Molecular Correlation Concept Research Articles

Ordered biochemical program of gene expression in cancer cells.

Our introduction of the molecular correlation concept and the key enzyme concept and the use of biologically meaningful tumor models and control systems resulted in the discovery of an ordered pattern of enzymic and metabolic imbalance and the elucidation of the linkage with transformation and progression. We showed that the biochemical and enzymic pattern of alterations was the result of a reprogramming of gene expression that was both quantitative and qualitative and was characteristic to neoplasia, since no similar pattern of imbalance was observed in any of the control normal, regenerating, or differentiating tissues. Important aspects of gene logic were identified. These include demonstration of operation of reciprocal control of activities of opposing key enzymes and antagonistic pathways of synthesis and catabolism in pyrimidine, purine, ornithine, and carbohydrate metabolism and recently in signal transduction. The extent of increase in the activities of key enzymes of pyrimidine and purine biosynthesis related to the absolute activity of the enzymes in resting liver. The qualitative alterations in gene expression included the isozyme shift of key regulatory enzymes. We identified a segment of gene expression that is essential for neoplasia. We pointed out the selective advantages that reprogramming of gene expression confers to cancer cells. Understanding these alterations in the enzymology and biochemistry of cancer cells made it possible to identify potentially sensitive targets for anticancer chemotherapy. In recent clinical studies we targeted the increased IMP dehydrogenase activity in leukemic blast cells by an inhibitor drug, tiazofurin, and achieved 77% responses, including complete remissions.

Targets and markers of selective action of tiazofurin

1. 1. The molecular correlation concept proposed that IMP dehydrogenase activity should be a sensitive target of chemotherapy. This hypothesis received support from an array of evidence. IMP dehydrogenase has the lowest activity in purine biosynthesis; it is the rate-limiting enzyme in GTP production; the enzymic activity is transformation- and progression-linked; it is elevated in all examined animal and human neoplastic cells. The activity of GMP synthetase and the concentrations of GMP and dGTP were increased in cancer cells. Whereas guanine salvage has a high potential activity, the low guanine content may well curtail actual salvage capacity. Ribonucleotide reductase activity was two orders of magnitude lower than that of IMP dehydrogenae. 2. 2. Tiazofurin, a C-nucleoside, had marked cytotoxicity on hepatoma cells in vitro and was the first drug that as a single agent profoundly inhibited the proliferation of the subcutaneously inoculated solid hepatoma 3924A in the rat. 3. 3. The impact of tiazofurin administration in hepatoma cells was revealed in a cascade of biochemical alterations involving primary, secondary and tertiary targets and markers of this drug action. The primary target was IMP dehydrogenase where the active metabolite of tiazofurin, TAD, was thought to be absorbed to the NADH site of the enzyme. As a consequence, the enzymic activity declined rapidly to about 30–40% and returned to normal range by 36 to 48 hr after injection. The secondary targets and markers are the profoundly decreased pools of guanylates (GMP, GDP, GTP). Concurrently, the concentrations of IMP and PRPP were increased 8- to 15-fold. The elevated IMP pools were attributed to the de-inhibition of the AMP deaminase activity subsequent to the decline in GTP concentration. The rise in PRPP pools was attributed to the selective inhibition of GPRT and HPRT activities by the high IMP pool which did not affect APRT activity. This interpretation is supported by the 6- to 8-fold increase in the concentrations of guanine and hypoxanthine and the lack of change in the adenine pools in the hepatomas after tiazofurin administration. The marked drop in NAD concentration which was drug dose- and time-dependent is attributed to the competition for NAD pyrophosphorylase activity by the precursors of NAD and tiazofurin monophosphate. The tertiary targets were dominated by the profound alterations in the concentrations of the dNTPs. This was characterized by a rapid and persistent drop (for 3 days) of the dGTP pool. The concentrations of dATP and dCTP also declined, but these alterations were less pronounced and the pools returned to normal after 2 days. In contrast, dTTP concentrations were continuously increased. The decline in the dNTP pool is attributed to desaturation by the decreased GDP concentration of ribonucleotide reductase. The increased dTTP concentration is attributed to the thymidine kinase activity which is highly elevated in the hepatoma. This interpretation is supported by the presence of markedly enlarged pools of thymidine and thymine in the hepatomas of tiazofurin-treated rats. 4. 4. The determinants of selectivity of tiazofurin action in hepatoma and liver entail the high concentrations of TAD observed in the hepatoma after tiazofurin injection. This is attributed to the high ratios of the activities of NAD pyrophosphorylase/phosphodiesterase in the tumors. Moreover, the hepatomas had low concentrations of NAD which decreased further after tiazofurin injection; there was no change in the liver. The IMP concentration in the hepatoma was markedly lower than that of the liver. The biochemical response to tiazofurin was much more pronounced in the hepatoma, whereas in the liver there was no change in the concentrations of guanylates and only a minor increase in IMP concentration. 5. 5. The results emphasize the differences in the biochemical phenotypes and responsiveness to tiazofurin of sensitive and non-sensitive tissues. The identification to tiazofurin of sensitve and non-sensitive tissues. The identification of targets and markers of tiazofurin suggest that for rapid screening of tiazofurin sensitivity the most meaningful indicators should be the drug-induced elevations in the concentrations of TAD and in the ratios of IMP/GTP or IMP/dGTP. Further indicators include a markedly depressed NAD concentration and a strongly elevated PRPP pool in the sensitive tissues after tiazofurin treatment.

