acids.

2012 CANCER RESEARCH VOL. 32

Table 2 Oxidation of ' *C-labeled fatty acids by liver and hepatomas (12) Values are given in microatoms fatty acid carbon oxidized per g tissue per hr at 37Â°.

Substrate Tissue CO,-"1 C Acetoacetate-'4C

Butyrate-3-14CLiverHepatomasWell-differentiatedPoorly Â±0.74.6 Â±210

Â±1.90.1 Â±60.05 differentiated4.5 Â±0.125 Palmitate-l-'4CLiverHepatomas

Well-differentiatedPoorly differentiated6.911.00.524120.05

DISCUSSION Isozyme Alterations as Anomalies of Gene Expression. The similarity of the results of these studies on isozymes to certain Metabolic Significance of Isoenzyme Alterations. The Morris immunological properties of tumors is striking. In cancer, hepatomas have taught us that tumors of great diversity can antigens that characterize adult differentiated tissues disappear arise from a single cell type, and the foregoing data make it (14), whereas new tumor-specific antigens appear (56); and it evident that this diversity is paralleled by diversity in is now well established that, in many instances, these new molecular patterns. Cancer as a disease is characterized by the antigens are identifiable as fetal proteins (1, 6,22-24, 29, 39, loss of host control over cellular proliferation. It is significant 73, 79). Obviously, these are opposite sides of the same coin: that among the molecular alterations in these neoplasms are an aberration of gene expression characterized by the loss of losses of enzymes that are under host regulation and are geared proteins normally present in adult, differentiated tissue; and to liver function and their replacement by enzymes with the acquisition of proteins normally low or absent in adult altered molecular structures that are not under host regulation tissue but present in fetal tissue. and are geared to the efficient utilization of metabolic fuels. Is this misprogramming of genetic information causal to the Many attempts have been made in the past to find neoplastic transformation? Our isozyme studies discount this meaningful relationships between the proliferative behavior of possibility, since several highly differentiated hepatomas tumors and their enzyme activities, but these have largely been exhibit isozyme patterns essentially identical with those of fruitless. It is now clear that mere assays of total enzyme liver. It is possible, however, that the neoplastic activity do not reveal profound alterations of isozyme activity transformation initiates an injury to some part of the which are likely to affect the metabolism and growth of tumor mechanism that switches genes on and off in differentiated cells. Thus far, alterations of isozyme pattern in relation to tissues, so that aberrations of protein synthesis now are these biological properties have not been explored extensively permissive but not obligatory. in other nonhepatic tumor systems; however, what little data In considering the underlying cause of the expression of have been obtained leave no reason to doubt that the same embryonic proteins in tumors, another question arises as to phenomena apply; and it seems to be likely as a general whether this is programmed or fortuitous. These embryonic proposition that loss of regulatory enzymes that are manifestations have been described in such terms as determinants of metabolic processes is a key to the unbridled derepressive-dedifferentiation (23), retrogenetic expression proliferation of cancer cells. (23, 24, 73), retrodifferentiation (7), blocked ontogeny (54), These data also illustrate cogently how ideas of the role of etc., implying a more or less systematic reversion to an aerobic glycolysis in neoplasia require change with emergence embryonic state; that is, a reversal of normal ontogeny. Our of new depths of understanding. Originally, the high glycolysis isozyme data do indicate some order in the observed was considered a sine qua non of cancer, a necessary source of alterations. For example, you will recall that many energy for cells that have lost respiratory capability. Later it well-differentiated hepatomas have largely lost isozymes of the was regarded as an inevitable accompaniment of the neoplastic differentiated liver cell but do not exhibit a resurgence of the transformation, although its significance was blunted by the fetal isozyme; but when resurgence of the fetal form does knowledge that (a) glycolysis is highly regulated and under occur, there is always a loss of the normal hepatic isozyme. appropriate conditions normal tissues may exhibit high These observations suggest that certain genes may have to be glycolytic activity, and (b) tumors have generally an intact, switched off before others are switched on. However, in functioning respiratory system. It is now evident that some general the alterations seem to be sporadic and unpredictable, tumors may utilize glucose poorly and that, within the and therefore suggest a disordered rather than a programmed spectrum of rat hepatomas, high aerobic glycolysis is not mechanism of gene activation (93). intrinsic to the neoplastic transformation but rather is a result This conception of a disordered rather than a programmed of a late stage of dedifferentiation, accompanied by and reversion is strongly bolstered by a large and ever-growing probably resulting from alterations in the activities of specific body of clinical literature pointing to bizarre aberrations of isozymes. protein synthesis in certain tumors, resulting in the ectopie

