George Weber

Malignant transformation-linked imbalance: decreased xanthine oxidase activity in hepatomas.

Xanthine oxidase was decreased 2- to 10-fold in all examined rat hepatomas irrespective of the malignancy; growth rate and degrees of histological differentiation of the neoplasms. The affinity to substrate (KM=6-8 muM) and the pH optimum (8.0) of the liver and hepatoma enzymes were the same. The reprogramming of gene expression, as manifested in the decreased activity of this key purine metabolizing enzyme, appears to be specific to neoplastic transformation. Since glutamine PRPP amidotransferase activity was increased but the opposing enzyme, xanthine oxidase, was decreased in all the hepatomas, the reprogramming of gene expression results in an imbalance that favors synthesis against catabolism. This enzymatic imbalance should confer selective advantages to the cancer cells.

Molecular mechanisms in the antiproliferative action of quercetin

A single treatment with quercetin (5.5 microM), a plant flavonoid, activated both apoptosis and differentiation programs in K562 human leukemia cells. K562 cells expressed commitment to apoptosis after 1 h exposure, however, at least 12 h of drug exposure was needed to induce differentiation. Early (1 h) down-regulation of the c-myc and Ki-ras oncogenes and rapid reduction of inositol-1,4,5-trisphosphate (IP3) concentration (IC50 = 9 microM, 1 h incubation) are part of the antiproliferative action of quercetin and appear to relate to induction of differentiation and/or apoptotic program of K562 leukemia cells treated with quercetin.

Synchronous behavior pattern of key glycolytic enzymes: Glucokinase, phosphofructokinase, and pyruvate kinase

Abstract In thyroid hormone deficient rats, the hepatic concentration of FAD was significantly reduced. This reduction appeared to be due to decreased activity of hepatic flavokinase, which was also observed under these conditions. Administration of large doses of riboflavin to hypothyroid rats restored the concentration of FAD to normal, probably by producing greater saturation of the enzyme with its substrate. Riboflavin also had a slightly stimulatory effect upon flavokinase activity. Similarities between riboflavin deficiency and thyroid hormone deficiency were noted with respect to the hepatic levels of FAD, the activity of several flavoprotein enzymes, and the response to correction of the deficiency. The feature common to both conditions appears to be reduced formation of FAD from riboflavin. In hyperthyroid animals, hepatic FAD levels were not increased above normal, despite a substantial increase in flavokinase activity. The results provide further evidence for an apparent upper limit in the hepatic concentration of FAD. The increase in flavokinase produced by thyroxine, like increases in activity of certain flavoprotein enzymes, was not blocked by treatment with actinomycin-D.

Role of ribonucleotide reductase in expression of the neoplastic program

Abstract Evidence is presented for the tight linkage of ribonucleotide reductase activity with normal and neoplastic proliferation. A sensitive and reproducible assay was worked out to measure CDP reductase activity in rat in normal liver and various tissues, hepatomas of different growth rates, kidney tumors and sarcoma and tissue culture cells of hepatoma 3924A. In the standard assay, linear kinetics were obtained and the reductase activity of the rat liver was 23 ± 3 pmol CDP metabolized per hr/mg protein. When hepatoma 3924A tissue culture cells that had accumulated in plateau phase were replated, allowed to go through lag and log phases and again into the plateau phase during a 96-hr period, ribonucleotide reductase activity rose at 6 hr after cells were plated, the activity was maintained at high levels during the first 48-hr period, and returned to the resting level at 72 and 96 hr. This rise was earlier than that of 6 other enzymes of pyrimidine de novo and salvage pathways (thymidine kinase, CTP synthetase, orotidine-5′-phosphate decarboxylase, orotate phosphoribosyltransferase, uridine phosphoribosyltransferase, and uridine-cytidine kinase). The rise in reductase activity was synchronous with the increase in incorporation of cytidine and deoxycytidine in the hepatoma cells. The reductase activity was markedly elevated in kidney tumors (31-fold) and in sarcoma (60-fold) as compared to the kidney cortex and muscle, respectively. In 14 lines of transplantable solid hepatomas, reductase activity was increased from 6.2- to 326-fold of that of normal rat liver. The rise in reductase activity positively correlated with the growth rate of the hepatomas; the behavior of CDP reductase was both transformation- and progression-linked. Reductase activity was also high in differentiating and regenerating liver; thus, it also was linked with normal proliferation. However, the elevation in activity was more marked in the rapidly-growing solid hepatoma 3924A (97-fold) than in normal tissues with the same replicative rate, such as regenerating (56-fold) or differentiating (46-fold) liver. Reductase activity was also high in organs of active cell renewal (thymus, bone marrow, spleen and intestine). Since in the solid hepatomas the levels of the substrate for the reductase, the ribonucleoside diphosphates, were generally unaltered, the marked elevation observed in the concentration of deoxynucleoside triphosphates may be attributed primarily to the early and marked rise in CDP reductase activity.