Increased activity of thymidine kinase isozyme in human colon tumor.

Thymidine kinase (TK), the salvage pathway enzyme, and its isozymes were examined in normal mucosa and carcinoma of the colon in 13 patients. In colon tumor TK activity increased to 328% of activity of normal colon. The colon TK isozymes were separated from the normal mucosa and carcinoma by DEAE-cellulose column chromatography. The TK isozyme eluted from the column by the elution buffer alone without NaCl was markedly higher (12-fold) in activity in carcinoma than in normal mucosa of the colon and is not affected by dCTP. The TK isozyme unaffected by dCTP has also been reported to be clearly involved in DNA synthesis. This isozyme, whose molecular weight is 100 000 by means of high performance liquid chromatography, is thought to be involved in DNA replication. The results indicate the applicability of the molecular correlation concept to carcinogenesis of colon.

Enzymes of purine metabolism in cancer

In cancer cells, a marked imbalance in the enzymic pattern of purine metabolism is linked with transformation and/or progression. In chemically-induced, transplantable hepatomas in rat, the specific activities of the anabolic enzymes, IMP dehydrogenase, GMP synthetase, adenylosuccinate synthetase, adenylosuccinase, AMP deaminase and amidophosphoribosyltransferase, increased to 13.5-, 3.7-, 3.1-, 1.8-, 5.5- and 2.8-fold, respectively, of those in normal liver. Activities of the catabolic enzymes, inosine phosphorylase, xanthine oxidase and uricase, decreased to 19, 10 and 4%, respectively. This enzymic imbalance was specific to hepatic neoplasia, since no similar pattern was observed in differentiating or regenerating liver. Most enzymic alterations were present also in chemically- and virus-induced animal tumors, in human kidney, liver and colon carcinomas, and in human colon carcinoma xenografts. The molecular correlation concept applies to purine biochemistry and an important segment of neoplastic gene expression was identified in the behavior of key purine-metabolizing enzymes.

