(CANCER RESEARCH 43, 3466-3492. August 1983]

Biochemical Strategy of Cancer Cells and the Design of Chemotherapy: G. H. A. Clowes Memorial Lecture1

George Weber

Laboratory for Experimental Oncology, Indiana University School ol Medicine, Indianapolis, Indiana 46223

Introduction the enzymic and metabolic pattern of cancer cells (97, 98). Three main propositions were made: An understanding of the biochemical strategy of the cancer 1. The biochemical strategy of the genome in neoplasia can cell is the via regia for the design of enzyme pattern-directed be identified by elucidating the pattern of gene expression as anticancer chemotherapy. The development and impact of novel revealed in the activity, concentration, and isozyme program of approaches that provided us with paradigms for problem solving key enzymes. in these two central areas of cancer research will be emphasized 2. Activities of key enzymes and metabolic pathways and in this presentation. I will underline the essence and substance concentrations of strategic metabolites are stringently linked with of our conceptual and experimental advances towards an un neoplastic transformation and progression; those not stringently derstanding of the biochemical strategy and the logic of gene linked do not yield a pattern. expression in cancer cells and identify a shared enzymic and 3. The linking of enzymic and metabolic imbalance with trans metabolic program in various animal and human neoplasms. formation and progression can be identified in a spectrum of With the pinpointing of the selective advantages that the altered tumors of graded malignancy, proliferative rate, and degree of gene expression confers to cancer cells, sensitive targets and differentiation. ordering principles have emerged for the rational design of Linking of Biochemical Strategy with Biological Behavior in enzyme pattern-targeted chemotherapy (97, 98). Neoplasia. To identify the pattern of biochemical imbalance and its linking to neoplastic transformation and progression, three Biochemical Basis of Chemotherapy types of relationships were postulated and verified by the molec ular correlation concept. The classification referred to the behav All cancer cells are transformed. Thus, they have a biological ior of activities of key enzymes and overall metabolic pathways commitment to continued replication which is manifested in a and of the concentrations of strategic metabolites (90, 97, 98, biochemical program of quantitative and qualitative imbalance. 109). Cancer cells are also characterized by the capacity for a gradual Progression-linked Alterations: Biochemical Expression of escalation in expression of neoplastic properties, i.e., progres the Different Degrees of Malignancy. In Class 1 are the bio sion, an amplification of the biochemical imbalance. chemical parameters that increase or decrease in a graded Drug treatment should induce a metabolic perturbation in fashion in correlation with tumor malignancy. These are strin cancer cells that would selectively interfere with the biochemical gently linked markers of neoplastic progression. program of the commitment to replication and result in the death The malignant program may be expressed in different degrees of cancer cells. The host cells should adapt and recover from that range from mild through advanced to the full-blown pattern. toxicity. This graded expression of the neoplastic program in the different In the following, an account will be given of the main ideas and tumor lines in a spectrum of neoplasms of the same cell type is experimental observations that led to the formulation of the termed "progression." Thus, the activities of key enzymes that present integrated concept of the biochemical strategy of cancer are stringently linked with the biological behavior should be cells. expressed in parallel with the degrees of tumor malignancy. In our laboratory, the introduction of three approaches pro Testing these predictions demonstrated that the activities and vided instruments for discovery: (a) the molecular correlation concentrations of key enzymes increased and those of the concept; (b) the key enzyme concept; (c) the use of meaningful opposing enzymes decreased in concordance with the malig biological models and controls. nancy of the different tumor lines. With the increase in the expression of malignancy, i.e., proliferation rate, the enzymic and metabolic imbalance becomes more and more pronounced. The Molecular Correlation Concept Transformation-linked Alterations: The Shared Programs In 1961,1 first introduced the ideas of the molecular correlation of Cancer Cells. In Class 2 are parameters that increased or concept as a theoretical and experimental method for discovering decreased in all hepatomas, irrespective of the degrees of neo the pattern of biochemical imbalance and its linking with neo plastic progression. These are ubiquitous changes, and they are plastic transformation and progression (90). This approach pro stringently linked, all-or-none markers of transformation. vided precise, testable predictions for anticipated alterations in Neoplasia is manifested by transformation that is heritable and characterizes every tumor cell. Since there is a shared program of commitment to proliferation in all cancer cells, displayed as 1This paper is based on the 22nd Annual G. H. A. Clowes Memorial Lecture, the malignant transformation, the activities of certain enzymes presented on April 30, 1982, at the 73rd Annual Meeting of the American Associ and the concentrations of strategic metabolites should exhibit ation for Cancer Research, St. Louis, Mo. The investigations of the author and his associates have been supported by USPHS Grants CA-13526 and CA-05034. the same type of alterations in all tumors in a spectrum of Received April 20, 1983. neoplasms. Such all-or-none alterations, stringently linked with

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Downloaded from cancerres.aacrjournals.org on September 29, 2021. © 1983 American Association for Cancer Research. G. H. A. Clowes Memorial Lecture transformation, do occur in tumors. Thus, the activities of certain and is linked with progression. key enzymes are increased, and the activities of other enzymes Thus, the reprogramming of gene expression in neoplasia is functionally opposed to them are decreased in all the neoplasms. reflected in both quantitative and qualitative alterations. "Repro Coincidental Alterations: The Apparent Diversity. In Class gramming" of gene expression is the appropriate term because 3 are alterations that fluctuated without relation to malignancy. the essential neoplasia-linked part of the program is displayed in These coincidental changes in gene expression are not strin an ordered, meaningful, and integrated enzymic and metabolic gently linked with neoplasia. imbalance. Alterations in certain enzymic activities that showed no relation to transformation or progression in the tumor spectrum proved The Key Enzyme Concept to be due to the behavior of non-key enzymes that are present in excess. Regulation of their activities is not linked with neopla The concept of key enzymes was developed in this laboratory sia. along with methods for identifying such enzymes (97, 98). Earlier Ordered Pattern versus Diversity and Randomness. The studies on tumor metabolism which failed to detect a meaningful behavior of the activities of key enzymes revealed an ordered pattern were due, in part at least, to the poor choice of enzymes transformation- and progression-linked reprogramming of gene and other biochemical variables. Our introduction of the concept expression. The meaningful pattern of enzymic and metabolic of key enzymes provided an approach to clarify the strategy of imbalance was first identified in the spectrum of hepatomas of gene expression in normal and neoplastic cells (92, 93, 97, 98, different proliferative rates. It was recognized that not all en 114,121). zymes should be expected to be linked to transformation and Observations in carbohydrate metabolism and conceptual ad progression because the integration of gene expression operates vances led to the recognition that not all enzymes should be through the control of a relatively small number of key enzymes. expected to be stringently linked with transformation and pro Some alterations showed no pattern, since they involved en gression because the integration of gene expression operates zymes or metabolites present in an excess. Such apparent through the stringent control of a relatively small number of diversity and randomness indicate coincidental alterations in enzymes (97, 98). I termed these key enzymes because the gene expression that are not stringently linked to the core of regulation of the rate and direction of the flux of opposing and neoplasia. What is important about cancer is ordered; what is competing synthetic and catabolic pathways is achieved through not is the random element and the diversity (97, 98). their control (97, 98). The Key Enzymes. The term "pacemaker" or rate-limiting Operational Advantages of the Molecular Correlation Con cept: A Unifying Approach in Cancer Biochemistry. The op enzyme is a narrower one, denoting the enzyme that has the erational advantage of the molecular correlation concept is its lowest activity in a sequence of metabolic steps in one pathway; testability; it provided predictions that could be verified or dis the key enzymes include the rate-limiting ones. The main criteria proved. That a theory must be testable to be useful, and thus for identifying key enzymes include their biological role, place in susceptible to verification or refutation, is in line with the views the pathway, and regulatory properties (97, 98). The key en expressed in other research areas by Popper (68), Burnet (11), zymes usually have relatively low activities, govern one-way and Medawar (60). metabolic reactions, and frequently are the first or the last in The molecular correlation concept has been a useful concep reaction sequences; they may be pathways in themselves or tual tool to recognize and overcome what Holton (31) called "the operate on both sides of reversible reactions with a one-way negative, or enslaving, role of presuppositions." In cancer bio enzyme opposed by another one-way enzyme. Key enzymes chemistry, such presuppositions implied that the enzymic pattern are frequently the targets of feedback or multiple regulation, of the cancer cell was either random or ordered. The molecular exhibit allosteric properties, or are interconvertible enzymes and correlation concept determined that the enzymic and metabolic may have an isozyme pattern (97, 98). The activities and amount pattern of cancer cells has both ordered and random compo of key enzymes are frequently subject to nutritional and hormonal nents. Mayr (59) pointed out, "Some sets of data may not have regulation. The behavior of activities, amount, and isozyme pat significance until certain concepts are clarified or principles es tern of key enzymes is stringently linked with transformation and tablished." In the biochemistry of cancer research, some ordering progression in cancer cells. Examples include: in pyrimidine principles have now been introduced, resulting in an integration metabolism, rc., bamoyl-phosphate synthetase II, CTP synthe- of the apparent diversity in the behavior of enzymes, metabolic tase, ribonucleotide reducíase, DNA polymerase, dihydrothy- pathways, and nucleotides into a meaningful picture. mine dehydrogenase; in purine metabolism, amidophosphoribo- Gene Logic in Neoplasia. With the conceptual and experi syltransferase, xanthine oxidase, IMP dehydrogenase; in carbo mental approaches of the molecular correlation concept, impor hydrate metabolism, glucokinase-hexokinase, 6-phosphofructo- tant aspects of gene logic have been uncovered in cancer cells, kinase, pyruvate kinase, glucose-6-phosphatase, glucose-6- (a) Reciprocal regulation of the activities of antagonistic key phosphate dehydrogenase. enzymes and overall metabolic pathways of synthesis and deg The Non-Key Enzymes. These are enzymes that apparently radation amplifies the quantitative changes in the enzymic and do not become limiting in metabolic flux. Such enzymes are metabolic capacity, (b) The extent of rise in key enzymic activities usually characterized by the following properties: they catalyze and nucleotide concentrations is a function of their levels in the reversible equilibrium reactions and are shared by both synthetic resting normal liver. The lower the activities and concentrations and catabolic pathways; they exhibit high activities; and they are in normal liver, the greater is the increase in the rapidly prolifer present in excess. Their activities are usually not markedly ating hepatomas. (c) In the isozyme shift, the replacement of the altered by nutritional and endocrine regulation and do not cor isozymes that respond to nutritional and hormonal regulation by relate with transformation or progression in neoplasia. Examples nonresponsive, Iow-Km isozymes is characteristic of cancer cells include: in pyrimidine metabolism, dihydroorotate oxidase; in

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Downloaded from cancerres.aacrjournals.org on September 29, 2021. © 1983 American Association for Cancer Research. G. Weber purine metabolism, GMP kinase; and in carbohydrate metabo drogenase) is in line with the capacity for the direct oxidative lism, phosphohexoseisomerase, lactate dehydrogenase, 6-phos- pathway which increased in hepatomas, as determined by iso phogluconate dehydrogenase. tope studies in slices (90, 92). The stringently linked correlation Testable Predictions. It was an important advance in our of thymidine kinase activity (measured in extracts) with hepatoma conceptual approach when we recognized the key enzymes growth rate was paralleled with the transformation and progres because testable predictions could be made regarding their sion linked rise in the behavior of the overall synthetic pathway expected behavior in neoplastic transformation and progression. measured in tissue slices by the incorporation of thymidine to Analysis of behavior and properties of the key enzymes and their DNA. Conversely, the decline of the activity of the rate-limiting relation to transformation and to progression in a spectrum of enzyme of thymidine degradation, dihydrothymine dehydrogen neoplasms has permitted identification of the linking of biochem ase (assayed in extracts), was paralleled by the decrease in ical alterations with the biological behavior of tumors. activity of the degradation of thymidine to CO2, measured in liver Information Value of Enzyme Activity Determinations. The and hepatoma tissue slices (23, 97, 98, 123). The stepped-up following information may be expected from assaying key en- activity of CTP synthetase (assayed in extracts) is reflected in zymic activities in normal and cancer cells. the increased concentration of CTP (measured in freeze-clamp 1. Under optimum assay conditions, when the only limiting tissue samples), and both parameters were transformation and factor is the amount of enzyme, it is assumed that the elevated progression linked (34, 47). The increase in GMP concentration or lowered specific activity is an accurate reflection of the enzyme reflects the elevated activity of GMP synthetase (10, 34). The amount. The validity of this assumption was confirmed by im- progression-linked depletion in the concentration of L-glutamine munotitration for 5 enzymes from different metabolic pathways in the hepatoma spectrum is paralleled with the rise in the activity (19, 50, 79, 87, 88) (see below). of enzymes involved in glutamine utilization in the tumors (Table 2. Measurement of enzymic activity may be utilized in probing 8). Similar correlations have been noted in relating the decrease the pattern of gene expression in normal and cancer cells (97, of activities of urea cycle enzymes with the decrease in urea 98). Determination of enzyme concentration demonstrates quan cycle activity and the increase of activities of key enzymes of titative alterations in gene expression. In the hepatoma model polyamine biosynthesis with the elevation of metabolites of poly- system, it has revealed a reprogramming of gene expression in amine production (122, 131). different metabolic areas. Such correlations can be expected only if the activities of key 3. By measuring the isozyme pattern, qualitative aspects of enzymes, particularly the rate-limiting ones, are compared with gene expression can be elucidated. Our studies indicated that the activity of the relevant metabolic pathway. For instance, the there was a definite pattern, which I named the isozyme shift activity of the rate-limiting enzyme, hexokinase, correlated with (97, 98). The shift was evidenced by the loss of the liver-type, the behavior of glycolysis; however, the activities of phospho high-Km enzymes that were strongly regulated by nutritional and hexoseisomerase or lactate dehydrogenase, which are present hormonal controls. These were largely or completely replaced by manyfold in excess in the liver, provided no correlation or pattern. muscle-type isozymes that had low Km and high affinity for 5. Since key enzymes are targets of chemotherapy, determi substrate and were less or not at all subject to physiological nation of the activity of such enzymes after drug treatment may (nutritional and endocrine) regulation and feedback signals and provide useful information on the extent of inhibition achieved by required no activator. Examples of these in the hepatomas are the anticancer therapy (17, 120). A lack of response or the the replacement of glucokinase with hexokinase (127, 128), liver- presence of enzymic activities markedly higher than those usually type pyruvate kinase with muscle-type isozyme, liver-type AMP observed in that type of tumor may indicate the emergence of deaminase chiefly by the muscle-type enzyme (36), and liver- resistance due to enzyme overproduction. type, high-Km uridine kinase with Iow-Km uridine kinase isozymes. 6. Since enzymes, particularly salvage ones, play a role in Important work was carried out in the isozyme area by Wein- activation of certain antimetabolites, in case of development of house and his associates that illuminated the isozyme pattern of drug resistance decrease or deletion in activities of such activat hepatomas and other critical areas of cancer biochemistry (126- ing enzymes might reveal the mechanism of drug resistance 128). which includes gene amplification. On the other hand, the pres 4. Key enzymes are indicators of the activity of the overall ence of such activating enzymes indicates the feasibility of metabolic pathway. To test the validity of this assumption, we treatment with a drug that required activation by the enzyme. compared the activities of key enzymes and their behavior with 7. Since 43 enzymic activities were recognized as transfor that of the relevant overall metabolic pathways and with concen mation linked, these enzymes may be used as markers of trations of end products, intermediates, key metabolites, and neoplastic transformation and should provide assistance in the nucleotides. There was good agreement between the alterations biochemical diagnosis of neoplasia. in the behavior of the key enzymic activity and in that of the 8. Since 28 enzymic activities were recognized as progression activity of the metabolic pathway (90, 92). In the hepatoma linked, these enzymes may be utilized as markers of tumor spectrum, the increased activities of key enzymes of glycolysis malignancy, and they may be of assistance in the biochemical measured in extracts were paralleled by an increase in glycolysis; grading of malignancy. conversely, the decrease in activities of the key gluconeogenic enzymes was reflected in the decrease in the gluconeogenic Biological Model Systems pathway. The activities of these pathways were measured in liver and hepatoma tissue slices by determining the conversion In 1955, we showed that during feeding of a hepatocarcino- of glucose to pyruvate and lactate and by the production of genic dye to rats the activity of liver glucose-6-phosphatase glucose from pyruvate (84). The elevated activity of a key enzyme steadily decreased and was absent in the resulting primary of pentose phosphate production (glucose-6-phosphate dehy hepatocellular carcinomas (103). Other investigators, including