OCTOBER 1972 2013

Downloaded from cancerres.aacrjournals.org on September 30, 2021. © 1972 American Association for Cancer Research. Sidney Weinhouse production of polypeptide hormones (9, 19, 25,40, 58). This represents a dynamic balance between synthesis and obviously is not dedifferentiation, but rather, as Matsushima et degradation (11, 27, 65). The enzymatic mechanisms of al. (45) termed it, disdifferentiation. intracellular protein degradation are as yet almost totally Despite the striking association of neoplasia with anomalous unexplored. gene expression, its significance to cancer needs cautious Whether the synthesis of inappropriate protein is measured interpretation, since similar alterations are sometimes observed as isozymes or antigens, or by hormone assay, cancer is in nonneoplastic tissues. Neither antigens nor isozymes are lost evidently associated with a breakdown in those mechanisms or gained absolutely but are altered in amounts. Liver marker that control the orderly expression of genes in differentiated enzymes, which are drastically lowered in poorly tissues. These are astonishing findings when viewed in the light differentiated hepatomas, also vary normally in activity under of the rigid control exercised by these mechanisms in normal dietary and hormonal influences. Fetal antigens such as tissues and point to a possibly crucial molecular lesion in a-fetoglobulin are detectable in normal tissues and may rise neoplasia. Obviously, we need to know much more about not only in cancer, but also in regenerating liver (1, 72) in normal differentiation before we can understand its disorder in hepatitis and cirrhosis (1), and also in "preneoplastic" liver cancer. Although our knowledge of cellular differentiation is shortly after carcinogens are fed (37, 72, 87). The gs antigen, still rudimentary, these are exciting days of rapid progress and coded by type C viruses, is found not only in tumor and fetal expanding perspectives in cancer research. With new ex tissues, but also in aged tissue (28).s During certain phases of perimental avenues constantly opening, these are pointing the cell cycle, cultured normal cells exhibit transient the way to a deeper understanding of differentiation and its alterations in surface structure, which are characteristic of disfunctions, which may well lie at the heart of the neoplastic transformed as well as embryonic cells (78). In our laboratory transformation. normal rat liver cells, prepared and cultured in vitro by various methods, invariably exhibit fetal isozyme patterns (E. Farber, D. Ballard, J. B. Shatton, and S. Weinhouse, unpublished REFERENCES work). In this connection, Tomkins et al. (44, 78) found that tyrosine aminotransferase is inducible in synchronized 1. Abelev, G. I. a-Fetoprotein in Oncogenesis and Its Association with Malignant Tumors. Advan. Cancer Res., 14: 295-358, 1971. cultured hepatoma cells in the Gj and S phases of the cell 2. Adelman, R. C., Ballard, F. J., and Weinhouse, S. Purification and cycle, whether or not steroid inducer is present, but requires Properties of Rat Liver Fructokinase. J. Biol. Chem., 242: inducer during the rest of the cycle. These findings also suggest 3360-3365, 1967. that cells, whether normal or malignant, may under certain 3. Adelman, R. C., Morris, H. P., and Weinhouse, S. Fructokinase, conditions undergo some sort of transient loosening of a Triokinase, and Aldolases in Liver Tumors of the Rat. Cancer Res., normally rigid gene readout mechanism, possibly during DNA 27:2408-2413, 1967. replication or repair. Nevertheless, a crucial distinction is that 4. Aisenberg, A. C. The Glycolysis and Respiration of Tumors. New this disfunction of gene control is permanent, irreversible, and York: Academic Press, Inc., 1961. progressive in cancer. 