Biochemical strategy of hepatomas

An insight into the ordered pattern of enzymatic and biochemical imbalance of cancer cells was made possible, at least in part, by the molecular correlation concept, the concept of key enzymes, and the use of biological model systems. With these approaches the pattern of gene expression in neoplasia can be studied in terms of the activities of key enzymes and their linking with neoplastic transformation and progression. An ordered pattern of biochemical imbalance was recognized in carbohydrate, pentose phosphate, purine, pyrimidine, DNA, and polyamine metabolism and in other biochemical areas. Current work is directed to clarifying the enzymology and metabolic pattern of thymidine metabolism and CTP biosynthesis since these areas are of particular importance in selective chemotherapy and rescue. The activities of key enzymes in thymidine metabolism have been correlated with the growth rates in a spectrum of hepatomas. Increases in the activities of four key enzymes in CTP biosynthesis appear to be specific to neoplasia because no similar pattern was observed in the normal adult resting or regenerating liver or in the fetal and developing liver. The overall pattern discovered in transplantable rat hepatomas applies to other rodent tumors. It is of particular importance that the pattern of biochemical imbalance is also applicable to human hepatocellular carcinomas. With the recognition of the ordered pattern of reprogramming of gene expression in hepatomas, the path is open for the development of sensitive assays for biochemical diagnosis of liver tumors and for a rational design of selective chemotherapy and rescue.

Biochemical behavior of MC‐29 virus‐induced transplantable chicken hepatoma

Studies were performed to test the applicability of the molecular correlation concept developed in chemically induced transplantable rat hepatomas to virally induced transplantable hepatoma in chicken. In a comparison of the activities of carbohydrate, purine, and pyrimidine key enzymes in rat and chicken hepatomas, similar patterns were observed in rodent and avian tumors. These results show that the enzymatic imbalance elucidated in chemically induced rat hepatomas is applicable to the virus-induced hepatoma in chicken, independent of the nature of the carcinogenic agent and the species.

Degradation of Pyrimidines — Enzymes, Localization and Role in Metabolism

Summary Pyrimidines can be degraded (1), reductively; (2), oxydatively by attack of oxygen in the position 5 or 6 of the pyrimidine ring; (3), by oxydative demethylation of thymine; (4), by decarboxylation of orotic acid to uracil; (5), by reductive degradation of orotic acid. The reductive degradation is the prevailing degrading process in nature. It is catalyzed by uracil reductase, dihydropyrimidinase and β-ureidopropionase. Properties and location of enzymes are described. Distribution and significance of pyrimidine degradation in different organisms are presented. Its presence in animals is preferentially discussed with respect to correlation between anabolic and catabolic reactions in terms of the “Molecular Correlation Concept”. Some plants use reverse reactions of pyrimidine degradation for biosynthesis of some pyrimidine-containing secondary products.

Enzymatic markers of neoplastic transformation and regulation of purine and pyrimidine metabolism