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Goldfarb and Pugh (24), in an excellent recent study, confirmed 1. Since all were of the same cell type, hepatocellular carci these observations. In 1957, we were the first to show the nomas, they provided the 2 biological properties we wished to absence of an enzymic activity in transplantable cancer cells characterize, (a)All tumors were composed of transformed cells when we demonstrated that glucose-6-phosphatase activity was and therefore testable for the behavior of the activities of enzymic absent in the chemically induced, transplantable, rapidly growing and metabolic parameters that were altered in all tumors. This Novikoff hepatoma (105). We also reported that in the Novikoff permitted the discovery of transformation-linked alterations, (b) hepatoma glucose-6-phosphatase activity could not be induced Since the tumor spectrum consisted of neoplasms with increas by starvation or steroid treatment which increased the enzymic ing degrees in the expression of malignancy (increased prolifer activity in normal and host liver (105). These studies were the ative rate and decreased differentiation), the biochemical prop first to indicate the lack of responsiveness of a tumor enzyme- erties that were linked with neoplastic progression could be forming system to nutritional or hormonal regulation. identified. In 1959, it was reported that the Dunning hepatoma had some 2. Another advantage was that most of these tumors con glucose-6-phosphatase activity (67). Since the Dunning had a sisted of 80 to 95% neoplastic cells with relatively little stroma, slower growth rate and was better differentiated than the Novi a cellular population much more homogeneous than that of koff hepatoma, it seemed to me that the different growth rates primary tumors. and malignancy of tumors should be manifested also in grada 3. Because the tumors were of different growth rates, the tions in the expression of biochemical properties (106). To test spectrum permitted elucidation of the biochemical basis of pro this idea rigorously, there was a need for hepatocellular carci liferation rate. nomas of different growth rates, degrees of differentiation, and 4. Since responsiveness of human neoplasms to chemother malignancy. The first of such to come to my attention was the apy is determined in part at least by their growth rate (136), chemically induced transplantable hepatoma 5123D which (in these slowly and rapidly growing rat hepatomas offered an 1960) was a slowly growing, well-differentiated tumor. The bio opportunity to clarify the biochemical basis of drug responsive chemistry of this tumor when compared with that of the Novikoff ness. hepatoma confirmed the expectation I expressed in 1959 that a 5. The slowly growing hepatomas are useful models for the spectrum of tumors of different malignancy would be helpful in human disease which is a major clinical challenge to chemother understanding the biochemistry of cancer cells (106). Subse apy. On a global scale, liver tumors are the most common quently, other hepatomas of different malignancy became avail neoplasms (9). able, and from these I assembled a spectrum of hepatocellular 6. The biological and biochemical properties of many of the carcinomas in which the molecular correlation concept was different tumor lines were remarkably unchanged from genera tested (115). tion to generation. This stability was due, in large part, to the Most of these tumors I obtained originally from Dr. Harold P. fact that the tumors were originally transplanted and carried in Morris, who was then at the National Cancer Institute and later the laboratory of Dr. H. P. Morris. The stability or the variation in at Howard University. It is a pleasure and a privilege to acknowl the different tumor lines can be determined by karyotypic, en edge the harmonious, productive collaboration with Dr. Morris zymic, or histological assays, and these can be related to the that spanned over 20 years, involved many thousands of these proliferative rate of the tumors. tumors, and resulted in 50 joint publications. The Morris hepa 7. An important operational advantage of the liver tumor tomas were made available to me and to about 60 laboratories spectrum is the availability of appropriate, meaningful control in the United States and around the world, and they have been tissues (normal, regenerating, and differentiating liver). Dediffer- of immense use in many aspects of cancer research. Through entiated tumors for which no control tissue is available (Ehrlich producing, maintaining, and distributing these tumors, many of tumor, HeLa cells, etc.) are of limited interpretive value in this them in collaborative research, Dr. Morris has made an imperish context. able contribution to cancer research. 8. An advantage of much interest to biochemists and phar The Measure of Malignancy in the Tumor Spectrum. In the macologists is that from many of these hepatomas an almost hepatoma spectrum, growth rate is used as a measure of the unlimited quantity of tumor material is available, which is helpful degree of malignancy. The rate of proliferation was quantitated in enzyme purification and in pharmacology. Because of its by several independent techniques: biological (tumor size and uniform growth rate and retention of a high percentage of viable volume); cytological (mitotic count); histological (degree of differ cells, hepatoma 3924A is particularly suitable for freeze-clamp entiation); and biochemical (thymidine index). The malignancy of studies. the tumor lines was ranked also by time required in weeks for 9. It might be argued that the progression-linked alterations the neoplasms to reach a uniform diameter of 1.5 cm (97, 98). occurred only in the proliferating cells that are part of the heter All methods yielded the same ranking orders for the tumors (61, ogeneous cell populations of these tumors. This would suggest 62). Most of these solid tumor lines showed remarkable stability that the enzymic and metabolic alterations do not occur in all which indicated a stable gene pool and permitted good reprod- cells, but only in those in the growth fraction; i.e., the enzymic ucibility of the studies in many laboratories (97, 98). The mathe increases would indicate merely the presence of an expanding matical relation between tumor-proliferative rate and the extent growth fraction. In some of the hepatomas, the growth fraction of alterations in biochemistry is measured by correlation coeffi was measured (54, 78). If such reasoning were valid, then the cients. biochemical assays would provide only an indirect way of meas Operational Advantages of the Hepatoma Spectrum as a uring the growth fraction. Model System for Testing the Molecular CorrelationConcept. This argument is refuted by extensive documentation that a The spectrum consisted of lines of transplantable solid hepato number of enzymic and metabolic changes occur to about the mas, each with a different proliferative rate. same extent in every tumor examined in the spectrum, including

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Downloaded from cancerres.aacrjournals.org on September 29, 2021. © 1983 American Association for Cancer Research. G. Weber the slowest growing (with the lowest growth fraction) and the rats. This model also provides a control for rapid proliferative most rapidly growing (highest growth fraction). Such transfor rate in the postnatal rat. We noted that livers should not be used mation-linked alterations indicate that the biochemical changes before 6 days after birth because in the rat the fetal liver is are present in all or nearly all cancer cells in the various tumors. largely a hemopoietic organ; the overwhelming proportion of 10. Since the most rapidly growing hepatomas required 1 hemopoietic cells to liver cells in fetal rat liver was documented week, whereas the slowest growing ones took 52 weeks, to in histological sections (104). Many of the enzymic changes from reach a diameter of 1.5 cm, the hepatoma spectrum provided an fetal to postnatal livers represent not altered enzymic activities array of neoplasms of vastly different proliferative rates and but shifts in the cellular population (97, 98, 104). degrees of differentiation. In the very slowly growing hepatomas, the threshold alterations, and in the most rapidly growing tumors, Generalization the extreme expression of biochemical imbalance can be identi fied. Such an unparalleled model system has been most useful To determine whether the biochemical pattern of enzymic and in cancer research, and its maintenance and availability to all metabolic imbalance originally discovered in the chemically in investigators remain of great scientific importance. duced, transplantable hepatomas of the rat does or does not apply to other neoplasms in animals and in humans, a series of Determination of Specificity to Neoplasia, Role of Control experimental neoplasms and primary human tumors was exam Tissues, and the Power of Interpretation ined. The Kidney Spectrum. Because kidney is the only organ other Determination of specificity of the biochemical pattern to the than liver that has strong gluconeogenesis and high glucose-6- neoplastic liver was made feasible by the ready availability of phosphatase activity, I asked Dr. Morris to produce renal cell appropriate controls: normal, resting liver; regenerating liver; and carcinomas for my laboratory. In these tumors, we tested the differentiating, developing liver. With these systems, it was pos hypothesis whether the decrease in gluconeogenesis and in sible to elucidate quantitative and qualitative differences between activities of gluconeogenic enzymes also occurs in renal neopla the metabolism of hepatomas and that of control tissues. The sia where glycolysis and gluconeogenesis operate as opposing spectrum of hepatomas of different growth rates and the array pathways. The biological properties and histology of these tu of control tissues vastly increased the power of interpreting mors were reported (63); and in a series of papers, we elucidated whether the biochemical changes were due to neoplastic trans the enzymology, nucleotide content, and metabolism of kidney formation and progression or to rapid growth rate or different tumors (33, 99, 107,110,132). degrees of differentiation which could be readily determined from Colon Carcinomas and Muscle Sarcoma. The applicabilityof the control tissues. Detailed characterizations have been pub the biochemical imbalance discovered in hepatomas was studied lished (92, 93, 96-98, 104, 105, 121, 124), and a concise in various other animal model systems; these include a chemically description of the control system is provided below because it induced muscle sarcoma and a series of chemically induced bears on the interpretation proposed. colon tumors in mouse and rat (102, 111 ). Control, Normal Tissues. The enzymic and metabolic phe- Human Neoplasms. To test the applicability to human malig notype of the hepatomas was compared primarily with that of nancy of the biochemical alterations observed in chemically the normal liver of rats of the same sex, strain, age, weight, and induced tumors in animals, primary neoplasms in human were nutritional status as the tumor-bearing ones. Animals were kept investigated with emphasis on primary hepatocellular carcinoma in individual cages with Purina fox chow and water available ad (114), renal cell carcinoma (33, 69, 99, 107), colon carcinoma libitum and were killed between 9 and 11 a.m. Rats were under (30, 113), lung neoplasia (25, 118), and leukemic cells (7). Xen- regulated cycles of light and darkness. Extensive background ografts of colon carcinomas of different proliferative rates in the studies were carried out in normal animals to establish to what nude mouse were also studied (108). The special problems in extent nutritional or hormonal variation may impact on the liver obtaining comparable human normal tissues and the precautions enzymic and metabolic activity. The liver of the tumor-bearing that we take to achieve valid comparisons were outlined (33, rat is not a suitable control tissue, because it was shown that it 99). was subject to numerous unpredictable artifacts. Host liver should be used as a control for hepatomas of different growth Pyrimidine Metabolism: Transformation- and Progression- rates only in clearly specified exceptions. linked Imbalance in Cancer Cells Regenerating Liver. Since the various liver tumor lines had different rates of proliferation, it was essential to clarify whether The biochemical strategy of cancer cells in pyrimidine and DMA the biochemical pattern was due to neoplastic alteration and metabolism is expressed in the behavior of the activities of progression or whether it reflected rates of proliferation that opposing key enzymes and pathways of synthesis and catabo- occur in normal liver. The biochemical phenotype of the prolifer lism and of the concentrations of crucial nucleotides and de- ating normal liver was elucidated by studying the regenerating oxynucleotides. liver at different time periods after partial hepatectomy in adult Characteristic Features of Normal Liver Pyrimidine Metab rats. The liver of sham-operated normal rats was used as a olism. The special features of pyrimidine metabolism in normal control. The proliferation rate of the regenerating liver at 24 hr rat liver include the following. after operation is similar to that of hepatoma 3924A. 1. The de novo synthetic pathway assembles UMP from pre Differentiating Liver. Since the hepatoma spectrum consisted cursors through 9 reactions. Carbamoyl-phosphate synthetase of tumor lines of different proliferative rates and degrees of II is the first rate-limiting enzyme of UMP biosynthesis. The first differentiation, it was necessary to determine the behavior of 3 and the final 2 enzymes are each on a complex protein (43, biochemical parameters in the differentiating liver of developing 45). With the exception of dihydroorotate dehydrogenase which

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REGULATION PATHWAY ENZYMES HEPATIC ACTIVITY (nmol/hr/mg protein)

UDP Kinase 400,000

UMP Kinase 44,000

OMP DC 34

OPRT 47

MITO CHONDRION DHO DH 200

PRPP DIHYDROOROTATE

DHOase 45

450

GPSn

Chart 1. Intracellular distribution of enzymes of de novo CTP synthesis in liver cells and the targets of antimetabolite action. A cytoplasmic protein which has 3 enzymic activities initiates the pathway, and a protein with 2 enzymic activities completes the series of steps leading to UMP biosynthesis. In addition to these 2 cytoplasmic complex proteins, comprising 5 enzymic activities, there is also dihy- Chart 2. Comparison of the specific activities of liver enzymes of UTP de novo droorotate oxidase which is located on the outside of the inner mitochondrial biosynthesis indicates that the first enzyme, carbamoyl-phosphate synthetase II, is membrane. From UMP, 2 reactions are catalyzed by kinases, yielding UDP and the rate-limiting one. The activity of the subsequent enzyme, CTP synthetase, is 5 UTP. The subsequent enzyme, CTP synthetase, forms CTP; they are all cytoplasmic nmol/hr/mg protein; this is the rate-limiting enzyme of CTP biosynthesis. UDP enzymes. OMP DC, orotidine-5'-monophosphate decarboxylase; OMP, orotidine kinase, nucleosidediphosphate kinase; OMP DC, orotidine-5'-monophosphate de 5'-monophosphate; OPRT, orotate phosphoribosyltransferase; OA, oxidase; DHO carboxylase; OMP, orotidine 5'-monophosphate; OPRT, orotate phosphoribosyl OH. dihydroorotate dehydrogenate; DHO, dihydroorotate; DHOase, dihydroorotase; transferase; DHO DH, dihydroorotate dehydrogenase; DHOase, dihydroorotase; CA~asp, carbamoylaspartate; ATCase, aspartate carbamoyltransferase; PALA, N- ASP, aspartate; P, phosphate; CPS //, carbamoyl-phosphate synthetase II. (phosphonacetyl)-L-aspartate; CA~P, carbamoylphosphate; CP-synth. II, carba- moyl-phosphate synthetase II.

is in the mitochondria, the other enzyme activities are in the cytosol (Chart 1). From UMP through actions of UMP and UDP kinases and CTP synthetase, the nucleotides UDP, UTP, and 2600-iZ CTP are synthesized. The rate-limiting enzyme of the overall 4OO- »'I process of de novo CTP biosynthesis is CTP synthetase (Chart '3Q200-1!o. "S 2). o23BOO900i- 2. From the pyrimidine nucleotides, UDP and CDP, ribonucle- otide reductase yields dUDP and dCDP. From subsequent syn

thetic steps of the de novo pathway, dCTP and dTTP are Charts. Pyrimidine biosynthesis. Comparison of the specific activities of the produced which, in the presence of dGTP and dATP, derived rate-limiting and salvage enzymes involved in the synthesis of dTMP (left), CTP from the purine-synthetic pathways, provide the substrates for (middle), and UMP (right) in normal rat liver. TdR, thymidine. DMA polymerase for the biosynthesis of DNA. The rate-limiting synthetic enzyme of the entire de novo pyrimidine, purine, and kinase produces CDP, the immediate precursors for CTP and also for CDP reductase from which the dCDP2 and, through DNA synthesis is ribonucleotide reductase which has orders of magnitude lower activity than any other enzyme in these meta subsequent steps, dTMP may be formed; (e) deoxycytidine bolic areas (85) (see below and Table 1). kinase salvages deoxycytidine by routing it directly to the heart 3. The salvage enzymes provide powerful alternative salvage of deoxynucleotide synthesis by forming dCMP, a precursor of routes to produce the key metabolites, UMP, CTP, and dTMP dCTP and dTMP; (r) of high significance is the salvage of (Charts 3 and 8; see below): (a) uridine is salvaged by uridine- cytidine kinase; (b) uracil is recycled by uridine phosphorylase 2 The abbreviations used are: dNTP, deoxynucleotide triphosphate; PRPP, phos- which produces uridine; (c) uracil is also recycled in a one-step phoribosylpyrophosphate (5-phosphoribosyl 1-pyrophosphate); XMP, xanthosine monophosphate; APRT, adenine phosphoribosyltransferase; HPRT, hypoxanthine process by the uracil phosphoribosyltransferase reaction; (d) phosphoribosyltransferase; GPRT, guanine phosphoribosyltransferase; LD»,50% uridine-cytidine kinase salvages cytidine to yield CMP, and CMP lethal dose.

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Downloaded from cancerres.aacrjournals.org on September 29, 2021. © 1983 American Association for Cancer Research. G. Weber thymidine by thymidine kinase; this provides an alternative path the rapidly growing hepatomas, they were 5.7- to 9.5-fold higher way for the production of the key deoxynudeotide precursor, than in normal control liver. The molecular correlation concept dTMP. classified the behavior of this enzyme as transformation linked 4. The pyrimidine-synthetic pathways are opposed by the 2 because the activity increased in all the tumors, even the slowest main catabolic pathways that degrade thymidine and undine to ones, and it was progression linked because it correlated signif C02 and ammonia. The rate-limiting catabolic enzyme is dihydro- icantly with the proliferation rate of the neoplasms (1, 3). thymine dehydrogenase which also catalyzes the dihydrouracil Carbamoyl-phosphate synthetase II exists as a multienzyme dehydrogenase step. Recently, this enzyme was purified in this complex with aspartate carbamoyltransferase and dihydrooro- laboratory (81). It is important for an understanding of the bio tase, the second and third enzymes of de novo uridylate synthe chemical strategy of cancer cells that in normal liver the capacity sis (see Ref. 3). In analyzing 12 to 17 hepatoma lines, we found of the pyrimidine-degradative pathway is markedly higher than that there was a display of an integrated program in which the that of the rate-limiting enzymes of the synthetic pathways (23). activities of other enzymes of de novo DTP synthesis (aspartate The Neoplastic Program of Pyrimidine Metabolism. In the carbamoyltransferase, dihydroorotase, orotate phosphoribosyl- neoplastic liver cells, there is a program expressed in pyrimidine transferase, and orotidine-5'-phosphate decarboxylase) in metabolism which entails an increase in the activities of key creased (123). The activities of the first 3 enzymes were trans enzymes of de novo biosynthesis and of salvage pathways formation and progression linked, and those of the final 2 en concurrent with a decrease in activities of key enzymes of zymes were transformation linked (1, 3, 123). The activity of pyrimidine degradation (23, 58, 82, 94, 95, 97, 98, 121, 123). dihydroorotate dehydrogenase did not yield a pattern. Here, characteristic examples will be given of the behavior of In 13 hepatomas of vastly different proliferative rates, we the activities of enzymes that are transformation and progression showed that the specific activities of UDP kinase (nucleosidedi- linked. phosphate kinase; ATP:UDP phosphotransferase) increased sig The specific activity of carbamoyl-phosphate synthetase II in nificantly (1.6- to 3.9-fold) in all neoplasms (Chart 5). Since the 17 rat hepatomas was significantly increased above the activity activity was elevated even in the slowest growing, most differ observed in control livers. The rise in enzyme activity correlated entiated hepatomas, but without a relation with proliferative rate, positively with the increase in the proliferative rates of the tumors, this enzymic behavior was classified as transformation linked yielding a Spearman's rank correlation coefficient (rs)of 0.924, (130). significant at the 1% level (Chart 4). In the slowly growing UDP kinase provides the substrate, DTP, for the rate-limiting hepatomas, the increases in activities were 1.3- to 2.9-fold; in enzyme of CTP biosynthesis, CTP synthetase, the specific activ ity of which is transformation and progression linked in the hepatoma spectrum (47). CTP synthetase activities in 14 tumors with slow and medium growth rates increased 1.6- to 4.4-fold 4 and in rapidly growing hepatomas 5.4- to 10.6-fold over the ?'lî activities of the normal liver, yielding a significant Spearman's rank correlation coefficient (47). i The most profound increase in an enzymic activity measured

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Chart 4. The transformation- and progression-linked behavior of the activity of Charts. Transformation-linked increase in activity of undine 5'-diphosphate carbamoyl-phosphate synthetase II in the hepatomas of different growth rates. (UDP kinase) in hepatomas of different growth rates. Means of specific activities Mean spécifieactivities are plotted as percentages of the normal liver value. Bars, are plotted as percentages of the normal liver value. Bars, S.E. ', values significantly S.E. *. values significantly different from that of the normal liver (p < 0.05). different from that of the normal liver (p < 0.05).

3472 CANCER RESEARCH VOL. 43

Downloaded from cancerres.aacrjournals.org on September 29, 2021. © 1983 American Association for Cancer Research. G. H. A. Clowes Memorial Lecture thus far was that of ribonucleotide reducíaseinthe hepatomas. enzymes. The specific activities of the rate-limiting enzymes of Elford et al. (21) reported a marked rise of the reductase activity de novo UMP, CTP, and dTMP synthesis range from 23 pmol to in hepatomas; the increase paralleled the tumor growth rates. 9 mol/hr/mg protein in the liver, whereas the salvage enzymes However, the relationship with the activity of the normal liver could synthesize 0.8 to 156 nmol products. In the rapidly growing could be determined only when Takeda and Weber (85) improved hepatoma, the increased activities of the rate-limiting enzymes the enzyme assay to achieve a sensitivity which was capable of of de novo synthesis could produce 4.2 to 86 whereas salvage detecting and measuring precisely CDP reductase activity in enzymes could synthesize 11 to over 1000 nmol/hr/mg protein. normal, resting adult rat liver (23 pmd/hr/mg protein). The strin This is especially striking for the activity of uridine-cytidine kinase gent linkage of CDP reductase activity with neoplastic prolifera that is increased 2- to 3-fdd in all the examined slow and tion is shown in Chart 6. The reductase activity increased 7.7- intermediate growth rate hepatomas and 5- to 6-fold in the rapidly to 15-fokJin the slowly growing hepatomas and 123- to 325-fold growing ones. These are transformation-linked increases. The in the rapidly growing tumors. Thus, the behavior of CDP reduc specific activities of uridine phosphorylase and uracil phosphori- tase activity was both transformation and progression linked. bosyltransferase increased in a similar fashion (123). The activity DNA polymerase, which has the second lowest activity in of deoxycytidine kinase increased in all the hepatomas, and in pyrimidine synthesis, occupies a strategic role in the immediate the rapidly growing hepatomas 12- to 18-fold over that in normal utilization of dNTPs for DNA biosynthesis. Laszlo and his asso control liver (27) (Chart 7). This is important because deoxycyti ciates showed that the activity of this enzyme was markedly dine kinase channels the nucleoside deoxycytidine directly into increased in the hepatoma spectrum and that the rise correlated the deoxynucleoside monophosphate pools, circumventing the positively with the increase in hepatoma growth rate (6, 66). An ribonucleotide reductase reaction (Chart 8). increase in correlation with hepatoma growth rate was also Thymidine metabolism is most relevant to DNA synthesis (51, observed in the activity of RNA polymerase which utilizes the 97, 98). Thymidine is salvaged by thymidine kinase, providing an ribonucleoside triphosphates for RNA biosynthesis (18). alternate route for dTMP production for DNA biosynthesis. Thy The Salvage Enzymes: Transformation- and Progression- midine may be degraded by thymidine phosphorylase and linked Increase. An importantcomponentin the biochemical through the activity of the rate-limiting enzyme of thymidine strategy of cancer cells poses a major problem in the design of chemotherapy, namely, that the activities of all the salvage 2600 enzymes increased in pyrimidine metabolism (Chart 3). It is i particularly relevant that in pyrimidine production the activities of the salvage enzymes were higher than those of the rate-limiting

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70 50 30 10 7 5 Normal liver HEPATOMA GROWTH RATE (WEEKS) Hepatomas in order of increasing growth rate Chart 6. Transformation- and progression-linkedbehaviorof the activity of CDP Chart 7. Transformation- and progression-linkedbehavior of the activity of the reductase(ribonucleosidediphosphate reductase)in hepatomasof different growth salvage enzyme, deoxycytidine kinase, in hepatomas of different growth rates. rates. Mean specificactivities are plotted as percentagesof the normal liver activity. Means of specific activities are plotted as percentages of the normal liver activity. 8ars, S.E. All activities were significantly different from that of the normal Sver(p Bars, S.E. All enzymic increases were significantly different from the value of the < 0.05). normal liver (p < 0.05).