5. Aisenberg, A. C., and Morris, H. P. Energy Pathways of Hepatoma 5123. Nature, 191: 1314-1316, 1961. Because of the elegance of the Jacob and Monod model, we 6. Alexander, P. Foetal "Antigens" in Cancer. Nature, 235: 137-140, tend to think of gene expression only at the transcriptional 1972. stage, that is, in terms of activation and inactivation of 7. Anderson, N. G., and Coggin, J. H., Jr. Models of Differentiation, structural genes by the action of specific repressers Retrogression and Cancer. Proceedings of the First Conference on synthesized by regulator genes. However, the cells of animals Embryonic and Fetal Antigens in Cancer. Oak Ridge National are highly structured; and gene expression involves many Laboratory, pp. 7-37, 1971. posttranscriptional events, including transport from nucleus to 8. Atkinson, D. E. Adenine Nucleotides as Stoichiometrtc Coupling cytoplasm, selective attachment to membranes, and Agents in Metabolism and as Regulatory Modifiers: The Adenylate posttranslational protein modification (78), any of which may Energy Charge. In: H. J. Vogel (ed.), Metabolic Regulation, Ed. 3, be crucial determinants of whether or not a gene is expressed Vol. 5, pp. 1-22. New York: Academic Press, Inc., 1971. as either antigenic or isozymic activity. Hormones, such as 9. Bailey, R. E. Periodic Hormonogenesisâ€”A New Phenomenon. Periodicity in Function of a Hormone-Producing Tumor in Man. J. insulin and glucagon, and the cortical steroids and cyclic adenosine 3',5'-monophosphate also play a part in regulation Clin. Endocrino!., 32: 317-327, 1971. 10. Ballard, F. J., and Oliver, I. T. Ketohexokinase Isozymes of of gene expression (78); but mechanisms remain conjectural. Glucokinase and Glycogen Synthesis from Hexoses in Neonatal Rat Moreover, proteins are also degraded in eukaryotic cells, and it Liver. Biochem. J., 90: 261-268, 1964. is important to recognize that the level of any specific protein 11. Barker, K. L., Lee, K-L., and Kenney, F. T. Turnover of Tyrosine Transaminase in Cultured Hepatomas Cells after Inhibition of 5The significance of fetal antigens, not only to cancer, but also to Protein Synthesis. Biochem. Biophys. Res. Commun., 43: embryonic development in general, has been broadened by discovery 1132-1138, 1971. (28) that group-specific antigens coded by type C oncogenic RNA 12. Bloch-Frankenthal, L., Langan, J., Morris, H. P., and Weinhouse, S. viruses are expressed during embryonic development. According to the so-called oncogene hypothesis, the RNA virus genome is transmitted Fatty Acid Oxidation and Ketogenesis in Transplantable Liver Tumors. Cancer Res., 25: 732-736, 1965. vertically in an integrated or latent state, and gs antigen, for which it codes, is expressed during embryonic development, without the 13. Bonney, R. J., and Potter, V. R. Reversible Change to Fetal appearance of infectious virus particles. The antigen then disappears Isozyme Patterns in Regenerating and Precancerous Liver. during postnatal life and reappears in the tumors or with increasing age Federation Proc., 31: 500, 1972. of animals. 14. Boyse, E. A., and Old, L. J. Some Aspects of Normal and