Enzymes that were identified as transformation-linked in the spectrum of hepatomas of different growth rates in the rat were used as markers of malignancy for liver tumors in rat and in man. The applicability of the enzymatic pattern was examined in kidney tumors of the rat, in lymphomas of the mouse and in hypernephroma-type kidney tumors in human. The impact of altered gene expression, as manifested in the enzymic imbalance, was further investigated by determining the concentration and behavior of purine and pyrimidine ribonucleotides in normal and neoplastic liver. The activity of transformation-linked enzymes was also studied in different organs of the rat and mouse to elucidate whether the activity might prove to be a predictor of the changes in activity observed in the different neoplasms. 1. 1.|In the hepatoma spectrum AMP deaminase and CTP synthetase activities were increased in all examined tumors and the rise correlated positively with the growth rate of the neoplasms. 2. 2.|A comparison of the in vivo concentrations (0-min sample) of ribonucleotides revealed that the level of adenine nucleotides was lower in rapidly-growing hepatoma 3924-A than in normal control liver. A further outstanding difference in ribonucleotide levels is the 5-fold increase in CTP content along with an overall elevation of the concentration of cytidine nucleotides. This biochemical imbalance in the hepatoma is attributed, in part at least, to the increased activity of UDP kinase and CTP synthetase in these tumors. 3. 3.|When ischemia was imposed, in normal liver there was a breakdown of the adenine, uridine, guanine and cytidine nucleotide triphosphates and a concurrent rise in the nucleoside monophosphate contents. It was particularly striking that the level of IMP increased 75-fold in 10-min ischemia. In contrast, no change was observed on ischemia in the hepatoma. When the hepatoma was injected with iodoacetamide and then subjected to ischemia the nucleoside triphosphates decreased as in the normal liver. 4. 4.|In measuring the activities of key carbohydrate, pentose phosphate, purine and pyrimidine enzymes we observed that frequently the high activity observed in the rat thymus was a predictor that the enzyme activity would be markedly increased in the rapidly growiing tumor. This relationship did not hoold without exception and did not apply to the mouse. 5. 5.|The integrated pattern of enzymatic alternations observed in rat, mouse and human neoplasms is specific to cancer cells, as no similar enzyme activity pattern was observed in the various normal organs and tissues examined in rat, mouse and human. 6. 6.|The present work demonstrates the applicability of the molecular correlation concept and much of the altered biochemical pattern discovered in the rat hepatoma spectrum to other tumors in rat, to lymphomas in mice and to primary kidney tumor in humans. Thus, increased activities of pyruvate kinase, glutamine PRPP amidotransferase, IMP dehydrogenase, AMP deaminase, adenylosuccinase and UDP kinase and decreased activity of xanthine oxidase were markers of malignancy in the three mouse tumors tested, plasma cell myeloma, Mecca lymphosarcoma and P1534 lymphocytic leukemia. In 15 cases of human hypernephromas, hexokinase and pyruvate kinase activities increased whereas those of glucose 6-phosphatase and fructose 1,6-diphosphatase decreased. Activities of glucose 6-phosphate dehydrogenase and glutamine PRPP amidotransferase increased, whereas that of xanthine oxidase decreased. IMP dehydrogenase, adenylosuccinase and AMP deaminase activities were elevated. Thus, these enzymes could be used in the biochemical diagnosis of human primary kidney tumors of the hypernephroma type. The biochemical imblance is similar to that observed in transplanted hepatomas and kidney tumors in the rat. 7. 7.|The applicability of the enzymatic imbalance observed in rodents to human tissue underlines the importance of such enzymatic markers in neoplastic transformation and these enzymes should be targets in the design of selective chemotherapy of neoplastic diseases.

Key enzymes of IMP metabolism: Transformation- and proliferation-linked alterations in gene expression