AUGUST 1983 3473

Downloaded from cancerres.aacrjournals.org on September 29, 2021. © 1983 American Association for Cancer Research. G. Weber catabolism, dihydrothymine dehydrogenase, for eventual catab- The activities of the overall metabolic pathways, both synthetic olism to CO2 and ammonia. Since thymidine phosphorylase is an (incorporation of thymidine into DMA) and catabolic (degradation equilibrium enzyme, the fate of thymidine depends on the balance of thymidine to CO2), were measured in liver and hepatoma of the activities of thymidine kinase and dihydrothymine dehydro slices. The rise in the activity of the synthetic pathway closely genase. In all hepatomas. thymidine kinase activity increased in paralleled the increase in thymidine kinase activity, and the parallel with the proliferative rate of the tumors (123). In the decline in the activity of the catabolic pathway closely paralleled rapidly growing hepatomas, the activity of thymidine kinase was the decrease in dihydrothymine dehydrogenase activity (23,123). elevated to over 300-fold that of the normal liver. In contrast, the The opposing behavior of the activities of the key antagonistic decreased activity of dihydrothymine dehydrogenase in all he enzymes and overall pathways of pyrimidine synthesis and ca patomas correlated negatively with the proliferative rate of the tabolism is an example of reciprocal regulation of metabolic tumors (121, 123). Thus, the behavior of the kinase and the pathways and key enzymes. It is in line with the opposing dehydrogenase was transformation and progression linked behavior of antagonistic enzymes and pathways in other meta (Chart 9). bolic areas, including glycolysis and gluconeogenesis, purine synthesis and degradation (97, 98, 123, 125). Since the activity of the catabolic pathway was orders of magnitude higher than that of the synthetic one, the decrease in thymidine catabolism was as important as the rise in utilization of thymidine for DNA synthesis (23, 123). The decrease in the activity of the degra- dative pathway in hepatomas was accounted for, in part at least, by the decrease in the activity of the rate-limiting enzyme, dihydrothymine dehydrogenase (121), and by the decline in the concentration of NADPH (77), the cofactor for the dehydrogen ase reaction. These observations reveal the ordered pattern of pyrimidine Hepatoma 86 56 metabolic imbalance in cancer cells. The reciprocal changes in

De novo the activities of opposing key enzymes and overall synthetic and catabolic pathways should amplify the metabolic imbalance. The Charts. Pyrmudine enzymic activities of de novo and salvage pathways in altered biochemical phenotype indicates the operation of a genie rapidly growing hepatoma 3924A. Enzymic activities are given in nmol/hr/rng program that is part of the pleiotropic strategy of cancer cells. protein. UR K, undine kinase; CY K, cytidine kinase; dCY K, deoxycytidine kinase; TdR K. thymidine kinase; Gin, glutamine; C-P synth. II, carbamoyl-phosphate Biochemical Commitment to Neoplastic Replication in Py synthetase II; synth., synthetase. rimidine Metabolism. The biochemical strategy of cancer cells

Chart 9. Reciprocal behavior of activities of opposing key enzymes, thymidine (TdR) kinase and dihydrothymine (DHT) dehydrogenase (as sayed in cytoplasmic extracts), and of synthetic and catabolic pathways of thymidine measured by the incorporation of thymidine into DMA and by the degradation of thymidine ot COZ (in tissue slices). The activities were expressed as percentages of values of liver of control normal rats.

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3474 CANCER RESEARCH VOL. 43

Downloaded from cancerres.aacrjournals.org on September 29, 2021. © 1983 American Association for Cancer Research. G. H. A. Clowes Memorial Lecture is expressed in pyrimidine metabolism in the integrated imbal strategies of the cancer cell as displayed in pyrimidine and DNA ance of the activities of key enzymes and metabolic pathways metabolism (Chart 10). of de novo and salvage biosynthesis and in the decline in the activities of the key enzymes and the overall activity of pyrimidine Furine Metabolism: Transformation- and Progression-linked degradation (thymidine to CO2). The integrated imbalance is Imbalance in Cancer Cells summarized in Chart 10. It is important to note that the targets of experimental and clinical anticancer drugs are usually the key Characteristic Features of Normal Liver Furine Metabolism. enzymes of the de novo synthetic pathways. It is a purpose of In liver purine metabolism, the following special features are this presentation to bring into focus the need to block also relevant (see Charts 14 and 15 and Table 2). salvage biosynthesis by inhibiting the activity of specific salvage 1. The de novo synthetic pathway assembles from precursors enzymes or the transport of salvage nucleoside precursors. through 10 reactions the key purine nucleotide, IMP, from which Table 1 shows the specific activities in normal liver of the adenylosuccinate, AMP, ADP, ATP, XMP, GMP, GDP, and GTP synthetic and catabolic enzymes of pyrimidine and DNA metab are produced. AMP may be converted to IMP by AMP deami- olism. The enzymes with the lowest activities in liver of normal nase; a purine nucleotide cycle operates in the liver. The key adult rat were elevated to the highest extent in the rapidly purine nucleotide, IMP, may be produced through (a) de novo growing hepatoma. In contrast, the synthetic enzymes with the biosynthesis, (ù)the purine nucleotide cycle, and (c) the salvage highest activity in normal liver had the smallest extent of rise in reaction catalyzed by hypoxanthine-guanine phosphoribosyl- the rapidly growing tumor. It is important that the rate-limiting transferase activity (see below). enzyme of thymidine or uridine degradation, dihydrothymine 2. From GDP and ADP, through ribonucleotide reducíase, dehydrogenase, had much higher activity than the rate-limiting dGDP and dADP are produced from which the kinase synthe synthetic enzyme of the de novo pathway, CDP reducíase, or sizes dGTP and dATP. From these purine dNTPs, in ine presence that of the enzyme with the second lowest activity, DNA polym- of dCTP and dTTP produced by ihe pyrimidine-synthetic path erase. Thus, in resting liver, the metabolic balance should favor ways, DNA polymerase synthesizes DNA. GTP and ATP, in the the catabolism of uridine and thymidine. The strategy of the presence of CTP and DTP, are converted by RNA polymerase cancer cell entails not only marked transformation- and progres into RNA. The rate-limiting enzyme for the de novo biosynthesis sion-linked increases in the activities of the key synthetic en of IMP is the first one, amidophosphoribosyltransferase; in the zymes but also transformation- and progression-linked de utilization of IMP for adenylate biosynthesis, it is adenylosuccin creases in the activity of the rate-limiting catabolic enzyme. ate synthetase, whereas for the utilization of IMP for guanylate These integrated multienzyme alterations reveal the enzymic production it is IMP dehydrogenase which also has the lowest

Chart 10. Biochemical strategy of cancer cells as revealed in the integrated reprogram- ming of gene expression manifested in the im balance of activities of key enzymes of pyrimi dine de novo and salvage biosynthetic and of degradative pathways. Tapered, thick arrows, enzymic activities that are increased with tumor proliferative rate (progression-linked altera tions); interrupted arrows, those that are de creased; straight thick arrows (e.g., UDP kinase, orotidine-5 ' -monophosphate decarboxylase), enzymic activity that is increased in all the tu mors (transformation-linked); dashed arrow, (e.g., thymidine phosphorylase), enzyme that is decreased in all the neoplasms. Arrows with normal thinness, no relation found with trans formation and progression (e.g., dihydroorotate oxidase; Class 3) or the behavior has not yet been determined (e.g., CDP kinase). Key en zymes are targets of anticancer clinical and experimental drugs, as indicated. BUdR, bro- modeoxyuridine; ARA-C, 1-/J-D-arabino-furano- syteytosine; Cfl, cytidine; CdR, deoxycytidine; 5-FU, 5-fluorouracil; OMP, orotidine 5'-mono- DHiydropyrlmldlna» phosphate; PALA, N-(phosphonacetyl)-L-aspar- tate.

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AUGUST 1983 3475

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i aoie i Comparisonof activitiesenzymesin of pyrimidine- and DNA-syntheticand -catabolic _Data liver and in rapidly growing hepatoma Joo «~values.are expressed as specific activity and as percentages of the normal liver '°for In calculations, the values 200 mg of protein per g, wet weight, of tissue »Enzymichomogenates and 80 mg of protein per g for supernatant fluids were used. activities were those determined in this laboratory and in other~Rapidly centers. = _ 250 213...•i,206196 2«3 |»*- j150«-t-135T•Õ-ti(-E-) \-Î-1 >growing ¿ 2nin•f--inXANTHINE i-1Normal r^f-,!1"-t-166•*•121*•i-121*230 •""(pmol/hr/mgliver hepatoma |< ~1Enzymes 3683F(% Õ°Anabolic EC no. protein) of liver) ~Ribonucleotideenzymes Po ' 5° i¿£DNA reducíase 1.17.4.1 23 18,348 udTMPsynthasepolymerase 2.7.7.7 56 5,806 2.1.1.b 180 2,860 % 100- dTMP kinase 2.7.4.9 420 7,000 2 Deoxycytidine kinase 2.7.1.74 800 1,400 Thymidine kinase 2.7.1.21 900 3,920 CTP synthetase 6.3.4.2 5,500 1,122 Carbamoyl-phosphatesynthe- 2.7.2.9 10,000 950 tase IIi~rlPMP

0-UV^IVlr Hpamrnacp 1 ? 000 7SO ~ JUUracil Utídnlüldíil, I¿.UUU / 760Omithine-5'phosphoribosyltransferase 2.4.2.9 19,000 j£decarboxylase-monophosphate 4.1.1.23 34,000 889 OXIDASE7t— >Orotate Ì: ~3ase phosphoribosyltransfer- 2.4.2.10 47,000 599 ,00.Uridine |^ -sUridine-cytidinephosphorylase 2.4.2.3 164,000 671 <">-Dihydroorotasekinase 2.7.1.48 156,000 694 >o 3.5.2.3 246,000 418 >* 6o- ct50 ,8"*! Aspartate carbamoyltransferase 2.1.3.2 448,000 706 f- o UDP kinase 2.7.4.6 444,000,000 298 <*"°"" r^*i

20-Catabolic .*r*T-ï-r*iK10 , 11 0_175—1001100^_••1Dihydrouracilenzymes >; fcH° Normal((-Ureidopropionasedehydrogenase 1.3.1.2 26,000 9 21i - 5 § g"SS1"MEDIUM93 S Õ 9 * S s1* liverThymidine 3.5.1.6 144,000 ™SLOWUMS I B*RAPID f.Dihydropyrimidinasephosphorylase 2.4.2.4 234,000 3127siAMIDOPHOSPHORIBOSYLTRANSFERASE256 * 3.5.2.2 276,000 *•Hepatomas in order of increasina arowth rate Chart 11. Reciprocalbehavior of key synthetic and catabolic enzymes in hepa- activity of all the purine enzymes. tomas. Transformation-linked increase in activity of amidophosphoribosyltransfer- 3. The highly active salvage enzymes provide alternative ase and transformation-linked decrease in activity of xanthine oxidase in hepato- routes to the de novo synthesis to produce the strategic purine mas. Enzymic activities were calculated per average cell and expressed as means of percentagesof the values of normal liver. Bars, S.E. All enzymic alterations were nucleotides, IMP, AMP, and GMP. Two salvage enzymes act to significantlydifferent from the activity of the normal rat liver. recycle 3 purine nucleoside precursors (hypoxanthine, adenine, and guanine) to nucleotides in the presence of PRPP. APRT Table 2 converts adenine to AMP. Because the activities and the Kms of Activities of enzymes of the anabolic and catabolic pathways of purine metabolism the other salvage enzyme, hypoxanthine-guanine phosphoribo All hepatoma activities were significantlydifferent from liver values (p < 0.05). syltransferase, differed with hypoxanthine or guanine as sub strates, for convenience of discussion the activities are reported liver hepatoma separately as HPRT and GPRT (112). (nmol/hr/mg 3683F (% of EnzymesSynthesisIMP no.1.2.1.146.3.4.16.3.4.42.4.2.144.3.2.23.5.4.62.7.4.81.2.3.21.7.3.32.4.2.1Normal 4. The de novo and salvage pathways of IMP synthesis are protein)23336602881,7306,09010064538,800Rapidliver)1,35054830828017543312110419 opposed by the catabolic pathway. IMP is degraded to uric acid dehydrogenaseGMP and, in the rat, to allantoin through the subsequent actions of synthetaseAdenytosuccinate 5'-nucleotidase, inosine phosphorylase, xanthine oxidase, and syn thetaseAmKtophos uricase (Table 2). In rat liver, the activity of the rate-limiting bosyl-transferaseAdenytosuccinaseAMPpriori catabolic enzyme, xanthine oxidase, is higher than that of the rate-limiting enzyme of cte novo biosynthesis, amidophosphori- deaminaseGMP bosyltransferase (71, 72). kinaseCatabolismXanthine

The Neoplastic Program in Purine Metabolism oxidaseUricaseInosine

De Novo IMP Biosynthesis and Degradation. The neoplastia phosphorylaseEC program entails a transformation-linked increase in the activity of amidophosphoribosyltransferase (71) (Chart 11) and a trans catabolism. Thus, in all hepatomas, the activities of 5'-nucleoti- formation- and progression-linked rise in that of formylglycinam- dase, inosine phosphorylase, xanthine oxidase (Chart 11), and idine ribonucleotide synthetase.3 By contrast, there is a transfor uricase (46, 72, 119) (Chart 16; Table 2) were decreased. mation-linked decrease in the activities of the enzymes of IMP De Novo Guanylate Biosynthesis from IMP. In the utilization of IMP for de novo guanylate biosynthesis, the activities of IMP 3W. L. Elliott and G. Weber, unpublishedobservation. dehydrogenase (Chart 12) and GMP synthetase increased in all

3476 CANCER RESEARCH VOL. 43

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for which GDP-mannose is required (133) and also might limit 1400- the synthesis of cyclic guanosine 3'-5'-monophosphate. GTP is an important factor in protein synthesis, and its role in DNA 1200-j?*>U-;; biosynthesis has recently been pointed out (13). GTP is an activator of CTP synthetase (47) and adenylosuccinate synthe ^<^«M9)ro•i-r*r-r-r-J[N<«ote9>Õ1mcoteMRAPID"--.-.-ftase (36). Thus, a drop in its concentration might curtail the 1000-ll8.2 biosynthesis of CTP and ATP. Conversely, because GTP is an inhibitor of AMP deaminase (36), a decrease in GTP pools would 800-W deinhibit AMP deaminase activity with a subsequent drain on tt)1^»g AMP pools and a rise in IMP concentration. High levels of IMP 600-s could impair the fidelity of DNA transcription by blocking certain

CIs400-A.o"O steps in the proofreading mechanisms. GTP is an activator of ribonucleotide reducíase (85) and promotes the conversion of GDP inlo dGTP; a marked decline in the GTP and GDP pools

S200-_Q could limit Ihe synthesis of dGTP which in turn would result in an imbalance in the concentration of the 4 dNTPs, hence limiting DNA biosynthesis. De Novo Adenylate Biosynthesis from IMP. In the utilization _JmJem•M•Normal of IMP for de novo adenylate biosynthesis (Chart 16), the activi I ¡ver-Jt,j.lyase) increased in all the hepatomas examined, showing transformation-linked behavior (Chart 13) (36). The AMP Chart 12. Transformation- and progression-linked increase in the behavior of produced may be utilized by the kinases for the biosynthesis of the activity of IMP dehydrogenase in hepatomas of different growth rates. Mean specific activities are plotted as percentages of the normal liver values. Bars, S.E. ADP and ATP and by adenylate kinase (15) to achieve a rapid All hepatoma activities were significantly higher than those of the normal liver (p < equilibrium of adenylates. AMP may also be recycled to yield 0.05). IMP by AMP deaminase activity which was markedly increased in the hepatomas and was transformation and progression linked the examined hepatomas, and the rise correlated positively with (36) (Table 2). the hepatoma proliferation rate (35, 38). The behavior of IMP Salvage Activities for Production of AMP, IMP, and GMP. dehydrogenase and GMP synthetase is transformation and pro In normal liver, the specific activities of the salvage enzymes gression linked and indicates an increased capacity for the APRT, HPRT, and GPRT (498, 460, and 1470 nmol/hr/mg production of XMP, GMP, GDP, and GTP in hepatomas (35, 38, protein, respectively) were 6.6- to 21-fold higher than that of 119). IMP dehydrogenase specific activity was markedly in amidophosphoribosyltransferase (70 nmol/hr/mg protein) (Chart creased in leukemic blast cells, and it showed good correlation 14) (74, 112). Thus, in resting liver, the capacity of salvage with the leukemic blast cell count (7). This confirms in human enzymes for recycling the bases was much higher than that of leukemic cells the positive correlation between IMP dehydrogen the rate-limiting de novo synthetic enzyme. A further metabolic ase activity and cell proliferation first discovered in the hepatoma spectrum (35, 38). Increased activity of amidophosphoribosyl- 250 transferase was also observed in leukemic cells (7). The purine salvage enzyme activities did not exhibit a pattern. The activity of GMP kinase is in an excess because it is orders A 200 — il of magnitude higher than that of IMP dehydrogenase and GMP synthetase; it showed no pattern, and we place it in Class 3 (10). The increased capacity to produce GDP provides the sub *l strates for ribonucleotide reducíasewhich generates dGDP and 150 — rfl Õu with the subsequent action of the kinase produces dGTP, a u ir* i precursor of DMA. GTP is also a precursor, with the other 1 nucleoside triphosphates, of RNA. The increased capacity of the de novo guanylate-synthetic pathway, which we have discov ered, is of importance for the biochemical program of cancer cells because of the following functions. The transformation- and progression-linked increase in IMP dehydrogenase and GMP synthetase activities and the elevation in GMP and dGTP con centrations indicate a close linkage with the core of the neoplastic program (10, 34, 35). From the numerous biological functions of the metabolites in the guanylate pathway, the following may be | SLOW) MEDIUM I RAPID I emphasized. GTP is a precursor of RNA, and GTP-dependent functions include 5'-"cap" formation in mRNA (80) and the pro Hepatomas in order of increasing growth rate duction of small nRNA species (135). GTP is also involved in Chart 13. The transformation-linked increase in the activity of adenylosuccinase promotion of polymerization of microtubules. A decrease in GTP in hepatomas. Mean specific activities are plotted as percentages of the normal liver activity. Bars, S.E. All enzyme activity increases were significantly different concentration might curtail the assembly of certain complex lipids from the values of the normal liver (p < 0.05).