2014 CANCER RESEARCH VOL. 32

Abnormal Cell Surface Genetics. Ann. Rev. Genet., 3: 269-290, 39. Lengerova, L. Expression of Normal Histocompatibility Antigens 1969. in Tumor Cells. Advan. Cancer Res., 16: 235-272, 1972. 15. Chance, B. In: Ciba Foundation Symposium on the Regulation of 40. Lipsett, M. B. Humoral Syndromes Associated with Non-endocrine Cell Metabolism. Boston: Little, Brown and Co. 1959. Tumors. Ann. Internal Med., 61: 733-756, 1964. 16. Criss, W. E. A New Pyruvate Kinase Isozyme in Hepatomas. 41. Lo, C. H., Cristofalo, V. J., Morris, H. P., and Weinhouse, S. Biochem. Biophys. Res. Commun., 35: 901-905, 1969. Studies on Respiration and Glycolysis in Transplanted Hepatomas 17. Criss, W. E. A Review of Isozymes in Cancer. Cancer Res., 31: of the Rat. Cancer Res., 18: 1-10, 1968. 1523-1542, 1971. 42. Lynen, F. The Aerobic Phosphate Requirement of Yeast. The 18. DiPietro, D. L., Sharma, C., and Weinhouse, S. Studies on Glucose Pasteur Effect. Ann. Chem., 546: 120-141, 1941. Phosphorylation in Rat Liver. Biochemistry, /: 455-462, 1962. 43. Markert, C. L., and Moller, F. Multiple Forms of Enzymes: Tissue, 19. Eliei, L. P. Non-Endocrine Secreting Neoplasms: Clinical Ontogenetic, and Species Specific Patterns. Proc. Nati. Acad. Sci. Manifestations. Cancer Bull., 20: 37-39, 1968. U.S.,45: 753-763, 1959. 20. Elwood, J. C., Lin, Y. C., Cristofalo, V. J., Weinhouse, S., and 44. Martin, D. W., Jr., Tomkins, G. M., and Bresler, M. A. Control of Morris, H. P. Glucose Utilization in Homogenates of the Morris Specific Gene Expression Examined by Synchronized Mammalian Hepatoma 5123 and Related Tumors. Cancer Res., 23: 906-913, Cells. Proc. Nati. Acad. Sei. U. S., 63: 842-849, 1969. 1963. 45. Matsushima, T., Kawake, S., Shibuya, M., and Sugimura, T. 21. Farina, F. A., Adelman, R. C., Morris, H. P., Lo, C. H., and Aldolase Isozymes in Rat Tumor Cells. Biochem. Biophys. Res. Weinhouse, S. Metabolic Regulation and Enzyme Alterations in Commun., 30: 565-570, 1968. Morris Hepatomas. Cancer Res., 28: 1897-1900, 1968. 46. Morris, H. P. Some Growth Morphological and Biochemical 22. Fishman, W. H. Inglis, N. R., and Green, S. Regan Isoenzyme: A Characteristics of Hepatoma 5123 and Other Transplantable Carcinoplacental Antigen/'". Cancer Res., 31: 1054-1057, 1971. Hepatomas. Progr. Exptl. Tumor Res., 3: 370-411, 1963. 23. Gold, P. Embryonic Origin of Human Tumor-Specific Antigens. 47. Morris, H. P. Studies on the Development, Biochemistry and Progr. Exptl. Tumor Res., 14: 43-58, 1971. Biology of Experimental Hepatomas. Advan. Cancer Res., 9: 24. Gold, P., and Freedman, S. O. Specific Carcinoembryonic Antigens 227-302, 1965. of the Human Digestive Tract. J. Exptl. Med., 122: 467-481, 48. Morris, H. P., and Wagner, B. P. Induction and Transplantation of 1965. Rat Hepatomas with Different Growth Rate (Including Minimal 25. Goodall, C. M. A Review: On Para-Endocrine Cancer Syndromes. Deviation Hepatomas). In: H. Busch (ed.), Methods in Cancer Intern. J. Cancer, 4: 1-10, 1969. Research, Vol. 4, pp. 125-152. New York: Academic Press, Inc., 26. Grossbard, L., and Schimke, R. T. Multiple Hexokinases of Rat 1968. Tissues. Purification and Comparison of Soluble Forms. J. Biol. 49. Newsholme, A. E., and Gevers, W. Control of Glycolysis and Chem., 247: 3546-3560, 1966. Gluconeogenesis in Liver and Kidney Cortex. Vitamins Hormones, 27. Hershko, A., and Tomkins, G. M. Studies on the Degradation of 25: 1-79, 1967. Tyrosine Aminotransferase in Hepatoma Cells in Culture. J. Biol. 50. Nordmann, Y., and Schapira, F. Muscle Type Isozymes of Liver Chem., 246: 710-714, 1971. Aldolase in Hepatomas. European J. Cancer, 3: 247-250, 1967. 