The purpose of this investigation was to test the concept that key enzymes of IMP synthesis, degradation and utilization may show characteristic patterns in proliferative conditions. In the synthetic pathway of IMP the first enzyme committed to de novo purine biosynthesis, glutamine PRPP amidotransferase, was measured. In the pathway of IMP catabolism the rate-limiting enzyme, xanthine oxidase, and uricase, the final enzyme, were examined. In the synthetic utilization of IMP, IMP dehydrogenase, the enzyme involved in channeling IMP into the production of XMP and adenylosuccinate synthetase, the enzyme channeling IMP into SAMP biosynthesis, were studied. Investigations were also carried out on GMP synthetase and adenylosuccinase. The enzymes of IMP metabolism were examined in normal and neoplastic liver proliferation, such as in differentiating and regenerating liver and in a spectrum of hepatomas of different growth rates. The following main observations were made. 1. 1.|In differentiating liver the specific activities of IMP dehydrogenase and amidotransferase were high after birth and decreased during development to the levels observed in the adult rat liver. In contrast, the adenylosuccinate synthetase and xanthine oxidase activities were low after birth and rose to the adult levels during development. 2. 2.|In a spectrum of hepatomas of different growth rates the specific activity of IMP dehydrogenase was 2- to 3-fold increased in the slow growing hepatomas and it increased in parallel with tumor growth rates, reaching 12- to 13-fold increases in the most rapidly growing neoplasms. The behavior of IMP dehydrogenase belongs to Class 1 as grouped by the molecular correlation concept. 3. 3.|The activities of adenylosuccinate synthetase and glutamine PRPP amidotransferase were increased in all hepatomas to approximately 2- to 4-fold, and those of the adenylosuccinase 1.2- to 2-fold of the levels observed in the corresponding normal rat livers. In contrast, the activities of the catabolic enzymes, xanthine oxidase and uricase, were decreased in all hepatomas to 2- to 10-fold of the activity of the relevant control rat livers. The ratios of amidotransferase/xanthine oxidase were markedly increased in all hepatomas and they reached 20- to 27-fold increases in those of rapid growth rate. The behavior of adenylosuccinate synthetase, adenylosuccinase, amidotransferase, xanthine oxidase and uricase belongs to Class 2 of the molecular correlation concept where enzymes are grouped that exhibit alterations that occur in all hepatomas irrespective of growth rate and degree of differentiation. 4. 4.|Since the alterations in the activities of adenylosuccinate synthetase, adenylosuccinase, amidotransferase, xanthine oxidase and uricase are expressed in all the hepatomas, even in the slowest growing, most liver-like tumors, this reprogramming of gene expression appears to be linked with the malignant transformation per se. However, these enzyme activities do not signal a linkage with the degrees in the expression of malignancy and growth rate. In contrast, the activity of IMP dehydrogenase is an indicator both of malignant transformation and growth rate. 5. 5.|In the regenerating liver IMP dehydrogenase specific activity rapidly increased to 500% in 6 to 18 hours; it slowly returned towards normal range in 96 hours after operation. Amidotransferase specific activity reached a peak of 165% of the sham-operated value at 48 hours after operation and then returned to normal range by 96 hours. The activities of SAMP synthetase, adenylosuccinase, xanthine oxidase and uricase did not change in regeneration. Thus, IMP dehydrogenase appears to be the earliest enzyme to rise in the regenerating liver, whereas the elevation of amidotransferase is a delayed and a minor one. The increase in activities of these two enzymes in the regenerating liver is in the range of the activity observed in the newborn rat liver. However, the activities are markedly lower than those observed in the very malignant hepatomas that exhibit the same growth rate as the regenerating liver. 6. 6.|The discriminating power of the biochemical pattern of enzymes of purine metabolism indicates that the imbalance in the pattern of alteration of the key enzymes of IMP synthesis, degradation and utilization is specific to malignancy. The pattern of these enzyme activities also provides quantitative and qualitative discriminants for the identification of regenerating and differentiating liver and for distinguishing the enzyme patterns from those observed in normal and rapidly growing neoplastic liver. 7. 7.|These experiments indicate the operation of an antagonistic behavior for the key synthetic enzymes, glutamine PRPP amidotransferase and IMP dehydrogenase, that are high in differentiation, regeneration and neoplasia as compared to the rate-limiting catabolic enzyme, xanthine oxidase, that is low in differentiation and in neoplasia and is unaltered in the regenerating liver. These observations are in line with earlier studies that demonstrated an antagonistic behavior for the opposing key enzymes of gluconeogenesis and glycolysis and of the synthetic and catabolic pathways of the de novo and salvage pathways of pyrimidine metabolism.

IMP dehydrogenase, an enzyme linked with proliferation and malignancy

PREVIOUS studies conducted in this laboratory demonstrated that in a spectrum of liver tumours with different growth rates there is an imbalance in the activities of key enzymes and competing pathways of carbohydrate, pyrimidine, DNA and ornithine metabolism1–4. Recent investigations on purine metabolism showed that in the hepatomas there was an increased capacity in the de novo pathway of biosynthesis of inosine 5′-monophosphate (IMP), as reflected in the increased activity of glutamine PRPP amidotransferase (EC 2.4.2.14) and a decrease in IMP catabolism5,6. These observations directed our attention to the metabolic fate of IMP because this purine nucleotide is at a strategic position in purine metabolism (Fig. 1). Control at such branching points is exerted primarily by modification of the activity or of the rate of synthesis of the first enzymes of the divergent pathways. In this instance, in normal conditions, a balance is maintained by both positive and negative feedback effects. Thus ATP is required for GMP biosynthesis and GTP for AMP biosynthesis; conversely, GMP and AMP each inhibit their own production7. In normal or malignant proliferation, or in response to a hormone stimulus, changes in control frequently involve reprogramming of gene expression. The theoretical framework and predictive value of generalisations about such biochemical changes (termed the molecular correlation concept) have been elaborated1–3.