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D«novo Salvag« D«novo Salvage D«novoSalvage Chart 14. Purine biosynthesis. Comparison of the specific activities of the rate- limiting and salvage enzymes involved in the synthesis of IMP (left), GMP (middle) and AMP {fight) in normal liver. advantage for the salvage pathways is that the affinity of the 3 salvage enzymes to PRPP is much better than that of amido- phosphoribosyltransferase (112). In rapidly growing hepatoma 3924A, the specific activity of the amidotransferase was increased 2.8-fold, whereas those of Chart 15. Enzymes of de novo and salvage pathways of purine synthesis in rapidly growing hepatoma 3924A. Activities of adenylosuccinate sythetase (SAMP APRT, HPRT, and GPRT were 170, 70, and 70% of normal liver synth.) and IMP dehydrogenase (IMP DH) are given in nmol/hr/mg protein. SAMP, values (112). With these changes, the activity of the amidotrans adenylosuccinate; PRA, phosphoribosylamine; HX, hexokinase. ferase was 193 nmol/hr/mg protein, whereas those of the sal vage enzymes were 4.4-, 1.7-, and 5.4-fold, respectively, of the cofactor, and various regulatory metabolite concentrations in the amidotransferase activity. For the common substrate, PRPP, for tissue at the prevailing pH (112), might be. Thus far, isotope data amidotransferase, the Kmwas 400 UM;whereas for APRT, HPRT, are in line with the enzymic capacities (129). Further work is and GPRT, the Km's were 2, 4, and 40 tuM, respectively (112). necessary to measure the activity of the metabolic fluxes which Accordingly, in the hepatoma, the activities of the salvage en should determine the in vivo contributions of the de novo and zymes were markedly higher and the affinity for PRPP was much salvage pathways. In the design and evaluation of chemotherapy, better than of the rate-limiting enzyme of de novo synthesis, the effectiveness of inhibitors of enzymes of the de novo pathway amidotransferase. may depend, in part at least, on the activities of salvage enzymes A further advantage conferred to cancer cells by the repro- which could furnish the essential metabolites through highly gramming of gene expression in purine metabolism is in the active one-step recycling processes. behavior of the activity of the enzymes involved in hypoxanthine It is noteworthy that in the hepatomas the activities of key metabolism. In normal liver, the activity of the salvage enzyme, enzymes of de novo biosynthesis increased markedly, whereas HPRT, was 460 nmol/hr/mg protein, whereas that of the degra- the changes in activities of the salvage enzymes were minor. dative enzyme, xanthine oxidase, was 100 nmol/hr/mg protein, This may relate to the fact that in normal rat liver the purine yielding a ratio of 4.6 in favor of the synthetic capacity. In salvage enzymes have high activities and high affinity for their hepatoma 3924A, with HPRT activity being slightly lower than in substrates. liver, but with xanthine oxidase activity decreased to 10%, a Biochemical Commitment to Neoplastic Replication in Pur ratio of 33 was observed. This ratio should strongly favor utili ine Metabolism. The biochemical strategy of cancer cells in zation of hypoxanthine for salvage also because the Kms for purine metabolism is expressed in the integrated imbalance of hypoxanthine of HPRT and xanthine oxidase were similar, at 3 the activities of the key enzymes and metabolic pathways of de to 7 UM. Similar shifts occur in the ratios of GPRT and guanine novo and salvage biosynthesis, and in the decrease in the deaminase activities in liver and hepatoma (112,117). activities of the key enzymes and overall activities of purine The relationship of activities of key enzymes of de novo and catabolism (Chart 16). The attacking points of experimental and salvage synthesis of the strategic purine nucleotides, AMP, IMP, clinical anticancer drugs are usually the key enzymes of the de and GMP, is shown in Charts 14 and 15. In hepatoma 3924A, novo synthetic pathways. It is an important objective of this under optimum kinetic conditions, the amidotransferase could paper to point out the need also to block salvage biosynthesis synthesize 193 nmol IMP per hr per mg protein. By contrast, by inhibiting the activity of specific salvage enzymes or the HPRT could provide 325 nmol IMP, and this enzyme has a 100- transport of salvage precursors. fold better affinity for PRPP than did amidotransferase. In the routing of IMP to GTP, the rate-limiting enzyme is IMP dehydro- The Pattern of Ribonucleotides and Deoxynucleotides in He genase which under optimum conditions could yield 23 nmol patomas XMP per hr per mg protein, whereas GPRT could produce 1020 nmol GMP per hr per mg protein. In the routing of IMP into The in vivo concentrations of AMP, ADP, and ATP were first adenylate synthesis, the rate-limiting enzyme, adenylosuccinate measured in tumors in the application of the freeze-clamp tech synthetase, could synthesize 88 nmol adenylosuccinate per hr nique to kidney neoplasms in studies that I carried out in Oxford, per mg protein; however, the salvage enzyme, APRT, could yield United Kingdom, in collaboration with H. A. Krebs, Marion 951 nmol AMP per hr per mg protein (112). Stubbs, D. H. Williamson, and others (132). We observed a As we have pointed out, the enzyme activities measured under decrease in the ATP concentration of the transplantable rat renal optimum kinetic conditions indicate only an approximation of cell carcinomas. Similar results were soon obtained in my labo what actual cellular activities, which depend on the substrate, ratory in hepatomas of different growth rates (124). Subsequent

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Adenyllte ¡ kinaie

AdenylMticcinate

Adenylocuccinate Chart 16. Biochemical strategy of cancer syothetaM cells as revealed in the integrated reprogram- ming of gene expression manifested in the im balance of the activities of key enzymes of purine de novo and salvage biosynthesis and of — — the degradative pathway. The arrows are as laaeAICRPeyelo hydro explained in Chart 10. The key enzymes are 5 -Nucl«otida» the targets of clinical and experimental anti- cancer drugs. Adapted from Weber (97). 6-MP, 6-mercaptopurine; SAMP, adenylosuccinate. traoaformylaseAdeayloaaccinateSuccino- 6-TG, 6-thtoguanine; Ac., acid; FAICAR, formylaminoimidazolecarboximide ribonucleo- INOSINË tide; AICRP, aminoimidazolecarboxamide ri- bose phosphate; AICAR. aminoimidazolecar- boximide ribonucleotide; AIR. aminoimidazole- Hypoxaothiiie- AICRP ribonucleotide; AIRP. aminoimidazoleribonule- KUHnine Inoaine •ynthetaseAIRPcarbozylaMAIRP»ynthctaieFGARphoiphoriboiylyl,IHYPOXA•«•-| otide phosphate; FGAM, formylglycinamidine transferaee phoaphorylaae ribonucleotide; DT/C, dimethyltriazenoimida- AIRfAIRPtFGAM-~ zolecarboxamide; FGAR, formylglycine amido- ribonucleotide; MTX. methotrexate; GAR, gly cine amidoribonucteotide; PRA. phosphoribo- sylamine; DOW, diazooxonorteucine; R-5-P, ri- INE;ZrMINE bose 5-phosphate. -r*FGAR— ('MTX ~\IMP•ynthetaaeGARfor XANT| |¡VCID f-*.GARtPRA: BeS*» rayUranifera Xanthioe oxidaae GAR/ ^ Azaserine \ ,ynthetate f 6-MP.DON. ) URICNTH | \. Acivicin Amidnphosp hori bcisy rauferaMf^ "^* - - ^ t 4-^PRPPt

pyrophoiphatekiaa«/FAICAR*AICARSAICARÎCARBOXY ^R-5-P Tubercidin ALLANTOIN

Cordycepin

Form y cm >v PsicofuranineRibo«cpho«phate

investigations using high-pressure liquid chromatography con were transformation and progression linked (Chart 17). In rapidly firmed these results and extended the analysis to 16 ribonucle- growing hepatoma 3924A, the concentrations of dATP, dGTP, otides (34). In the hepatoma spectrum, the only elevation in the dCTP, and dTTP increased to 17-, 5-, 8-, and 12-fold of those ribonucleotide pools was the transformation- and progression- of normal liver. Chart 17 also shows that the concentrations of linked, rise in CTP concentration which was 4-fold increased in CDP, UDP, GDP, and ADP, substrates of the conversion of hepatoma 3924A. The concentration of GMP was elevated in a ribonucleotides to deoxynucleotides, were the same as in the transformation-linked fashion, and in hepatoma 3924A it in liver across the hepatoma spectrum. However, the pools of these creased 3.2-fold over the value of normal control liver. The ribonucleotides were greatly in excess of those of the dNTPs. ribonucleotides that participate in numerous metabolic reactions, The concentrations of the substrates of ribonucleotide reduc including the nucleoside diphosphates serving as substrates for íase,ADP, GDP, UDP, and CDP, were 1,060, 112, 210, and 56 the ribonucleotide reducíase reaction, occur in mw concentra nmol/g in liver and 778, 137, 249, and 50 nmol/g in hepatoma tions. In contrast, the dNTPs, which are primarily involved in 3924A (34). In the presence of this great excess of substrates, DNA production, are present in the liver in J/M concentrations; the conversion of the ribonucleotides to deoxyribonucleotides therefore, in tumors a major enlargement of the dNTP pools was apparently takes place only in traces in the resting liver due to expected. We determined that the increases in the dNTP pools the very low activity of ribonucleotide reducíase. The transfor-

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In evaluating the significance of the alterations in enzymic activities, 2 assumptions were made: Assumption 1, that the activity of the enzyme measured in the presence of optimum substrate and cofactor and pH yielding linear kinetics was pro portionate with the amount of the enzyme; Assumption 2, that the amount of enzyme was an indicator of the gene expression under the specified steady state conditions. Two independent methods were used to measure enzyme amount. Enzyme con centration was determined by (a) assay of enzymic activity where strict proportionality between activity and added amount of enzyme was ensured (kinetic evidence) and (b) ¡mmunotitration of the enzyme protein amount by specific antienzyme serum (immunotogical evidence). Key enzymes were selected for each of the 4 vitally important metabolic pathways that are profoundly altered in activity in rapidly growing hepatoma 3924A. The key enzymes were highly purified from rat liver and hepatoma 3924A and were used to produce antisera in rabbits. The specificity of the antiserum for each enzyme was determined, and immunoti tration was carried out. The concentrations of a key enzyme of glycolysis (6-phosphofructokinase), of pentose phosphate pro duction (glucose-6-phosphate dehydrogenase), of de novo purine Slow M«d.Fast M Slow Med. Fast Slow Med. Fast Slow Mpd Fast biosynthesis (amidophosphoribosyltransferase), and of de novo Liver HepatoMS Liver HepaUMHS Liver Hepatomas Liver Hepatovas pyrimidine biosynthesis (CTP synthetase) were elevated. This was shown both by assay of enzyme activity and by immunoti tration of the amount of enzyme protein (19, 79, 87, 88) (Chart 18). In addition, an important enzyme of pyrimidine salvage, thy midine kinase, was examined. This enzymic activity in slow, BEHAVIOR OF RIBONUCLEOSIOC OIPHOSPHATE POOLS intermediate, and rapidly growing tumors (hepatomas 16, 7787, Chart 17. Progression-linked increase in the pools of dNTPs in hepatomas of and 3924A) increased 5-, 16-, and 30-fold, and a 4-, 15-, and different growth rates. It is noteworthy that the concentrations of the nudeoside diphosphates did not change across the hepatoma spectrum. *, alterations signifi 20-fold excess of antiserum was required to neutralize 50% of cantly different (Sig. ditt.) from those observed in normal (Norm.) Bver (p < 0.05). the thymidine kinase activities. We conducted that the elevated Data plotted from Jackson ef al. (34). Med., medium. thymidine kinase activity in rat hepatomas of different growth

mation- and progression-linked increase of the deoxyribonucleo- tide pools in hepatomas of different proliferative rates emerges in the presence of an unchanged amount of the ribonudeoside diphosphates. The stringent linkage of ribonucleotide reducíase activity with the rise of hepatoma proliferation rate and with the increase in the levels of dNTPs suggests that the increase in reducíase activity accounts for the channeling of the ribonude- otides to the deoxynucleotide pools (85). The elevated activity of reducíase (85), along with the increased activities of PRPP i synthetase, CTP synthetase, thymidine kinase, and IMP dehy-

drogenase, provide an enzymic explanation for the expansion of *M 40C 800 'timi i/NI, the dNTP pools (116, 117). In the presence of an excess con ANTISERUM (pi) ANTISERUU (gli centration of the substrates, the ribonudeoside diphosphates, PHOSPHOFRUC TOK IN ASE Ci P DEHVDROCCMASC the rise in the activity of the enzyme, ribonudeotide reductase, is responsible for the increased product formation, the striking enlargement in the dNTP pods.

Reprogramming of Gene Expression in Neoplastic Cells: The Evidence

It was proposed that the enzymic and metabolic pattern of è - ordered transformation- and progression-linked alterations in O « M M M 100 II« tumor cells was a manifestation of the reprogramming of gene ANTISCRUU (pii expression (97, 98). This interpretation of the altered phenotype GLUT AMINE PRPP AMIOOTRAMSFERASt of cancer cells is based on demonstration of changed concentra Chart 18. Evidence for reprogramming of gene expression in cancer eels. increased concentrations of key enzyme amounts in carbohydrate, pentose phos tions in the end products of gene expression: the amount of phate, purine. and pyrimidine metabolism G 6-P dehydrogenase; glucose-6-phos specific catalytic proteins, the enzymes. phate dehydrogenase.

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rates reflected the concordant increase in the concentration of Table 4 the thymidine kinase protein (50). Discriminatingbetween power rapidly of growing biochemical neoplastic, pattern newborn, in pur/ne and metabolism: regenerating key differencesliver The molecular events that resulted in this heritable multien- zyme change in the phenotype are of the greatest interest (125). The molecular basis of the reprogramming of gene expression geneexpressionIMPMarkers of (6days that resulted in the integrated alterations in activities of over 40 old)337°52°116C191°125256*112C137°124e48"1173C1364°ofing (24hr)495"113C115e118e96124°60"115e148100e91e96e109°150"resting enzymes is under investigation. dehydrogenaseAdenylosuccinate synthe-taseAdenylosuccinaseAMP

Specificity to Cancer Cells of Enzymic and Metabolic Pattern deaminaseGMP The developing and differentiating liver and regenerating and kinaseGMP synthetaseAdenosine normal liver in adult rats provided appropriate control models. kinaseAdenosine Although they have similar growth rates, these control tissues deaminaseAmidophosphoribosyl-transferaseFormylglycinamidine can be distinguished readily both from neoplastic liver and from each other by their enzymic and metabolic pattern (96-98). ribo-nucleotide Examples of the discriminating power of the biochemical pattern synthetaseInosine phosphorylaseXanthme are shown in Tables 3 and 4. There are alterations that overlap oxidaseUneaseAmidotransferase/xanthineoxidaseNormalliver100a100100100100100100100100100100100100100'' with those in neoplastic tissues; however, the enzymic and metabolic imbalance is characteristic of neoplasia, since there is no overall pattern similar to that in neoplasia (34, 91, 92, 96-98, asratSpecific activities are expressed the normalRegeneratadult 104,110,121,124). livervalues.0 discriminant.cQuantitative Qualitative discriminant.Hepatoma(rapidlygrowing3683F)13503081754331215486420280660191042740percentagesNewborn Generalization: Presence of a Strongly Conserved Segment of Gene Expression in Animal and Human Neoplasms work brought evidence for a similar enzymic pattern in the Lewis lung tumor in mouse." It is striking that the enzymic imbalance Similar Pattern of Gene Expression in Different Types of was also observed in the MC-29 virus-induced, transplantable Tumors. An overall pattern of enzymic imbalance was discovered in chemically induced, transplantable rodent tumors, including hepatoma in chickens (49, 70, 83). We determined the presence of the pattern in human hepato- hepatomas, kidney tumors (33, 69, 97, 98), and sarcoma in rats (106); colon carcinomas in rats and mice (111); and myeloma, cellular (114) and renal cell carcinomas (33, 73, 99, 107, 110), in lymphosarcoma, and lymphocytic leukemia in mice (110). Current colon tumor xenografts of different degrees of differentiation and growth rates (108), and in human primary lung and colon ade- Table 3 nocarcinomas (16, 118). Recently, part of the pattern was also Discriminating power of biochemical pattern in pyrimidine and DNA metabolism: observed in human leukemias and lymphomas (7, 22). The key differences between rapidly growing neoplastic, newborn, and regenerating applicability of all or part of the metabolic imbalance was dem liver onstrated in 16 different animal and human neoplasias (Table 5). (rapidly Independence of Biochemical Program of Cancer Cells growing (6 days ating (24 from the Carcinogenic Agent. The altered enzymic pattern in expressionThymidineMarkers of gene liver100a1001001001001001001001001001001001001001003683F)3,900<0.111,500,0002509467068891,1222987606716947581,3703,920old)1,224°69°1,788e87"216e223e190e170148244e46°110°109°157e629ehr)910e64?1,450"100"146"137°165e200°100213e107°120"105"102"1,683erat, mouse, chicken, and human neoplasias identifies a strongly DNAThymidineto conserved segment of the altered gene expression in neoplastic CO2Thymidineto cells. Therefore, it appears that essential elements of the bio toCO2DNA/cellCarbamoyl-phosphateto DNA/thymidine chemical commitment to neoplastic transformation and progres sion have been identified. The biochemical program displayed is synthe taseIIAspartate independent from the carcinogen, since the tumors analyzed carbamoyltransfer-aseOrotidine-5 included those produced by chemical and viral agents and those -monophosphatedecarboxylaseCTP' that arose spontaneously, e.g., primary human neoplasia. The various carcinogens apparently trigger through a final common synthetaseUDP pathway the display of the enzymic and metabolic program that kinaseUracil phosphoribosyltransfer-aseUridine we have identified in different species and in tumors of different cell origin and histology. phosphorylaseUridine Tumor-specific Biochemical Markers. Just as there is a kinaseCytidine kinaseDeoxycytidine general biochemical imbalance shared by many different types kinaseThymidine of tumors, there are also tumor type-specific biochemical mark kinase 487"5548°225,586e126"78"51ers that aid in pinpointing alterations that are characteristic of reductaseThymidineRibonucleotide 10010010010018,34854<1024 phosphorylaseDihydrouracil individual classes of tumors. An example is galactokinase activity dehydrogenaseDihydrothymine which increased in every human primary colon tumor, but de dehydrogenase 2,702"Regener3,359e creased in other neoplasms (16). Uridine phosphorylase activity Thymidinekinase/dihydrothy-mine 100Hepatoma17,200Newborn dehydrogenaseNormal increased in all other tumors examined but was low in colon a Specific activities (nmol/hr/mg protein) were expressed as percentage of carcinomas (16). In the sarcoma, the pools of uridylates were normal adult liver values. 6 Qualitative discriminant. c Quantitative discriminant. 4G. Weber, unpublishedobservations.