28. Huebner, R. J., Kelloff, G. J., Sarma, P. S., Lane, W. T., Turner, H. 51. Nowell, P. C., Morris, H. P., and Potter, V. R. Chromosomes of C., Gilden, R. V., Oroszlan, S., Meyer, H., Myers, D. D., and Peters, "Minimal Deviation" Hepatomas. Cancer Res., 27: 1561-1579, R. L. Group-Specific Antigen Expression during Embryogenesis of 1967. the Genome of the C-type RNA Tumor Virus: Implications for 52. Ono, T. and Weinhouse, S. (eds.). Isozymes and Enzyme Ontogenesis and Oncogenesis. Proc. Nati. Acad. Sei. U. S., 67: Regulation in Cancer. Gann Monograph 13, in press. 366-376, 1970. 53. Passoneau, J. V., and Lowry, O. H. The Role of 29. Hull, E., Carbone, P. P., GiÃœin,D., O'Gara, R. W., and Kelley, M. Phosphofructokinase in Metabolic Regulation. Advan. Enzyme G. a-Fetoprotein in Monkeys and Hepatoma. J. Nati. Cancer Inst., Regulation, 2: 265-276, 1964. 42: 1035-1544, 1969. 54. Potter, V. R. Environmentally-Induced Metabolic Oscillations as a 30. Ichihara, A., and Ogawa, K. Branched-Chain Amino Acid Challenge to Tumor Autonomy. Miami Winter Symposium, Vol. 2, Transaminases in Normal Rat Tissues and Hepatomas. Gann pp. 291-313. Amsterdam: North Holland Publishing Co., 1970. Monograph 13, in press. 55. Potter, V. R. Transplantable Animal Cancer. The Primary 31. Johnson, M. J. The Role of Aerobic Phosphorylation in the Pasteur Standard. Cancer Res., 21: 1331-1333, 1971. Effect. Science, 94: 200-202, 1941. 56. Prehn, R. T. Cancer Antigens in Tumors Induced by Chemicals. 32. Katunuma, N., Katsunuma, T., Tornino, L, and Matsuda, Y. Federation Proc., 24: 1018-1022, 1965. Regulation of Glutaminase Activity and Differentiation of the 57. Racker, E. Mechanisms in Bioenergetics, pp. 193-253. New York: Isozymes during Development. Advan. Enzyme Regulation, 6: Academic Press, Inc. 227-242, 1968. 58. Roof, B. S., Carpenter, B., Fink, D. J., and Gordon, G. S. Some 33. Katzen, H. M., and Schimke, R. T. Multiple Forms of Hexokinase Thoughts on the Nature of Ectopie Parathyroid Hormones. Am. J. in the Rat. Proc. Nati. Acad. Sei. U. S., 54: 1218-1225, 1965. Med., 50: 686-691, 1971. 34. Knox, W. E. Enzyme Patterns in Fetal, Adult and Neoplastic Rat 59. Rutter, W. J., and Weber, C. S. Specific Proteins in Tissues. Basel: S. Karger AG, 1972. Cytodifferentiation. In: Developmental and Metabolic Control 35. Knox, W. E., Farron, F., and Hsu, H. H. J. Fetal-Type Isozymes in Mechanisms and Neoplasia, University of Texas M. D. Anderson Rat Tumors. Proc. Am. Assoc. Cancer Res., 13: 34, 1972. Hospital and Tumor Institute at Houston, pp. 195-218. 36. Krebs, H. A. and Eggleston, L. V. The Role of Pyruvate Kinase in Baltimore: The Williams & Wilkins Co., 1965. the Regulation of Gluconeogenesis. Biochem. J., 94: 3c-4c, 1965. 60. Sato, K., Weinhouse, S., and Morris, H. P., Glycogen Synthetases 37. Kroes, R., and Weisburger, J. H. Precocious Appearance of a-Fetal and Phosphorylases in Rat Hepatomas. Proc. Am. Assoc Res., 13: Protein in Serum of Rats Given Carcinogens. Proc. Am. Assoc. 27, 1972. Cancer Res., 13: 53, 1972. 61. Schapira, F. Isozymes et Cancer, Pathol. Biol., 18: 309-315, 1970. 38. Latner, A., and Skillen, A. Isoenzymes in Biology and Medicine. 62. Schapira, F., Dreyfus, J. C., and Schapira, G. Anomaly of Aldolase New York: Academic Press, Inc., 1968. in Primary Liver Cancer. Nature, 200: 995-997, 1963.