Increased UDP kinase activity in rat and human hepatomas

PREVIOUS work1–4, (carried out in this laboratory with the molecular correlation concept as a conceptual and experimental approach resulted in an insight into the biochemical strategy of the cancer cell) much of it conducted with a model system of hepatomas of very different growth rates5, has revealed a gradually emerging metabolic imbalance that is linked with the increase in tumour growth rate1–4,6–12. Such biochemical alterations were manifested in progressive changes in the activities of opposing and competing key enzymes and opposing and competing metabolic pathways that correlated closely with tumour growth rate. These alterations in gene expression that seem to be linked with the increase in the expression of malignancy were manifested in increases in the activities of key glycolytic, pyrimidine and DNA synthesising enzymes4,7–12. Concurrently, there were progressive decreases in the activities of the key enzymes of gluconeogenesis, pyrimidine catabolism and the urea cycle4,7,8,10,12. In addition to this ‘malignancy-linked’ (growth-rate-linked) metabolic imbalance, we now show that the reprogramming of gene expression in cancer cells entails ‘transformation-linked’ alterations that are present in all hepatomas irrespective of growth rate or differentiation.

Ordered and specific pattern of gene expression in neoplasia

The present paper outlined the evidence for the specificity of the alterations observed in the neoplastic liver in terms of their ability to provide an integrated system of quantitative and qualitative discriminants that can clearly distinguish the neoplastic liver from the rapidly growing fetal, newborn, adult, or regenerating liver. This examination of the specificity of the imbalance of enzymes and metabolic pathways in hepatomas is based also on studies on the ordered nature of the biochemical pattern in hepatomas of different growth rates that has been documented in earlier publications. The evidence leads to the conclusion that in the model system of hepatomas of different growth rates and in a control system of rapidly growing differentiating and regenerating liver there is a biochemical pattern that is ordered and specific to neoplasia. The results examined in this paper deal with key enzymes and metabolic pathways in carbohydrate, pyrimidine, DNA and ornithine metabolism. Similar considerations should apply to other biological systems and control mechanisms. The application of the conceptual and experimental approaches of the molecular correlation concept to the cyclic AMP system is reported elsewhere.

Control of gene expression in carbohydrate, pyrimidine and DNA metabolism

Control of gene expression in carbohydrate, pyrimidine and DNA metabolism

Carbohydrate metabolism in cancer cells and the molecular correlation concept.

Carbohydrate metabolism in cancer cells and the molecular correlation concept.

The molecular correlation concept of neoplasia

We examined the intermediary metabolism and enzymology of a spectrum of liver tumors of different growth rates. The new results we presented along with those assembled from the literature indicate that a definite pattern of intermediary metabolism does exist. Special stress was laid on the identification of the biochemical parameters which correlate positively or negatively with the growth rate in the hepatomas. With this concept in mind a pattern was identified showing that the synthetic pathways of carbohydrate and lipid metabolism, and the breakdown pathways of protein and of nucleic acid metabolism decrease parallel with the increase in hepatoma growth rate. On the other hand, the catabolic pathway of carbohydrate metabolism increases progressively, whereas in protein and nucleic acid metabolism the anabolic pathways increase gradually parallel with the increase in hepatoma growth rate. An application of our concepts developed for carbohydrate metabolism interprets these findings as examples of antagonism between synthetic and breakdown pathways centering on opposing, key, one-way, rate-limiting reactions. The metabolic parameters and enzyme activities which do not correlate with the growth rate are usually identifiable as non-rate-limiting reactions or enzymes present in an excess. The metabolic pattern identified in the spectrum of liver tumors of different growth rates offers a framework for experimental testing and application of these concepts to other types of tumors and to chemotherapy.