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Tables Table6 Shared enzymie programs identified in 16 types of neoplasías Enzymic markers of transformation Rat Hepatomas of different growth rates8 The relationship of enzymic activities with transformation and progression was Renal cell carcinomas MK-1. MK-2, MK-3" determined in the rat hepatoma spectrum. Colon adenocarcinoma8 Sarcoma8 Metabolic areas Synthetic pathway Degradative pathway Mouse Colon adenocarcinomas8 Increased activity Decreasedactivity Plasmacell myeloma* Pyrimidineand Carbamoyl-phosphatesynthe- Thymidinephosphoryl- tasell" Mecca lymphosarcoma8 DNA ase Lymphocytic leukemia8 Aspartate carbamoyltransferase8 Dihydrothyminedehydro- Dihydroorotase8 genase Avian MC-29 virus-induced hepatoma" Orotate phosphoribosyltransfer- ase Human Hepatocellularcarcinoma0 Orotidine-5'-phosphate decar- Renalcell carcinoma0 boxylase Lung adenocarcinoma0 UDPkinase Colon carcinoma0 CTP synthetase" Colon xenografts" Ribonucleotidereducíase8 Malignant lymphomas0 DNA polymerase" Leukemias Uridine-cytidinekinase 8 Chemically induced, transplantable, Deoxycytidine kinase8 Thymidine kinase8 0 Viral-induced, transplantable. 0 Spontaneous, primary. 5'-Nucleotidase d Spontaneous, transplanted in nude mice. Purine and Amidophosphoribosyltransferase RNA Formylglycinamidineribonucleo- Inosine phosphorylase tide synthetase Xanthineoxidase markedly elevated; these pools were unchanged in other tumors. Adenylosuccinatesynthetase Uncase The sarcoma was also distinguished from other tumors by Adenylosuccinate lyase Adenylate kinase8 AMP deaminase8 increased GMP kinase activity which was unaltered in other IMP dehydrogenase3 neoplasms and by the decreased activities of adenylosuccinate GMP synthetase8 tRNA methylase lyase and AMP deaminase which were markedly increased in Arginyl-tRNA synthetase8 liver and kidney tumors in the rat. Pyruvate kinase activity was RNA polymerase8 decreased to 35% in sarcoma, but it was increased in all other types of tumors examined (102). Ornithine Ornithine decarboxylase Integrated Enzymic and Metabolic Program in Neoplasia Pentose Glucose-6-phosphatedehydro- Ornithinecarbamoyl and Chemotherapeutic Targets. For the rational design of phosphate genase transferase8 Transaldolase anticancer chemotherapy, it is essential to identify enzymic, PRPPsynthetase8 metabolic, and nucleotide targets in cancer cells that are con sistent elements in the biochemical commitment to neoplastic Decreasedactivity Increased activity replication. However, it has been claimed that there were no Carbohydrate Glucose-6-phosphatase8 Hexokinase" biochemical differences between normal and cancer cells and Fructose-1,6-diphosphatase8 6-Phosphofructokinase8 Phosphoenolpyruvatecarboxyki- Pyruvate kinase8 that if there were any differences they were merely quantitative, nase" not qualitative. These largely uninformed claims have been Pyruvate carboxylase" superseded by the striking advances in the elucidation of the Membrane Adenylate cyclase'* cAMP phosphodiester- enzymology, isozyme shift, and nucleotide pattern of cancer cells cAMP" ase" in the past 20 years [overviews were presented (8, 12, 48, 62, 8Also linked with progression. 89, 91-93, 95, 97, 98, 100, 114, 127, 128)]. It is informative to b cAMP, cyclic adenosine 3':5'-monophospnate. summarize the most relevant quantitative and qualitative differ ences between cancer cells and their homologous normal coun regulation of the activities of the opposing key enzymes and terparts (Tables 6 and 7). synthetic and catabolic pathways. In pyrimidine metabolism, We have identified 43 enzymes that are transformation linked there was an increase in activities of the synthetic enzymes and (activities altered in all cancer cells), and 26 of these are also a decrease in those of the catabolic ones, along with an elevation progression linked (activities gradually altered with tumor malig in the pools of CTP and of the dNTPs. In purine metabolism, the nancy) (Table 6). reprogramming of gene expression resulted in an increased The progression-linked alterations in the activities of key en potential to produce IMP, and particularly GMP, GDP, and GTP, zymes and metabolic pathways and in the pools of metabolites and a decreased capacity to degrade purines. The alterations in and nucleotides are summarized in Table 7. Over 70 biochemical carbohydrate metabolism, discussed elsewhere (100,128), pro markers have been pinpointed, most of them in this laboratory, vide increased activities of the key enzymes of glycolysis and as stringently linked quantitative correlates of neoplastic pro pentose phosphate production with a decrease in those of the gression. key gluconeogenic enzymes and gluconeogenesis. The alteration The qualitative changes, the isozyme shift, are expressed in resulted in a higher capacity to glycolyze and through the in the gradual decrease in the activity of liver-specific, regulatory, creased activities of glucose-6-phosphate dehydrogenase, high-Km isozymes and the increase in activity of the non-liver- transaldolase, and PRPP synthetase to produce pentose phos type, Iow-Km isozymes (19, 36, 48, 93, 97, 98,127, 128). phates and PRPP (19, 28, 29, 53, 67, 79, 84, 93,101,103,105, Selective Advantages of Pleiotropic Gene Conservation 106, 115, 116, 121, 124, 128). The enzymic and metabolic and Targets of Chemotherapy. In all metabolic areas examined imbalance in pyrimidine, purine, and carbohydrate metabolism in hepatomas, an imbalance was revealed in the reciprocal confers selective reproductive advantages to cancer cells. These

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Table? Progression-linkeddiscriminants: enzymatic and metabolic markers of the degrees of malignant transformation

Nucleic acid metabolism Carbohydrate metabolism Protein and amino acid metabolism Other metabolic areas Pyrimkline, RNA, and DMA synthesis Glucose catabolism (increased) Protein synthesis (increased) Membrane cAMP8 metabolism (increased) Hexokinase cAMP phosphodiesterase (in Carbamoyl-phosphate synthetase II Phosphofructokinase Amino acid incorporation into protein creased) Aspartate carbamoyltransferase Pyruvate kinase (alenine, aspártele, glycine, Adenylate cydase (decreased) Dihydroorotase serine, isoleucine. valine) CTP synthetase Glucose synthesis (decreased) Activity of postmicrosomal protein- Polyamine synthesis (increased) Ribonucleotide reducíase Glucose-6-phosphatase synthesizing system Omithine decarboxylase Thymidylate synthetase Fructose-1,6-diphosphatase Deoxycytidylate deaminase Phosphoendpyruvate carboxyki- Enzymes catabolizing amino acids (de Polyamine synthesis (decreased) Uridine-cytidine kinase nase creased) S-Adenosylmethionme synthetase Deoxycytidine kinase Pyruvate carboxylase Glutamate dehydrogenase Thymidine kinase Glutamate-oxaloacetate transami- Ketone body utilization (increased) DNA polymerase Specific phosphorylating enzymes (de nase Succinyl-CoA:acetoacetate-CoA DNA nucleotidyltransferases creased) Tryptophan pyrrolase transferase RNA polymerases Fructokinase Serotonin deaminase tRNA methylase Glucokinase 5-Hydroxytryptophan decarboxylase Lipid metabolism (decreased) CTP concentration a-Glycerophosphate dehydrogenase dCTP concentration Fnjctose metabolism (decreased) Enzymes degrading amino acids (in Hydroxymethylglutaryl-CoA syn- dTTP concentration Thiokinase creased) thase Atdolase Glutaminase Pynmidme catabolism (decreased) NADP content (decreased) Dihydrouracil dehydrogenase Isozyme shift (decreased) Amino acid content (decreased) NADP High-Km isozymes L-Glutamine NADPH Furine synthesis (increased) Formylglycinamidme ribonudeotide Isozyme shift (increased) Urea cycle (decreased) synthetase Low-Km isozymes Ormthine carbamoyltransferase IMP dehydrogenase GMP synthetase Ribose 5-phosphate utilization (in AMP deaminase creased) GMP concentration PRPP synthetase dGTP concentration dATP concentration

Purme catabolism (decreased) Adenylate kinase ATP * cAMP, cyclic adenosine 3':5'-monophosphate. advantages, although short term, result in a differential survival tabolites and precursors, or amino acids; (c) it is postulated that and reproduction of the neoplastia gene pool in the host. the selectivity of the drugs against cancer cells will be the higher The integrated enzymic imbalance in the cancer cells suggests the more extensive is the biochemical difference from normal that a number of genes involved in the production and regulation cells, because it is assumed that the marked metabolic imbalance of opposing key enzymes of carbohydrate, pentose phosphate, reveals an increased dependence on the enzyme or the process purine, pyrimidine, and other strategic metabolic pathways are involved; (d) because of the commitment to replication and closely coadapted during evolution. Thus, the pleiotropic repro- utilization of precursors and decreased responsiveness and gramming of gene expression may represent the program of adaptability of the neoplastic cells to regulatory signals, the one, or a complex, of master genes and integrative genomic amplified and stringently linked enzymic and metabolic pattern elements (125), since the isozyme pattern of key enzymes was in neoplasia is more vulnerable to drug-induced perturbations also stringently linked with the commitment of the cancer cells than that of the normal tissue which has a wider range of to transformation and progression. Until it becomes feasible to adaptability, repair, and recovery. use direct gene therapy, the most relevant components in the Selective toxicity is based on the following criteria: Criterion 1, neoplastic phenotype of the biochemical commitment to replica in the tumors there should be transformation-linked increases (a) tion, i.e., the key enzymes, should be prime targets of selective in activities of key enzymes, (b) in the pools of strategic nucleo anticancer chemotherapy. tides and deoxynucleotides, (c) in the influx of precursors (nu- cleosides, deoxynucleosides, amino acids, glucose, free fatty Design of Enzyme Pattern-targeted Chemotherapy acids) and/or a decreased concentration of strategic amino acids (glutamine) or coenzymes (NADP, NADPH); Criterion 2, en The design of chemotherapy that is directed against the en hanced selectivity is introduced by designing drug combinations zyme pattern of the neoplasm is based on the following consid to exploit the progression-linked pattern of enzymic and meta erations: (a) the enzymic and metabolic imbalance provides a bolic changes in the tumor that have conferred reproductive selective reproductive advantage for the cancer cells; (b) by advantages to the cancer cells. If the metabolic alteration was pinpointing the biochemical alterations that characterize the com important and stringently linked with the malignant replication, mitment of cancer cells for replication, we can identify targets the tumor cells would depend on it more intensively and the for anticancer drug treatment. Such biochemical targets are the perturbation of the biochemistry should be far more disruptive transformation- and progression-linked increased activities of key to the cancer cells than to normal ones. Criterion 3, in tumors enzymes and pools of nucleotides and the high metabolic re that retained a segment of gene expression characteristic of the quirements revealed in depleted concentrations of strategic me tissue of origin, drug combinations that are selectively synergistic

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Downloaded from cancerres.aacrjournals.org on September 29, 2021. © 1983 American Association for Cancer Research. G. Weber in cells containing the enzyme patterns characteristic both of TabteS proliferation or neoplasia and of the tissue of origin of the tumor Increased specific activities of glutamine-utilizing enzymes in tumors can be utilized (37, 39). Evidence was published from this labo Increase (% of control normal tissue values) ratory showing the feasibility of this approach (37, 39, 55-57, Carbamoyl- 118, 120). Amido- phosphate trans- GMP synthetase CTP syn- Tumors ferase synthetase II thetase Targeting Chemotherapy against Glutamine-utilizing En Rat3683FRat hepatoma zymes in Rat Hepatoma carcinoma,MK-3Ratrenal cell The principles developed for an enzyme pattern-targeted sarcomaHuman carcinomaHumanrenal cell chemotherapy have been tested in a series of investigations in colon tumor2122201345158548297218167183946495180518032910614307768181314 this laboratory (4, 5,37,39,55-57,118,120). One such program was designed on the basis of the imbalance we discovered in glutamine metabolism. This approach is now illustrated in some detail. To justify launching chemotherapy against an altered enzyme pattern in cancer cells, several criteria should be fulfilled. To design anti-glutamine-targeted chemotherapy, the following bio chemical alterations should be demonstrated in the cancer cell. The Drug Treatment Is Targeted against an Essential Part of the Neoplastic Biochemical Program. That a change in glutamine metabolism is an essential aspect of the biochemical program of neoplastic cells is suggested by the observation that the increases in the activities of enzymes involved in glutamine utilization were strongly conserved in different types of animal and human neoplasms. The specific activities of glutamine-utiliz- ing enzymes in purine synthesis (amidophosphoribosyltransfer- ase and GMP synthetase) and in pyrimidine synthesis (carba- moyl-phosphate synthetase II and CTP synthetase) increased in the series of rat hepatomas (120). The elevations of amidotrans- ferase activity were transformation linked, whereas those of the activities of the other 3 enzymes were both transformation and progression linked. The activities of the 4 enzymes also increased in renal cell carcinoma and sarcoma in rat and in human renal cell carcinoma and colon carcinoma (120) (Table 8). Increased Enzymic Activities Are Reflected in the Pattern Chart 19. Enzymic imbalance in glutamine synthesis, catabolism, and utilization in rapidly growing hepatoma 3924A. GMP syn., GMP synthetase; CTP syn., CTP of the Products and in the Metabolic Pathways. The impact of synthetase; carbamoyl-phosphate syn. II, carbamoyl-phosphate synthetase II; increased enzymic activities should be revealed in alterations in FGAR AT, phosphoribosylformylglycinamidine synthetase; PRPP AT, amidophos- the concentrations of strategic ribonucleotides. Freeze-clamp phoribosyltransferase (amidotransferase); glutamine syn., glutamine synthetase. studies determining the in vivo nucleotide pools in transplanted hepatoma 3924A showed that the concentration of CTP (the tration in hepatoma 3924A probably reflects decreased synthesis product of CTP synthetase) was increased 4-fold and that of by glutamine synthetase, increased catabolism by glutaminase, GMP (the product of GMP synthetase) was elevated 3.4-fold and stepped-up utilization by the glutamine-utilizing enzymes over the concentration of the normal liver (34). (44, 52, 120) (Chart 19). The wide margin of difference between Impact of the Altered Enzymic Pattern Is Expressed in the glutamine content in hepatoma and in other tissues, and the Pools of dNTP, the Immediate Precursors of DNA. The in biochemical imbalance in this metabolic area, suggest that selec creased activities of glutamine-utilizing enzymes and the enlarge tive toxicity by antiglutamine agents might be achieved against ment in the pools of the nucleotides produced are reflected in this tumor. the amplification of dNTP pools. The freeze-clamp studies in This chain of argument offers a rational design for chemother hepatoma 3924A indicated that the pools of dNTPs (the products apy and a rigorous quantitative approach where the drug targets of ribonucleotide reducíase) were markedly enlarged with the (a) the activities of the glutamine-utilizing enzymes and (b) the concentrations of dGTP, dATP, dTTP, and dCTP increasing ribonucleotide end products, GTP and CTP, can be measured. 4.8-, 17.7-, 12.1-, and 7.9-fold, respectively (34). Thus, the drug action and the extent of its effectiveness, as Pool of an Essential Metabolic Precursor in Cancer Cells, expressed in the concentrations of the dNTP pools, can be u-Glutamine, Is Depleted. This criterion requires evidence that determined and correlated with the tumor response to drug this is a biologically significant depletion in tumor cells in the treatment. concentration of relevant metabolic precursors as compared to Drug selectivity against hepatoma 3924A is anticipated be the levels found in normal tissues. In the freeze-clamp studies, cause, through the higher glutamine-utilizing enzymic activities the in vivo concentration of L-glutamine in hepatoma 3924A was and the enlarged pools of GTP, CTP, and dNTPs, the cancer 9-fold lower (0.5 mw) than that in liver (4.5 HIM) and lower than cells are committed to utilizing these biosynthetic capacities. The in any other rat tissues (2 to 5 ITIM).The low glutamine concen- low concentration of L-glutamine in this hepatoma should provide