OCTOBER 1972 2015

63. Schapira, F., and Gregori, C. Pyruvate Kinase de PHepatome du 78. Tomkins, G. M. Specific Enzyme Production in Eukaryotic Cells. Placenta et du Foie Foetal de Rat. Compi. Rend., 272. Advan. Cell Biol., 2: 299-322, 1971. 1169-1172, 1971. 79. Uriel, J. Transilory Liver Anligens and Primary Hepaloma in Man 64. Schapira, F., Reuber, M. D., and Hatzfeld, A. Resurgence of Two and Ral. Pathol. Biol., 17: 877-884, 1969. Fetal-Type Aldolase (A and C) in Some Fast-Growing Hepatomas. 80. Vessel!, E. S. (ed.). Multiple Molecular Form of Enzymes. Ann. Biochem. Biophys. Res. Commun., 40: 321-325, 1970. N. Y. Acad. Sci., 151: 1-689, 1968. 65. Schimke, R. J., and Doyle, D. Control of Enzyme Levels in Animal 81. Vessell, E. S., and Fritz, P. J. Factors Affecting the Activily, Tissue Tissues. Ann. Rev. Biochem., 39: 929-976, 1970. Dislribulion, Synlhesis and Degradalion of Isozymes. In: M. 66. Scrutton, M. C., and Utter, M. F. The Regulation of Glycolysis and Rechcigl, Jr. (ed.), Enzyme Synlhesis and Degradalion in Gluconeogenesis. Ann. Rev. Biochem., 37: 249-302, 1968. Mammalian Syslems, pp. 339-374. Basel: S. Karger AG, 1971. 67. Sharma, C., Manjeshwar, R., and Weinhouse, S. Effects of Diet and 82. ViÃ±uela,E., Salas, M., and Sols, A. Glucokinase and Hexokinase in Insulin on Glucose Adenosine Triphosphate Phosphotransferases of Liver in Relalion lo Glycogen Synlhesis. J. Biol. Chem., 238: Rat Liver. J. Biol. Chem., 238: 3840-3845, 1963. 1175-1177, 1963. 68. Sharma, R. M., Sharma, C., Donnelly, A. J., Morris, H. P., and 83. Walker, D. G. On the Presence of Two Soluble Glucose Weinhouse, S. Glucose-ATP Phosphotransferases during Phosphorylating Enzymes in Adult Liver and Ihe Development of Hepatocarcinogenesis. Cancer Res., 25: 193-199, 1965. One of These after Birth. Biochim. Acta, 77: 109-226, 1963. 69. Shatton, J. B., Morris, H. P., and Weinhouse, S. Kinetic, 84. Warburg, O. The Metabolism of Tumors. London: Arnold Electrophoretic, and Chromatographie Studies on Glucose-ATP Constable, 1930. Phosphotransferases in Rat Hepatomas. Cancer Res., 29: 85. Warburg, O. On Ihe Origin of Cancer Cells. Science, 123: 309-314, 1161-1172,1969. 1956. 70. Shugar, D. (ed.). Enzymes and Isozymes, Structure, Properties and 86. Warburg, O. On Respiralory Impairmenl in Cancer Cells. Science, Function. New York: Academic Press, Inc., 1970. 124: 269-270,1956. 71. Spoiler, P. D., Adelman, R. C., and Weinhouse, S. Distinclive 87. Walabe, H. Early Appearance of Embryonic a-Globulin in Ral Properlies of Nalive and Carboxypeptidase-treated Aldolases of Serum during Carcinogenesis wilh 4-DimelhyIaminoazobenzene. Rabbit Liver and Muscle. J. Biol. Chem., 240: 1327-1337, 1965. Cancer Res., 31: 1192-1194, 1971. 