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In the resting Effect of acivicin on nucleotide concentrations in hepatoma 3924A cells after 76 hr treatment liver, with its lower requirement for GIP, CTP, and the dNTP pools, and for the activities of the glutamine-utilizing enzymes, no drug added NucleotidesATPGTPCTPUTPdATPdGTPdCTPdTTPControl:(nmol/109cells)3373+ ofcontrol8762o46"152°12464Ö40"53" and because the higher levels of glutamine might protect these 98"741 enzymes, decreased sensitivity to the antiglutamine agent is +8904+ expected. 641654 ±107157 Enzyme Pattern-targeted Action of the Antiglutamine Agent, -t-4104+ 6147+ Acivicin 15250 The antiglutamine agent acivicin, L-(aS,5S)-«-amino-3-chloro- ± 27% Mean ±S.E. of 3 experiments. 4,5-dihydro-5-isoxazoleacetic acid, was isolated from Strepto- b Significantly different from control (p < 0.05). myces sviceus and exhibited antitumor activity against L1210 and P388 leukemias in mouse and human lung and in breast phate content should account for the depletion of the pools of xenografts in athymic mice (26). Neil ef al. (64, 65) showed that dGTP, dCTP, and dTTP. in L1210 cell culture acivicin blocked DNA synthesis and arrested Action of Acivicin on Glutamine-utilizing Enzymes in Hep cell cycle progression in early S phase; glutamine protected the atoma in Vitro. Purified CTP synthetase from hepatoma 3924A cells. Jayaram ef a/. (40) demonstrated that acivicin competitively was competitively inhibited by acivicin at K¡= 1 MM (57). The inhibited the activities of various bacterial and mammalian en activity of GMP synthetase in the crude extracts was inhibited zymes that catalyze the transfer of the amido group of L- by acivicin at K¡= 7 ^M; this was mixed inhibition. The other 2 glutamine. Further work showed that acivicin was a competitive glutamine-utilizing enzymes were competitively inhibited by aciv inhibitor of purified rat liver CTP synthetase (65). We tested the icin in hepatoma extracts (Table 10). Aoki ef al. (2) reported that approaches of enzyme pattern-targeted chemotherapy by eluci the activity of purified carbamoyl-phosphate synthetase II from dating the action of acivicin in tissue culture of hepatoma cells rat liver and hepatoma 3924A was competitively inhibited by and in vivo in rats carrying transplantable hepatoma 3924A (17, acivicin at K = 7 UM. The activity of amidophosphoribosyltrans- 57,120, 134). ferase, studied in crude extracts of liver and hepatoma, was Action of Acivicin in Hepatoma 3924A Tissue Culture Cells inhibited at K¡= 5 mw (120). These observations are in good and Protection by Nucleosides. Acivicin, on incubation for 7 agreement with those of Jayaram ef al. (40), who showed in days, killed hepatoma cells with an LDso of 1.4 /¿Masmeasured other tissues that a series of glutamine-utilizing enzymes were by clonogenic assay (57). When hepatoma cells were grown in competitively inhibited by acivicin. log phase, 72-hr incubation with acivicin (100 ^M) resulted in Inactivation by Acivicin of Glutamine-utilizing Enzymes in 55% growth inhibition, as measured by cell counts. Addition of Vitro and in Vivo and Selectivity. Our further studies threw pyrimidine or purine nucleosides (cytidine, deoxycytidine, guan- novel light on the action of acivicin. The inhibition of the enzymes osine) singly to the medium yielded no protection. The 3 nucleo was not reversible in vitro, yet this should have been so in case sides together provided complete protection against acivicin (57). of competitive inhibition unless we were dealing with a very These results supported the idea that the increased glutamine- tightly binding inhibitor. On in vivo injection of acivicin, the utilizing enzyme activities were essential for the hepatoma cells activities of the glutamine-utilizing enzymes markedly declined and that both purine and pyrimidine synthesis were involved in within minutes, and the decreased enzyme activities remained the program of commitment to neoplastic growth. The protection low for many hr after acivicin disappeared from the blood stream by nucleosides implied the presence of appropriate activities of (17, 120). These in vivo studies, to be outlined below, were not the salvage enzymes, uridine-cytidine and deoxycytidine kinases, compatible with a simple competitive inhibition and implied the and hypoxanthine-guanine phosphoribosyltransferase. The re operation of another mechanism. sult confirmed our conclusions, drawn from enzymic studies Investigations in vitro revealed that the purified or crude en outlined earlier in this paper, that the salvage pathways have a zymes were rapidly inactivated by addition of acivicin, and no vital role in determining the anticancer action of antimetabolites method that we used could reactivate them. Acivicin in vitro on the de novo pathways and that blockers of salvage enzymes selectively inactivated the glutamine-dependent activity of car or of nucleoside transport should be utilized in combination bamoyl-phosphate synthetase II from partially purified extracts chemotherapy. of hepatoma and liver, with an inactivation constant, Klnact,of 90 Action of Acivicin on Pools of Ribonucleotide Triphosphate fiM, and a minimum inactivation half-time, T, of 0.7 min. L- and dNTP in Hepatoma Cells in Tissue Culture. Treatment Glutamine (1 HIM) protected the enzyme from inactivation by 10 with acivicin for 16 hr decreased the concentrations of GTP and /UMacivicin. The ammonia-utilizing and aspartate carbamoyltrans- CTP to 62 and 46%, respectively, and accumulated that of UTP ferase activities of the enzyme complex were not inhibited by to 152% of controls; ATP level was not changed. The pools of acivicin. The inhibited synthetase II activity was not restored by dGTP, dCTP, and dTTP decreased to 64, 40, and 53% controls, (a) purification of the enzyme involving extensive dilutions with no change in the dATP pools (Table 9). The results were (1:1000) at 4 steps, (b) gel filtration of the enzyme-acivicin compatible with an explanation that acivicin inhibited the activity complex, (c) addition of a high concentration of L-glutamine (50 of GMP synthetase, resulting in depression of GTP. Furthermore, m«)to the assay mixture, or (d) freezing (in liquid nitrogen) and the drug inhibited CTP synthetase, yielding a decrease in con thawing the complex (2). centration of the product, CTP, and an accumulation of the In rats carrying transplanted hepatoma 3924A, injection of substrate, UTP. The resulting decrease in the nucleoside diphos- acivicin (25 mg/kg i.p.) caused a strikingly rapid decrease in the

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Table 10 Inhibition by acivicin of activities of glutamine-utilizingenzymes in hepatoma3924/4 con instandardcentration K„(mm)2.30.20.5assay(mM)20210K,(mM)5.0000.0050.001 EnzymesAmidotransferaseGMP ofinhibitionCompetitiveMixedCompetitive supernatantCrude synthetaseCTP supernatantPurified synthetase preparation Carbamoyl-phosphatesynthetase IIExtractCrudePurified preparationGlutamine0.017Glutamine 1Acivicin 0.007Type Competitive

activities of the glutamine-utilizing enzymes in the hepatoma. Significant declines were detected at 10 min after injection, and by 6 hr amidotransferase, CTP synthetase, and carbamoyl- phosphate synthetase II activities decreased to 34, 4, and 6%, respectively, of the controls. Aspartate carbamoyltransferase activity did not change (17,120) (Chart 20). With an acivicin dose

of 5 mg/kg, GMP synthetase activity in the hepatoma decreased o «o to 9% of the control at 6 hr after injection. All enzymic activities (J D-V/--CJ bottomed out at these low levels for 12 hr after injection and o then slowly started to return toward normal range by 48 to 72 hr (Table 11). Enzymic activities in the host liver also decreased,

but these changes were less pronounced than those in the 0 1 2 3 4 5 6 12 2» 36 «8 60 72

hepatoma; the pattern of return was similar to that in the hepa HOURS AFTER ACIVICIN INJECTION toma. Chart 20. Acivicin action in vivo on activities of glutamine-utilizing enzymes irs The rapid in vitro and in vivo inactivation of the 4 glutamine- hepatoma 3924A. utilizing enzymes (Table 11) may be explained by the observation that in bacterial systems acivicin acted as an active site-directed Table11 affinity analogue of u-glutamine (86). We postulated that the rapid In vivo effect of acivicin on enzyme activities in hepatoma 3924A Specific activities (nmol/hr/mg protein) are expressed as percentage of 0.9% action of acivicin in vivo was due to alkylation of the active NaCIsolution-injectedcontrols. Acivicindose, 25 mg/kg ¡.p.;dosefor GMP synthe center; thus, the L-glutamine concentration of the tissues might tase studies, 5 mg/kg. be a major determining factor in acivicin toxicity. Time after Action of Acivicin in Vivo on Ribonucleotide Pools in Hep acivicin in phoribosyl synthe synthe phosphate jectionControl10transferase1 tase55.9 tase56.5 synthetaseII82.0 atoma 3924A. These experiments were designed to elucidate 42 ±4.4a10087"56"32*21"34b35"46"74"99GMP±1.610075"31"9o13"35"8687CTP±2.610020"7"3"3"4"8"23"37"60"Carbamoyl-±8.61007250*13*6"6o17"35"6679 whether and to what extent the changes in enzymic activities were reflected in the concentrations of the relevant nucleotides. min0.5 At various time periods after acivicin injection, groups of rats hr1 hr2hr6hr12 bearing hepatoma 3924A were anesthetized, and tumors and host livers were freeze-clamped (Table 12). In the hepatomas, hr24 ATP and UTP concentrations remained unchanged. The GTP hr48 pool decreased to 56% of control 2 hr after injection, and the hr72 lowest value was reached at 6 hr (32%); the pool was still hrAmidophos a Mean ±S.E.of 4 or more rats in each group. significantly decreased at 24 hr, returning to normal range by 48 " Significantly different from controls (p < 0.05). and 72 hr. The CTP concentration decreased to 46% at 30 min after injection, and it reached the lowest level (2%) at 2 hr. CTP pool remained at less than 20% for 24 hr, and returned to normal Table 12 range at 48 and 72 hr after injection. In the host liver, the Effecf of acivicin on nucleoside triphosphate pools in hepatoma 3924A concentrations of ATP and UTP did not change, and those of The concentration of nucleotides is calculated in nmol/g. wet weight, and also expressed as percentage of 0.9% NaCI solution-injected controls. The acivicin GTP and CTP decreased in the same pattern as in the hepato dose was 25 mg/kg i.p. mas, but to a minor degree (17, 120). The differential selectivity Time after of acivicin impact on hepatoma and liver may be due, in part at acivicin in least, to the higher glutamine content of the host liver. jectionControl10 It is important that the decline in enzymic activities preceded ±87a100103857070776465117108GTP204+231009378"69"32*32o42*57118130CTP90±810012046"42o2"11"18"19"8989UTP190±511001271231041031221067798120 that in the pools of GTP and CTP (Chart 21). There was a min0.5 differential sensitivity to acivicin, with CTP synthetase and GMP hr1 synthetase being the most, and amidophosphoribosyltransferase hr2hr6hr12 the least, sensitive to the antiglutamine agent (Table 11). The K¡ for acivicin for these enzymes determined in vitro roughly corre hr24 lated with their in vivo sensitivity to this drug (Table 10). hr48 hr72 The mechanism of competitive inhibition could not play an hrATP793 important role in the biological action of acivicin, since the inhi * Mean ±S.E.of 4 or more rats in each group. bitions were not reversible. The decrease in enzymic activities 6 Significantly different from controls (p < 0.05).

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Table13 Synergistic in vivo action of acivicin and actinomycin on hepatoma 3924A Means of 10 rats in each group are given as percentage of controls. Treatment schedule and doses are as outlined in text. Tumor/body Body wt (% of Tumor volume ratio (% of con- Treatment initial) (% of control) trol) Control (0.9% NaCI solu tion)ActinomycinAcivicinAcivicin

-t- actinomycin10082«10174a10058"58a16o10078a67a22" " Significantly different from controls (p < 0.05). * Synergistic response. HOURS AFTER ACIVICIN INJECTION Chart 21. Effect of acivicin injection on plasma acivicin tevel, hepatoma CIP synthetase (CTP S) activity, and CTP concentration. ulation (Table 13) (120). was strikingly rapid, with significant declines detected at 10 min Enzymic and Nucleotide Targets and Biochemical Basis of after injection. The decreased enzymic activities observed after Synergistic Action of Acivicin and Actinomycin: Role of Sal in vivo injection of acivicin could not be reactivated in vitro by vage Pathways. Purine- and pyrimidine-biosynthetic processes incubation with high concentrations of L-glutamine or other meth from de novo and salvage pathways culminate in production of ods used. In the case of carbamoyl-phosphate synthetase II, ATP, GTP, UTP, and CTP. These nucleotides are substrates for activity could not be restored by purifying the enzyme through RNA polymerase, which synthesizes RNA. Acivicin selectively the steps that should have removed acivicin, unless it was very decreased concentrations of GTP and CTP with little change in tightly bound to the active center or the enzyme was inactivated. those of ATP and UTP. These observations and a report that The results indicated to us that inactivation of the enzyme by RNA polymerase activity was markedly increased in hepatoma acivicin was carried out by affinity labeling of the glutamine- 3924A (18) led us to design experiments using actinomycin which binding site (86). The action of acivicin was not limited to hepa selectively blocked the utilization of GTP and CTP by RNA toma, because similar in vivo inactivation of the synthetase II polymerase (32) (Chart 22). was observed in a chemically induced transplantable sarcoma It was remarkable that, although acivicin injection markedly carried in the rat. The activity of aspartate carbamoyltransferase decreased the activities of the key enzymes, amidophosphori- was unaffected (120). bosyltransferase and GMP synthetase, only the GTP levels de Combination Chemotherapy of Acivicin with Actinomycin creased, but ATP pools did not. In pyrimidine biosynthesis, the in Hepatoma 3924A in Vitro and in Vivo. The 4 ribonucleoside activities of carbamoyl-phosphate synthetase II and CTP synthe triphosphates are the substrates for RNA polymerase for syn tase were inhibited, but this was expressed only in the depletion thesis of RNA. RNA polymerase was a promising target because of CTP pools, and UTP concentrations were unchanged. The the activity was markedly increased in hepatoma 3924A (18). protection of ATP and UTP pools was tentatively attributed to Since the acivicin action was expressed in depletion of GTP and the contribution of the pyrimidine and purine salvage pathways. CTP pools, it was expected that actinomycin which inhibited This hypothesis was tested in the following experiments. RNA polymerase to utilize UTP and CTP would yield a synergistic In tissue culture studies, we showed that in hepatoma 3924A response (see Chart 21 ). This rationale led us to study the effects cells the salvage nucleosides, uridine, deoxycytidine, and guan- of acivicin and actinomycin separately and together in the culture ine together were able to protect the cancer cells from the action medium of hepatoma 3924A; cell kill was measured by colony of acivicin (57). This observation indicated the presence in the counts. The LD50values for acivicin and actinomycin were 1 and cells of adequate activities of the salvage enzymes, uridine and 0.01 ¿¿M,respectively,and the 2 drugs together were synergistic deoxycytidine kinases and GPRT. in killing hepatoma cells (120). In further investigations, we compared in liver and hepatomas In similar studies in hepatoma cells in culture, acivicin alone the activities of the key enzymes of de novo and salvage path decreased GTP and CTP concentrations, whereas actinomycin ways of pyrimidine and purine synthesis. The results (outlined alone caused a 5-fold increase in CTP pools and a 1.5-fold rise above) indicated that in the rapidly growing hepatoma 3924A the in UTP concentration. When acivicin and actinomycin were added salvage enzymes were present in high activities. Further impor to the cells together, the decrease in GTP and CTP pools caused tant determinants include the concentrations of salvage precur by acivicin was overcome by actinomycin which raised GTP and sors, substrates, and cofactors (uracil, uridine, cytidine, deoxy CTP pools 2- and 4-fold (120). These observations may explain, cytidine, thymidine, adenine, hypoxanthine, guanine, PRPP, ATP, in part at least, the synergistic cytotoxic action of actinomycin etc.), all of which impact on the contributions of the de novo and and acivicin in hepatoma cells. salvage pathways. A possible in vivo synergistic action of acivicin and actinomycin Thus, the inhibition of carbamoyl-phosphate synthetase II was was tested in rats inoculated with hepatoma 3924A tumor frag counterbalanced by the high uridine kinase activity in the hepa ments. Groups of rats were treated with acivicin, or with acti toma, which was sufficient to maintain the UTP pool at the nomycin, and with a combination of the 2 drugs. Treatment was prevailing tissue uridine level. Similarly, the inhibition of amido- started 24 hr after the tumor cells were inoculated, and volumes phosphoribosyltransferase activity was counterbalanced by the were calculated by measuring tumor diameters. The results high APRT activity which at the tissue adenine concentrations showed that combination chemotherapy yielded a synergistic was able to maintain the ATP pool. The decrease in GTP con inhibition of tumor growth, measured 14 days after tumor inoc- centration was attributed to the marked inhibition of GMP syn-

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with dipyridamole, which has the added advantage that it is a licensed drug in the United States Pharmacopoeia. Cytotoxic Action of Dipyridamole in Hepatoma 3924A Cells. Logarithmically growing hepatoma cells were incubated with dipyridamole, and survival was determined by clonogenic assay. In treatment schedules of 1 or 7 days, LDSOvalues were 87 and 20 tiM, respectively. These dipyridamole concentrations were higher than those required for the 50% inhibitory concentrations for blocking nucleoside incorporations which were 0.2 to 0.5 //M for uridine, cytidine, thymidine, and guanosine. Since we dem onstrated that dipyridamole was effective in killing hepatoma cells, we decided to investigate the biochemical mechanism of action. Dipyridamole Action on Cell Survival and DNA, RNA, and Protein Synthesis. In dose-response studies, 10 to 75 MM di 2. Enzymic and nucleotide targets in the synergistic action of acivicin and actinomycin and the role of salvage pathways. AT, amidotransferase; GMP pyridamole inhibited incorporation of uridine and thymidine but synth., GMP synthetase; CTP synth., CTP synthetase; Actino. D, actinomycin D; not that of formate, glycine, and leucine. Since dipyridamole C-P synth. II, carbamoyl-phosphate synthetase II. under these conditions inhibited the incorporation of pyrimidine and purine salvage nucleosides, its blocking of RNA and DNA synthesis may be attributed, in part at least, to inhibition of thetase. Whereas GPRT activity was high, apparently the tissue transport of salvage precursors. concentration of guanine was too low or the affinity for guanine With dipyridamole concentrations of 60 to 100 pM, the pools (40 /UM)was too high to permit replenishment of the GTP pool of ATP, GTP, CTP, and DTP decreased to 27, 50,19, and 23%, by this salvage enzyme. Tissue culture experiments showed that respectively, and 76% of the hepatoma cells were killed. This if guanine was supplied in adequate concentration the hepatoma raised the possibility that dipyridamole interfered with the bio cells were protected from acivicin; thus, sufficient GPRT activity chemical commitment of hepatoma cells by depleting the pools was present. The decrease in CTP concentration was an expres of the 4 ribonucleotides. sion of the marked inhibition of CTP synthetase, which apparently Synergistic Action of Dipyridamole and Acivicin on Hepa could not be overcome by the activities of cytidine and deoxy- toma Cells. When acivicin or dipyridamole was incubated with cytidine kinases; this may be attributed to low tissue concentra hepatoma cells, survivals of 64 and 65%, respectively, were tions of cytidine and deoxycytidine. In current work, we are observed. Combination of the 2 drugs yielded 11% survival, testing these assumptions by measuring the concentrations of indicating synergism. These results support our idea that com nucleosides in freeze-clamp samples of the hepatoma. bination chemotherapy with an antimetabolite of the de novo Another approach in determining the role of nucleosides in pathways (acivicin) and an inhibitor of the salvage pathways chemotherapy is by using an effective inhibitor of nucleoside (dipyridamole) might yield synergistic anticancer cytotoxic action. transport, dipyridamole (Persantin). In vivo administration of acivicin, dipyridamole, and the 2 drugs together in rats bearing s.c. transplanted hepatoma 3924A showed that acivicin decreased mainly the GTP and CTP pools, Effect of the Transport Inhibitor, Dipyridamole, on Hepatoma whereas dipyridamole depleted the concentrations of all 4 ribo 3924A Cells nucleotides. Combination chemotherapy with the 2 drugs yielded Experimental and clinical investigations indicated that inhibition summation, decreasing ATP, GTP, CTP, and DTP levels to 23, of the de novo synthetic pathways of pyrimidine and purine 14,15, and 29%, respectively, of the control. The decline in UTP metabolism by antimetabolites failed to yield lasting remissions concentration was a synergistic response (112). in neoplastic diseases. Our studies on experimental and primary These drugs also had a marked impact in the hepatoma on human neoplasms showed that along with increased activities the dNTP pools. Acivicin lowered dATP, dGTP, and dCTP pools of key enzymes of nucleic acid biosynthesis there also were without affecting dTTP concentration. Dipyridamole depleted elevations in the activity of the salvage enzymes of pyrimidine dGTP and dCTP pools to 52 and 22%, respectively. Acivicin and synthesis and maintenance of the high activities of purine salvage dipyridamole in combination yielded a summation, decreasing enzymes. From the studies of others and those presented in this the pools of dATP, dGTP, dCTP, and dTTP to 64, 34, 17, and paper, there is evidence that nucleosides can protect and rescue 44%, respectively, of the controls (112). The attacking points of cancer cells from the cytotoxic action of inhibitors of de novo acivicin and dipyridamole in pyrimidine biosynthesis are summa nucleic acid biosynthesis. Our chain of reasoning led us to rized in Chart 23. determine whether blockers of nucleoside transport would pro The results indicate that a protocol of combination chemo vide synergistic anticancer action (112, 134). therapy of effective antimetabolites (acivicin, tiazofurin, and oth Effect of Dipyridamole on Nucleoside Incorporation in Hep ers) and an inhibitor of nucleoside transport (dipyridamole) is atoma 3924A Cells. Recently, we showed that dipyridamole in productive, particularly because the antimetabolites are in Phase concentrations of 1CT6to 10~7 effectively blocked incorporation II clinical trials and dipyridamole has been used in clinical practice of purine and pyrimidine nucleosides into macromolecules in (for different therapeutic objectives) for many years. Investiga hepatoma 3924A cells (134). Our investigations demonstrated tions with these drugs throw light on their mechanism of action that dipyridamole was 9- to 27-fold more effective than was and on the relative contributions of efe novo and salvage path nitrobenzylthioinosine; therefore, all our studies were carried out ways in normal and neoplastic tissues in animals and in humans.

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1800—1

1400-

1000-

600-

200-

140- u < 120-

Z O 100- U u. O 4? SAUVAGE NUCLEOSIDES 80- Chart 23. Attacking points of acivicin and dipyridamole in pyrimidine biosyn thesis. 1, uridine-cytidine kinase; 2, deoxycytidine kinase; 3, thymidine kinase; 4, 60- CTP synthetase; 5, CTPase; 6, CDP reducíase;7, DNA polymerase; 8, carbamoyl- phosphate synthetase II. Drug Targeting against IMP Dehydrogenase and Biological 1)0- and Metabolic Impact

The drug targeting approach that we proposed on basis of enzymic imbalance ¡sa productive one. Robins wrote, "The

postulation of Weber that IMP dehydrogenase is a key enzyme I I I ! i I in neoplasia and therefore a sensitive target for cancer chemo 12 24 36 48 60 72 therapy has received considerable experimental support [98]. It HOURS AFTER TIAZOFURIN INJECTION would appear that the search for potential inhibitors of IMP Chart 24. Rapid response in IMP dehydrogenase (IMP DH) activity, IMP con dehydrogenase will continue to be a fruitful area for the design centration, and guanylate pools in hepatoma 3924A to injection of tiazofurin (200 and synthesis of potent antitumor agents [75]." One of the mg/kg i.p.). components synthesized, tiazofurin, proved to be a good inhibi istration.5 This is drug design targeted against a key enzyme, tor of IMP dehydrogenase, and in a series of brilliant studies IMP dehydrogenase, on the basis that its activity was markedly from the laboratories of Robins, Johns, Cooney, Jayaram, and increased in this hepatoma, an example of enzyme pattern- Glazer, it was shown that a number of tumors including the targeted chemotherapy. Lewis lung tumor were particularly sensitive to tiazofurin action It is only a matter of time, effort, and support that through (14,20,41,42,76). discoveries in the biochemical pharmacology of cancer cells In current work in my laboratory, in collaboration with Dr. rational therapy should be successful for all neoplastic diseases. Jayaram, we have shown that tiazofurin is a good inhibitor of the markedly increased activity of IMP dehydrogenase in hepa- toma 3924A. Tiazofurin killed hepatoma cells in tissue culture Conclusions with an LD50of 5 MM•Inanimals inoculated with hepatoma 3924A The introduction of the molecular correlation concept and the tiazofurin, as a single drug (150 mg/kg i.p. daily for 5 days), key enzyme concept and the use of biologically meaningful tumor caused an 85% inhibition of tumor growth as determined 14 models and control systems resulted in the discovery of an days after tumor transplantation. Our investigations demon ordered pattern of enzymic and metabolic imbalance and the strated that a single in vivo injection of tiazofurin (200 mg/kg) elucidation of the linkage with transformation and progression. caused a rapid decline in the activity of IMP dehydrogenase in With the conceptual and experimental guidance of the molecular the hepatoma and a concurrent 15-fold rise in the concentration correlation concept, we showed that the biochemical and en of IMP. Following the decline in IMP dehydrogenase activity, the zymic pattern of alterations was the result of a reprogramming pools of GMP and GTP markedly declined, and the concentration of gene expression that was both quantitative and qualitative of dGTP was depleted to lower than 20%. All parameters re and was characteristic to neoplasia, since no similar pattern of turned to normal after one single injection by 24 to 48 hr; imbalance has been observed in any of the control normal, however, the dGTP pool remained depressed for 72 hr. The regenerating, or differentiating tissues. marked decrease in GTP and particularly the sustained depletion Important aspects of gene logic were identified. These include in the dGTP pools may explain, in part at least, the chemother- demonstration of operation of reciprocal control of activities of apeutic action of tiazofurin on hepatoma 3924A (Chart 24). This is the first time that a marked therapeutic response was achieved 5M. S. Lui, M. A. Faderan, J. J. Liepnieks, E. Olah, H. N. Jayaram, and G. against rapidly growing hepatoma 3924A by single drug admin- Weber, to be published.

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opposing key enzymes and antagonistic pathways of synthesis sity of the biochemistry of cancer cells it is possible to perceive and catabolism in pyrimidine, purine, ornithine, and carbohydrate the pattern that characterizes the commitment of cancer cells to metabolism. It was established that the extent of increase in the replication. Inasmuch as the notion of causality and order per activities of key enzymes of pyrimidine and purine biosynthesis vades the approach I have outlined, it seems that the Ariadne related to the absolute activity of the enzymes in the resting thread is firmly in hand. Observations no longer need to range liver. Light was thrown also on qualitative alterations in gene over a diversity of metabolic changes in cancer cells; they can expression. These included the isozyme shift where key regula now be targeted to the transformation- and progression-linked tory enzymes, which were subject to nutritional and endocrine biochemical alterations which should have bearing on the rational regulation and had a high Kmfor their substrate, were replaced, design of selective anticancer chemotherapy. particularly in the rapidly proliferating hepatomas, with an iso zyme population that was less subject to nutritional and hor References monal regulation and had a low Kmfor their substrates. 1. Aoki, T., Morris, H. P., and Weber, G. Regulatory properties and behavior of The activities of the key enzymes in purine and pyrimidine activity of carbamoyl-phosphate synthetase II (glutamine-hydrolyzing) in nor metabolism in the resting liver were low in comparison with the mal and proliferating tissues. J. Biol. Chem., 257: 432-438, 1982. 2. Aoki, T., Sebolt, J., and Weber, G. In vivo inactivation of carbamoylphosphate high activity of the enzymes of the salvage pathways. In pyrimi synthetase II in rat hepatoma. Biochem. Pharmacol., 31: 927-932, 1982. dine metabolism, the activities of the de novo and salvage 3. Aoki, T., and Weber, G. Carbamoyl-phosphate synthetase (glutamine-hydro enzymes increased markedly in a transformation- and progres lyzing): increased activity in cancer cells. Science (Wash. D.C.) 272: 463- 465,1981. sion-linked fashion. In contrast, in purine biosynthesis only the 4. Ban, J., Olah, E., and Weber, G. Action of lycurim and pyrazofurin on activities of the key enzymes of de novo biosynthesis were hepatoma 3924A cells in culture. Cancer Treat. Rep. 66: 343-349, 1982. 5. Ban, J., Olah, E., and Weber, G. Cytotoxic and cell kinetic effects of 3,4,5- elevated, and those of the salvage pathways changed little. trihydroxybenzohydroxamic acid (VF 122) in hepatoma 3924A cells. Cancer The pattern of enzymic and metabolic imbalance was present Treat. Rep. 66: 2071-2080, 1982. in 16 different types of neoplasms, including chemically induced, 6. Baril, E. F., Jenkins, M. D., Brown, 0. E., Laszlo, J., and Morris, H. P. DNA polymerases I and II in regenerating rat liver and Morris hepatomas. Cancer transplantable tumors in rodents, virally produced tumors in Res., 33: 1187-1193,1973. chickens, and various neoplasias in humans. Two generalizations 7. Becher, H. J., and Lohr, G. W. Inosine 5'-phosphate dehydrogenase activity in normal and leukemic blood cells. Klin. Wochenschr., 57:1109-1115,1979. were proposed. Since the enzymic and metabolic imbalance was 8. Bergel, F. Chemistry of Enzymes in Cancer. Springfield, III.: Charles C present in these different types of tumors, a segment of gene Thomas, Publisher, 1961. expression that is essential for neoplasia has been identified. 9. Blumberg, B. S., and London, W. T. Hepatitis B virus pathogenesis and prevention of primary cancer of the liver. In: J. G. Fortner and J. E. Rhoads The biochemical phenotype of neoplasia was independent from (eds.), Accomplishments in Cancer Research, pp. 184-199. Philadelphia: J. the carcinogen, since it was present in chemically, virally, and B. Lippincott Co., 1982. 10. Boritzki, T. J., Jackson, R. C., Morris, H. P., and Weber, G. Guanosine 5'- spontaneously induced tumors in animals and humans. There phosphate synthetase and guanosine 5'-phosphate kinase in rat hepatomas fore, the biochemical imbalance originally discovered and docu and kidney tumors. Biochim. Biophys. Acta, 658: 102-110, 1981. mented most extensively in the spectrum of hepatomas of dif 11. Burnet, F. M. Immunological Surveillance, p. 127. New York: Pergamon ferent growth rates was generally applicable, in part or entirely, Press, 1970. 12. Busch, H. The Molecular Biology of Cancer. New York: Academic Press, Inc., to all tumors examined thus far. In addition, individual tumor 1974. markers have been noted for a number of neoplasms. 13. Cohen, M. B., Maybaum, J., and Sadee, W. Guanine nucleotide depletion and toxicity in mouse T lymphoma (S-49) cells. J. Biol. Chem., 256: 8713- With these and other concepts and observations outlined 8717, 1981. above, insight was gained into strategic aspects of the biochem 14. Cooney, D. A., Jayaram, H. N., Glazer, R. I., Kelley, J. A., Marquez, V. E., ical commitment of cancer cells to replication. It has become Gebeyehu, G., Van Cott, A. C., Zwelling, L. A., and Johns, D. G. Studies on the mechanism of action of tiazofurin metabolism to an analog of NAD with possible to pinpoint the selective advantages that reprogram- potent IMP dehydrogenase-inhibitory activity. Adv. Enzyme Regul., 21: 271- ming of gene expression conferred to cancer cells. In turn, 303, 1983. understanding these alterations in the enzymology and biochem 15. Criss, W. E., Litwack, G., Morris, H. P., and Weinhouse, S. Adenosine triphosphate: adenosine monophosphate phosphotransferase isozymes in istry of cancer cells made it possible to identify potentially sen rat liver and hepatomas. Cancer Res., 30: 370-375, 1970. sitive targets for anticancer chemotherapy. 16. Dentón, J., Lui, M. S., Aoki, T., Sebolt, J., Takeda, E., Eble, J. N., Glover, J. L., and Weber, G. Enzymology of pyrimidine and carbohydrate metabolism On the basis of these advances, it became feasible to design in human colon carcinomas. Cancer Res., 42: 1176-1183, 1982. an enzyme pattern-targeted chemotherapy. In this strategy, 17. Dentón, J., Lui, M. S., Aoki, T., Sebolt, J., and Weber, G. Rapid in vivo drugs were directed against immediate targets, activities of key inactivation of acivicin of CTP synthetase, carbamoyl-phosphate synthetase II, and amidophosphoribosyltransferase in hepatoma. Life Sci., 30: 1073- enzymes, to perturb the steady state of secondary targets, the 1080,1982. pools of ribonucleotides; then it became possible to assess the 18. Duceman, B. W., and Jacob, S. T. Transcriptionally active RNA polymerases effectiveness of chemotherapy by measuring the perturbation in from Morris hepatomas and rat liver. Biochem. J., 790: 781-789,1980. 19. Dunaway, G. A., Jr., Morris, H. P., and Weber, G. Comparative study of rat the pools of dNTP. By carefully correlating the extent of depres liver, muscle, and hepatoma 3924A phosphofructokinase isozymes. Cancer sion of the activities of key enzymes and the pools of nucleotides Res., 34: 2209-2216,1974. 20. Earte, M. F., and Glazer, R. I. Activity and metabolism of 2-0-D-ribofuranosyl- with the survival of cancer cells, determined by clonogenic as thiazole-4-carboxamide in human lymphoid tumor cells in culture. Cancer says and by the volume of tumors measured in tumor-bearing Res., 43: 133-137,1983. animals, the response to chemotherapeutic targeting could be 21. Elford, H. L., Freese, M., Passamani, E., and Morris, H. P. Ribonucleotide reducíaseand cell proliferation I. Variation of ribonucleotide reducíaseactivity quantitated. with tumor growth rale in a series of ral hepatomas. J. Biol. Chem., 245: Advances have been made in showing the role of nucleosides 5228-5233,1970. in determining anticancer action and the chemotherapeutic use 22. Ellims, P. H., Eng Gan, T., and Medley, G. Cylidine triphosphate synlhetase activity in lymphoproliferalive disorders. Cancer Res., 43:1432-1435,1983. of drugs that can synergistically inhibit activities of key enzymes 23. Ferdinandus, J. A., Morris, H. P., and Weber, G. Behavior of opposing and the transport processes in de novo and salvage pathways. pathways of Ihymidine utilization in differentiating, regenerating and neoplas- tic liver. Cancer Res., 37: 550-556,1971. Research in this and other laboratories in recent years dem 24. Goldfarb, S., and Pugh, T. D. The origin and significance of hyperplaslic onstrated that beyond the sheer complexity and apparent diver- hepalocellular islands and nodules in hepatic carcinogenesis. J. Am. Coll.