72. Stanislawski-Birenzwajg, M., Uriel, J., and Grabar, P. Association of 88. Weber, G., Banerjee, G., and Morris, H. P. Comparative Embryonic Anligens wilh Experimenlally Induced Hepalic Lesions Biochemistry of Hepalomas. I. Carbohydrate Enzymes in Morris in the Rat. Cancer Res., 27: 1990-1997, 1967. Hepaloma 5123. Cancer Res., 21: 933-937, 1961. 73. Stonehill, E. H., and Bendich, A. Retrogenetic Expression. The 89. Weber, G., Slamm, N. B., and Fischer, E. A. Insulin: Inducer of Reappearance of Embryonal Anligens in Cancer Cells. Nature, 228: Pyruvate Kinase. Science, 149: 65-67, 1965. 370-371,1970. 90. Weinhouse, S. Studies on Ihe Fale of Isolopically Labeled 74. Sugimura, T., Salo, S., and Kawake, S. The Presence of Aldolase C Melaboliles in the Oxidative Melabolism of Tumors. Cancer Res., in Rat Hepatoma. Biochem. Biophys. Res. Commun., 39: //: 585-591, 1951. 626-630, 1970. 91. Weinhouse, S. Oxidative Metabolism of Neoplastic Tissues. Advan. 75. Tanaka, T., Harano, H., Sue, F., and Morimura, H. Crystallization Cancer Res., 3: 269-325, 1955. Characterization and Metabolic Regulation of Two Types of 92. Weinhouse, S. On Respiratory Impairment in Cancer Cells. Science, Pyruvate Kinase Isolated from Ral Tissues. J. Biochem. Tokyo, 62: 124: 267-268, 1956. 71-91,1967. 93. Weinhouse, S. Respiralion, Glycolysis and Enzyme Alterations in 76. Tanaka, T., Imamura, K., Ann, T., and Tanuichi, T. Molecular Liver Neoplasms. Miami Winter Symposium, Vol. 2, pp. 462-480. Forms of Pyruvate Kinase and Phosphofruclokinase in Normal and Amsterdam: North Holland Publishing Co., 1970. Cancer Tissue. Gann Monograph 13, in press. 94. Wroblewski, F. (ed.). Multiple Molecular Forms of Enzymes. Ann. 77. Taylor, C. H., Morris, H. P., and Weber, G. A Comparison of Ihe N. Y. Acad. Sci., 94: 655-1030, 1961. Properlies of Pyruvate Kinase from Hepatoma 3924A, Normal Liver Muscle. Life Sci., 8: 635-644, 1969.

2016 CANCER RESEARCH VOL. 32

Downloaded from cancerres.aacrjournals.org on September 30, 2021. © 1972 American Association for Cancer Research. Glycolysis, Respiration, and Anomalous Gene Expression in Experimental Hepatomas: G. H. A. Clowes Memorial Lecture

Sidney Weinhouse

Cancer Res 1972;32:2007-2016.

Updated version Access the most recent version of this article at: http://cancerres.aacrjournals.org/content/32/10/2007.citation

E-mail alerts Sign up to receive free email-alerts related to this article or journal.

Reprints and To order reprints of this article or to subscribe to the journal, contact the AACR Publications Subscriptions Department at [email protected].

Permissions To request permission to re-use all or part of this article, use this link http://cancerres.aacrjournals.org/content/32/10/2007.citation. Click on "Request Permissions" which will take you to the Copyright Clearance Center's (CCC) Rightslink site.