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Toxicol., 1: 119-144,1982. 54. Looney, W. B., Mayo, A. A., Janners, M. Y., Mellon, J. G., Allen, P., Salak, 25. Greengard, 0., and Herzfeld, A. The undifferentiated enzymic composition of D., and Morris, H. P. Cell proliferation and tumor growth in hepatoma 3924A. human fetal lung and pulmonary tumors. Cancer Res., 37: 884-891,1977. Cancer Res., 37: 821-825,1971. 26. Hanka, L. J., Martin, D. G., and Neil, G. L. A new antitumor antimetabolite, 55. Lui, M. S., Jackson, R. C., Harkrader, J. R., and Weber, G. Rat hepatoma: («S,5S)-iv-amino-3-chloro-4,5-dihydro-5-isoxazoleaceticacid(NSC-163501): chemotherapy with lycurim and pyrazofurin. J. Naît.CancerInst., 68: 665- antimicrobial reversal studies and preliminary evaluation against L1210 668,1982. mouse leukemiain vivo. Cancer Chemother. Rep., 57: 141-148, 1973. 56. Lui, M. S., Jackson, R. C., and Weber, G. Enzyme pattern-directed chemo 27. Harkrader, R. J., Jackson, R. C., Ross, D. A., and Weber, G. Increasein liver therapy. Effects of anti-pyrimidinecombinationson the ribonucleotidecontent and kidney deoxycytidine kinase activity linked to neoplastic transformation. of hepatomas. Biochem. Pharmacol.,28: 1189-1195, 1979. Biochem. Biophys. Res. Commun., 96: 1633-1639, 1980. 57. Lui, M. S., Kizaki, H., and Weber, G. Biochemicalpharmacology of acivicinin 28. Heinrich, P. C., Morris, H. P., and Weber, G. Increased PRPP synthetase rat hepatoma cells. Biochem. Pharmacol.,37: 3469-3473,1982. activity in rapidly growing hepatomas. FEBS Lett., 42: 145-148,1974. 58. Maley, F., and Maley, G. F. Nucleotide interconversions. IV. Activities of 29. Heinrich, P. C., Morris, H. P., and Weber, G. Behavior of transaldolase (EC deoxycytidylate deaminase and thymidylate synthetase in normal rat liver 2.2.1.2) and transketolase (EC 2.2.1.1) activities in normal, neoplastic,differ and hepatomas. Cancer Res., 7:1421-1426,1961. entiating and regenerating liver. Cancer Res., 36: 3189-3197,1976. 59. Mayr, E. Populations,Speciesand Evolution,p. 2. Cambridge,Mass.: Harvard 30. Herzfeld, A., Legg, M. A., and Greengard, 0. Human colon tumors: enzymic UniversityPress, 1971. and histological characteristics. Cancer (Phila.),42: 1280-1283,1978. 60. Medawar, P. B. Induction and Intuition in Scientific Thought, p. 51. London: 31. Holton, G. Thematic presuppositions and the direction of scientific advance. Methuen and Co., 1969. In: A. F. Heath(ed.),ScientificExplanation,pp. 1-27. Oxford, U. K.: Clarendon 61. Miyaji, H., Morris, H. P., and Wagner, B. P. Histologicalstudy of some primary Press, 1981. and transplantable hepatic tumors in rats. Methods Cancer Res., 4: ISS 32. Hyman, R. W., and Davidson, N. Kinetics of the in vitro inhibitionof transcrip US, 1968. tion by actinomycin. J. Mol. Biol., 50: 421-428,1970. 62. Morris, H. P., and Wagner, B. P. Induction and transplantation of rat hepa tomas with different growth rate (including "minimal deviation" hepatomas). 33. Jackson, R. C., Goulding, F. J., and Weber, G. Enzymesof purinemetabolism in human and rat renal cortex and renal cell carcinoma. J. Nati. Cancer Inst., Methods Cancer Res., 41:125-152,1968. 62:749-754, 1979. 63. Morris, H. P., Wagner, B. P., and Meranze, D. R. Transplantable adenocar- 34. Jackson, R. C., Lui, M. S., Boritzki, T. J., Morris, H. P., and Weber, G. Purine cinomas of the rat kidney possessing different growth rates. Cancer Res., and pyrimidinenucleotide patterns of normal, differentiating and regenerating 30:1362-1369,1970. liver and of hepatomas in rats. Cancer Res., 40: 1286-1291,1980. 64. Neil, G. L., Berger, A. E., Bhuyan, B. K., Blowers, C. L., and Kuentzel, S. L. 35. Jackson, R. C., Morris, H. P., and Weber, G. Partial purification, properties Studies of the biochemicalpharmacology of the fermentation-derivedantitu- and regulation of inosine 5'-phosphate dehydrogenase in normal and malig mor agent, («S,5S)-rt-amino-3-chloro-4,5-dihydro-5-isoxazole-aceticacid(AT- nant rat tissues. Biochem. J., 766: 1-10, 1977. 125). Adv. Enzyme Regul., 77: 375-398, 1979. 36. Jackson, R. C., Morris, H. P., and Weber, G. Enzymes of the purine ribonu- 65. Neil, G. L., Berger, A. E., McPartland, R. P., Grindey, G. B., and Bloch, A. cleotide cycle in rat hepatomas and kidney tumors. Cancer Res., 37: 3057- Biochemicaland pharmacologicaleffects of the fermentation-derived antitu- 3065,1977. mor agent, («S,5S)-a-amino-3-chloro-4,5-dihydro-5-isoxazoleaceticacid(AT- 37. Jackson, R. C., and Weber, G. Enzyme pattern-directed chemotherapy: the 125). Cancer Res., 39: 852-856, 1979. effects of combinations of methotrexate, 5-fluorodeoxyuridineand thymidine 66. Ove, P., Laszlo, J., Jenkins, M. D., and Monis, H. P. Increased DNA on rat hepatoma cells in vitro. Biochem. Pharmacol.,25: 2613-2618,1976. polymerase activity in a series of rat hepatomas. Cancer Res., 29: 1557- 38. Jackson, R. C., Weber, G., and Morris, H. P. IMP dehydrogenase,an enzyme 1561, 1969. linkedwith proliferationand malignancy.Nature(Lond.),256:331 -333,1975. 67. Pilot, H. C., Fohn, C. H., Clark, W. H., Jr., and Farber, E. A comparative 39. Jackson, R. C., Williams, J. C., and Weber, G. Enzyme pattern-directed biochemical and morphologic study of some transplanted and primary rat chemotherapy: synergistic interaction of 3-deazauridine with o-galactosa- hepatomas. Proc. Am. Assoc. Cancer Res., 3: 52,1959. mine. Cancer Treat. Rep., 60: 835-843,1976. 68. Popper, K. R. The Logic of Scientific Discovery, p. 40. New York: Harper and 40. Jayaram, H. N., Cooney, D. A., Ryan, J. A., Neil. G., Dion, R. L, and Bono, Row, 1968. V. H. L-(aS,5S)-a-Amino-3-chloro-4,5-dihydro-5-isoxazole-aceticacid (NSC 69. Prajda, N., Donohue, J. P., and Weber, G. Increased amidophosphoribosyl- 163501): a new amino acid antibiotic with the properties of an antagonist of transferase and decreased xanthine oxidase activity in human and rat renal L-glutamine.Cancer Chemother. Rep., 59: 481-491, 1975. cell carcinoma. Life Sci., 29: 853-860,1981. 41. Jayaram, H. N., Dion, R. L., Glazer, R. I., Johns, D. G., Robins, R. K., 70. Prajda,N., Eckhardt, S., Suba, Z., and Lapis. K. Biochemicalbehaviorof MC- Srivastava, P. C., and Cooney, D. A. Initial studies on the mechanism of 29 virus-induced transplantable chicken hepatoma. J. Toxicol. Environ. action of a new oncolytic thiazole nucleoside, 2-fl-D-ribofuranosylthiazole-4- Health, 5: 503-508, 1979. carboxamide(NSC-286193). Biochem. Pharmacol.,37: 2371-2380,1982. 71. Prajda, N., Katunuma, N., Morris, H. P., and Weber, G. Imbalanceof purine 42. Jayaram, H. N., Smith, A. L., Glazer, R. I., Johns, D. G., and Cooney, D. A. metabolism in hepatomas of different growth rates as expressed in behavior Studies on the mechanismof action of 2-0-D-ribofuranosylthiazole-4-carbox- of glutamine PRPP amidotransferase (amidophosphoribosyltransferase,EC amide (NSC-286193) II. Relationship between dose-level and biochemical 2.4.2.14). Cancer Res., 35: 3061-3068, 1975. effects in P388 leukemiain vivo. Biochem. Pharmacol.,37:3839-3845,1982. 72. Prajda. N., Morris, H. P., and Weber, G. Imbalance of purine metabolism in 43. Jones, M. E. Regulationof pyrimidineand argininebiosynthesis in mammals. hepatomas of different growth rates as expressed in behavior of xanthine Adv. Enzyme Regul., 9: 19-49,1971. oxidase (EC 1.2.3.2). Cancer Res., 36: 4639-4646,1976. 44. Katunuma, N., Katsunuma, T., Towatari, T., and Tornino, I. Regulatory 73. Prajda, N., Morris, H. P., and Weber, G. Glutamine-phosphoribosylpyrophos- mechanisms of glutamine catabolism. In: S. Prusiner and E. R. Stadtman phate amidotransferase (amidophosphoribosyltransferase,EC 2.4.2.14) ac (eds.), The Enzymes of Glutamine Metabolism, pp. 227-258. New York: tivity in normal, differentiating and neoplastic kidney. Cancer Res., 39: 3909- Academic Press, Inc., 1973. 3914,1979. 45. Keppler, D., and Holstege, A. Pyrimidine nucleotide metabolism and its 74. Rhoads, A. R., and Morris, H. P. Enzymatic potential for preformed purine compartmentation. In: H. Sies (ed.), Metabolic Compartmentation, pp. 147- metabolism in hepatomas with different growth rates. J. Nati. Cancer Inst., 203. New York: Academic Press, Inc., 1982. 59:905-910, 1977. 46. Kizaki, H., Morris, H. P., and Weber, G. Behavior of inosine phosphorylase 75. Robins, R. K. Nucleosideand nucleotideinhibitors of inosinemonophosphate (EC 2.4.2.1) activity in normal, differentiating and regenerating liver and in (IMP)dehydrogenase as potential antitumor inhibitors. Nucleosidesand Nu- hepatomas. Cancer Res., 40: 3339-3344, 1980. cleotides, 7:35-44, 1982. 47. Kizaki, H., Williams,J. C., Morris, H. P., and Weber, G. Increasedcytidine 5'- 76. Robins, R. K., Srivastava, P. C., Narayanan,V. L., Plowman, J., and Paull, triphosphate synthetase activity in rat and human tumors. Cancer Res., 40: K. D. 2-(j-D-Ribofuranosylthiazole-4-carboxamide,anovel potential antitumor 3921-3927,1980. agent for lung tumors and métastases.J.Med. Chem., 25: 107-108, 1982. 48. Knox, W. E. Enzyme Patterns in Fetal, Adult and Neoplastic Rat Tissues. 77. Ross, D. A., Jackson, R. C., Morris, H. P., and Weber, G. Decreasedcontent Basel: S. Karger AG, 1972. of reduced and oxidized nicotinamide-adeninedinucleotide phosphate in rat 49. Kovalszky, I., Jeney, A., Asbot, R., and Lapis, K. Biochemistry and enzyme hepatomas. Cancer Biochem. Biophys., 6: 61-64, 1982. induction in MC-29 virus-induced transplantable avian hepatoma. Cancer 78. Sasaki, T., Morris, H. P., and Baserga, R. The cell cycles of three transplant- Res., 36: 2140-2145,1976. able Morris hepatomas. Cancer Res., 30: 788-793, 1970. 50. Lai, M.-H. T., and Weber, G. Increasedconcentration of thymidine kinase in 79. Selmeci,L. E., and Weber, G. Increasedglucose6-phosphatedehydrogenase rat hepatomas. Biochem. Biophys. Res. Commun., 777: 280-287,1983. concentration in hepatoma 3924A: enzymic and immunological evidence. 51. Lea, M. A., Morris, H. P., and Weber, G. Comparative biochemistry of FEBS Lett., 67: 63-67, 1976. 80. Shatkin, A. J. Capping of eucaryotic mRNA's. Cell, 9: 645-653, 1976. hepatomas VI. Thymidine incorporation into DNA as a measureof hepatoma growth rate. Cancer Res., 26: 465-469,1966. 81. Shiotani, T., and Weber, G. Purification and properties of dihydrothymine 52. Linder-Horowitz, M., Knox, W. E., and Morris, H. P. Glutaminaseand growth dehydrogenasefrom rat liver. J. Biol. Chem., 256: 219-224, 1981. rates of hepatomas. Cancer Res., 29:1195-1199,1969. 82. Sneider, T. W., and Potter, V. R. Deoxycytidylate deaminase and related 53. Lo, C., Cristofalo, V. J., Morris, H. P., and Weinhouse, S. Studies on enzymes of thymidine triphosphate metabolism in hepatomas and precan- respiration and glycolysis in transplanted hepatic tumors of the rat. Cancer cerous rat liver. Adv. Enzyme Regul., 7: 375-394, 1969. Res., 28: 1-10,1968. 83. Staub, M., Szpaszokukockaja, T., Bencsath, M., Antoni, F., and Lapis, K.

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DNA polymerase and thymidine kinase activities in MC-29 virus-induced Enzymatic markers of neoplastic transformation and regulation of Durine and transplantable hepatoma and the effect of cytostatic treatment on these pyrimidine metabolism. Adv. Enzyme Regul., 75: 53-77,1977. activities. Chem.-Biol. Interact., 41: 181-192, 1982. 111. Weber, G., Kizaki, H., Tzeng. D., Shkrtani, T., and Olah, E. Colon tumor: 84. Sweeney, M. J., Ashmore, J., Morris, H. P., and Weber, G. Comparative enzymology of the neoplastic program. Life Sci.. 23: 729-736,1978. biochemistry of hepatomas. IV. Isotope studies of glucose and fructose 112. Weber, G., Lui, M. S., Natsumeda, Y., and Faderan, M. A. Salvage capacity metabolism in liver tumors of different growth rates. Cancer Res., 23: 995- of hepatoma 3924A and action of dipyridamole. Adv. Enzyme Regul., 27: 1002, 1963. 53-69.1983. 85. Takeda, E., and Weber, G. Role of ribonucleotide reducíasein expression of 113. Weber, G., Lui, M. S., Takeda, E., and Dentón, J. E. Enzymology of human the neoplastic program. Life Sci.. 8:1007-1014,1981. colon tumors, ufe Sci., 27: 793-799, 1980. 86. Tso, J. Y., Bower, S. G.. and Zalkin, H. Mechanism of inactivation of glutamine 114. Weber, G.. Malt, R. A.. Glover, J. L. Williams, J. C.. Prajda, N., and Waggoner, amidotransferase by the antitumor drug L-(«S,5S)-«-amino-3-chloro-4,5-dih- C. D. Biochemical basis of malignancy in man. In: W. Davis and C. Maltoni ydro-5-isoxazoleacetic acid (AT-125). J. Biol. Chem., 255: 6734-6738,1980. (eds.). Biological Characterization of Human Tumours, pp. 60-72. Amster 87. Tsuda, M., Katunuma, N., Morris, H. P., and Weber, G. Purification, properties dam: Excerpta Medica, 1976. and immunotitration of hepatoma glutamine phosphoribosylpyrophosphate 115. Weber, G., and Morris, H. P. Comparative biochemistry of hepatoma. III. amidotransferase (amidophosphoribosyltransferase, EC 2.4.2.14). Cancer Carbohydrate enzymes in liver tumors of different growth rates. Cancer Res.. Res., 39. 305-311,1979. 23:987-994,1963. 88. Tzeng, D. Y., Sakiyama. S.. Kizaki, H., and Weber, G. Increased concentration 116. Weber, G., Olah, E., Dentón, J. E., Lui, M. S., Takeda, E., Tzeng, D. Y., and of CTP synthetase in hepatoma 3924A: immunological evidence. Life. Sci., Ban, J. Dynamics of modulation of biochemical programs in cancer cells. 28:2537-2543, 1981. Adv. Enzyme Regul., 79: 87-102, 1981. 89. Warburg, O. On the origin of cancer cells. Science (Wash. D. C.), 123: 309- 117. Weber, G., Olah, E., Lui, M. S., Kizaki, H., Tzeng, D. Y., and Takeda, E. 314,1956. Biochemical commitment to replication in cancer cells. Adv. Enzyme Regul., 90. Weber, G. Behavior of liver enzymes during hepatocarcinogenesis. Adv. 78:3-26, 1980. Cancer Res., 6. 403-494, 1961. 118. Weber, G., Olah, E., Lui, M. S., and Tzeng, D. Biochemical programs and 91. Weber, G. The molecular correlation concept: studies on the metabolic pattern enzyme-pattern targeted chemotherapy in cancer cells. Adv. Enzyme Regul., of hepatomas. Gann Monogr., 1:151-178,1966. 17:1-21,1979. 92. Weber, G. Carbohydrate metabolism in cancer cells and the molecular cor 119. Weber, G., Prajda, N., and Jackson, R. C. Key enzymes of IMP metabolism: relation concept. Naturwissenschaften, 55: 418-429,1968. transformation- and proliferation-linked alterations in gene expression. Adv. 93. Weber, G. The molecular correlation concept: ordered pattern of gene expres Enzyme Regul., 14: 3-24, 1976. sion in neoplasia. Gann Monogr. Cancer Res., 73: 47-77, 1972. 120. Weber, G., Prajda, N., Lui, M. S., Dentón, J. E., Aoki, T., Sebolt, J., Zhen, 94. Weber, G. Ordered and specific pattern of gene expression in neoplasia. Adv. Y.-S., Burt, M. E., Faderan, M. A., and Reardon, M. A. Multi-enzyme-targeted Enzyme Regul., 77: 79-102, 1973. chemotherapy by acivicin and actinomycin. Adv. Enzyme Regul., 20: 75-96, 95. Weber, G. The molecular correlation concept: recent advances and implica 1982. tions. In: H. Busch (ed.), The Molecular Biology of Cancer, pp. 487-521. New 121. Weber, G., Queener, S. F., and Ferdinandus, J. A. Control of gene expression York: Academic Press, Inc.. 1974. in carbohydrate, pyrimidine and DNA metabolism. Adv. Enzyme Regul., 9: 96. Weber, G. Biochemical strategy of the regenerating liver cell. In: R. Lesch 63-95,1971. and W. Reutter (eds.), Liver Regeneration after Experimental Injury, pp. 103- 122. Weber, G., Queener, S. F., and Morris, H. P. Imbalance in ornithine metabo 117. New York: Stratton Intercontinental Medical Book Corp., 1975. lism in hepatomas of different growth rates as expressed in behavior of L- 97. Weber, G. Enzymology of cancer cells, Part 1. N. Engl. J. Med., 296: 486- omithine carbamoyl transferase activity. Cancer Res., 32: 1933-1940,1972. 493, 1977. 123. Weber, G.. Shiotani, T., Kizaki, H., Tzeng, D., Williams, J. C., and Gladstone, 98. Weber, G. Enzymology of cancer cells, Part 2. N. Engl. J. Med., 296: 541- N. Biochemical strategy of the genome as expressed in regulation of pyrimi 551.1977. dine metabolism. Adv. Enzyme. Regul., 76: 3-19, 1978. 99. Weber, G. Enzymic programs of human renal adenocarcinoma. In: G. Sufrin 124. Weber, G., Stubbs, M., and Morris, H. P. Metabolism of hepatomas of different and S. A. Beckley (eds.). Renal Adenocarcinoma, pp. 44-50. Geneva: UICC growth rates in situ and during ischemia. Cancer Res., 37:2177-2183,1971. Publications, 1980. 125. Weber, G.. Trevisani, A., and Heinrich, P. C. Operation of pleiotropic control 100. Weber, G. Differential carbohydrate metabolism in tumor and host. In: M. S. in hormonal regulation and in neoplasia. Adv. Enzyme Regul., 72: 11-41, Amott, J. van Eys., and Y.-M. Wang (eds.). Molecular Interrelations of 1974. Nutrition and Cancer (UT System Cancer Center 34th Annual Symposium on 126. Weinhouse, S. Oxidative metabolism of neoplastic tissues. Adv. Cancer Res., Fundamental Cancer Research), pp. 191-208. New York: Raven Press, 1982. 3:269-325,1955. 101. Weber. G., Banerjee. G., and Morris, H. P. Comparative biochemistry of 127. Weinhouse, S. Glycolysis, respiration, and anomalous gene expression in hepatomas. I. Carbohydrate enzymes in Morris hepatoma 5123. Cancer Res., experimental hepatomas: G. H. A. Clowes Memorial Lecture. Cancer Res., 27. 933-937, 1961. 32:2007-2016,1972. 102. Weber, G., Burt, M. E., Jackson, R. C., Prajda, N., Lui, M. S., and Takeda, 128. Weinhouse, S. Changing perceptions of carbohydrate metabolism in tumors. E. Purine and pyrimidine enzymic programs and nucleotide pattern in sar In: M. S. Amott, J. van Eys, and Y.-M. Wang (eds.). Molecular Interrelations coma. Cancer Res., 43: 1019-1023, 1983. of Nutrition and Cancer (UT System Cancer Center 34th Annual Symposium 103. Weber, G., and Cantero, A. Glucose-6-phosphatase activity in normal, pre- on Fundamental Cancer Research), pp. 167-181. New York: Raven Press, cancerous, and neoplastic tissues. Cancer Res., 75:105-108,1955. 1982. 104. Weber, G., and Cantero, A. Glucose-6-phosphatase activity in regenerating, 129. Wheeler, G. P., Alexander, J. A., and Morris, H. P. Synthesis of purines and embryonic, and newborn rat liver. Cancer Res., 75: 679-684,1955. nucleic acids and catabolism of purines by rat liver and hepatomas. Adv. 105. Weber, G., and Cantero, A. Glucose-6-phosphate utilization in hepatoma, Enzyme Regul., 2: 347-370, 1964. regenerating and newborn rat liver, and in the liver of fed and fasted normal 130. Williams, J. C.. Morris, H. P., and Weber, G. Increased UDP kinase activity in rats. Cancer Res., 77: 995-1005, 1957. rat and human hepatomas. Nature (Lond.). 253: 567-569,1975. 106. Weber, G., and Cantero, A. Fructose-1,6-diphosphatase and lactate dehydro- 131. Williams-Ashman. H. G., Coppoc, G. L., and Weber, G. Imbalance in ornithine genase activity in hepatoma and in control human and animal tissues. Cancer metabolism in hepatomas of different growth rates as expressed in formation Res., 79:763-768,1959. of putrescine, spermidine and spermine. Cancer Res., 32:1924-1932,1972. 107. Weber, G., Goulding, F. J., Jackson, R. C., and EWe, J. N. Biochemistry of 132. Williamson, D. H., Krebs, H. A., Stubbs, M., Page, M. A., Morris, H. P., and human renal cell carcinoma. In: W. Davis and K. R. Harrap (eds.), Character Weber. G. Metabolism of renal tumors in situ and during ischemia. Cancer ization and Treatment of Human Tumors, pp. 227-234. Amsterdam: Excerpta Res., 30: 2049-2054,1970. Medica, 1978. 133. Wyngaarden, J. B., and Kelley, W. N. Disorders of purine and pyrimidine 108. Weber, G., Hager. J. C., Lui. M. S., Prajda, N.. Tzeng, D. Y., Jackson, R. C., metabolism. In: J. B. Standbury. J. B. Wyngaarden, and D. S. Fredrickson Takeda. E., and Eble. J. N. Biochemical programs of slowly and rapidly (eds.), The Metabolic Basis of Inherited Disease, Ed. 4, pp. 821-822. New growing human colon carcinoma xenografts. Cancer Res., 41: 854-859, York: McGraw-Hill Book Co., 1966. 1981. 134. Zhen, Y.-S., Lui, M. S., and Weber, G. Effects of acivicin and dipyridamole 109. Weber, G., Henry, M., Wagle, S. R., and Wagle, D. S. Correlation of enzyme on hepatoma 3924A cells. Cancer Res., 43:1616-1619,1983. 135. Zleve, G. W. Two groups of small stable RNA's. Cell, 25: 296-297, 1981. activities and metabolic pathways with growth rate of hepatoma. Adv. En zyme Regul., 2. 335-346,1964. 136. Zubrod. C. G. Chemical control of cancer. Proc. Nati. Acad. Sei. U. S. A. 69. 110. Weber, G., Jackson, R. C., Williams, J. C., Goulding, F. J., and Eberts, T. J. 1042-1047, 1972.

3492 CANCER RESEARCH VOL. 43

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George Weber

Cancer Res 1983;43:3466-3492.

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