Feedback Inhibition of Key Glycolytic Enzymes in Liver: Action of Free Fatty Acids

Increasing concentrations of sodium octanoate were progressively inhibitory to the activities of glucokinase, hexokinase, phosphofructokinase, and pyruvate kinase. Glucose-6-phosphate and 6-phosphogluconate dehydrogenases were also markedly inhibited. Other enzymes of carbohydrate metabolism such as lactate dehydrogenase, phosphohexose isomerase, and fructose-1,6-diphosphatase were not decreased. Among the key glycolytic enzymes, the inhibition of pyruvate kinase by the fatty acid was most marked. The biological significance of the inhibition of the key glycolytic enzymes is interpreted as a feedback inhibitory mechanism in regulation of fatty acid biosynthesis. The mechanism may function for rapid adaptation by which the organism can use the fatty acid level as a metabolic directional switch in decreasing glycolysis and turning on gluconeogenesis.

Regulation of enzymes involved in gluconeogenesis

Summary 1. A simplified scheme of gluconeogenesis was discussed in terms of the sequence of physiological reactions. The enzymes involved in gluconeogenesis were compared as activities expressed on a uniform basis. 2. In studying the effects of cortisone it was shown that the extent of induction of hepatic glucose-6-phosphatase activity was inversely related to the weight of the rats. In contrast, fructose-1,6-diphosphatase activity was increased to the same extent in all weight groups. 3. In dose response studies with cortisone, easily measurable enzyme increases were accomplished with doses of 2.5 mg and optimum induction was achieved with a dose of 10 mg/100 g rat. The potency of equal doses of cortisone, hydrocortisone, medrol and triamcinolone was compared and it was found that triamcinolone was the most effective in increasing gluconeogenic enzyme activities and liver glycogen and nitrogen levels. 4. In dose response studies with triamcinolone it was found that the minimum effective dose was 0.25 mg and optimum induction was achieved by 1 mg/100 g rat. 5. Time sequence studies demonstrated that a single injection of triamcinolone was capable of causing the continued synthesis of gluconeogenic enzymes for 6 days. 6. In short-term experiments it was possible to achieve statistically significant rises in 2–4 hr for both gluconeogenic enzymes by the use of triamcinolone. Cortisone was also capable of inducing a rise in fructose1,6-diphosphatase activity in 4 hr, but this hormone failed to increase hepatic glucose-6-phosphatase activity in 24 hr. 7. By the use of actinomycin and puromycin blocking of the cortisoneinduced increases, evidence was provided that the corticosteroid-induced increases in hepatic gluconeogenic enzymes were due to de novo protein synthesis. 8. Since the markedly increased activities of gluconeogenic enzymes in acute alloxan diabetes were inhibited by ethionine, puromycin, and actinomycin it was concluded that these enzyme increases were also due to de novo synthesis. 9. The protein synthetic inhibitors failed to block the increased levels of gluconeogenic enzymes in chronic alloxan diabetes, whereas the inhibitors were effective in animals chronically injected with cortisone. It was suggested that the difference between chronic diabetic and chronic cortisoneinjected rats lies in the fact that the diabetic rats had no insulin. 10. It was shown that insulin was able to inhibit the corticosteroidinduced increases in gluconeogenic enzyme activities. In sequence studies when animals were injected with insulin concurrently with triamcinolone administration over a 4-day period there was an immediate and sustained inhibition by insulin of the increases induced by triamcinolone in the activities of gluconeogenic enzymes and in nitrogen content. 11. In chronically diabetic rats insulin administration decreased liver glucose-6-phosphatase activity to normal in 6 days and fructose-1,6-diphosphatase activity in 10 days. Thus, insulin was capable of interfering with the synthesis of gluconeogenic enzymes in chronic alloxan diabetes, resulting in an inhibition which could not be accomplished by actinomycin, puromycin or ethionine. 12. The presented evidence is treated in the light of a tentative working hypothesis suggesting that insulin may act as the physiological repressor of a sequence of gluconeogenic enzymes in the liver.

Regulation of gluconeogenesis and glycolysis: Studies of mechanisms controlling enzyme activity

Abstract Various regulatory mechanisms functioning at the enzyme activity and enzyme biosynthetic level in carbohydrate metabolism were explored. The regulatory influences operate on receptor sites on the enzyme molecules and on the enzyme-forming systems. These enzymatic control mechanisms were examined as the basis for rapid and chronic regulation of gluconeogenesis and glycolysis by hormones and regulatory signal molecules. This work presented the effects of NADH, free fatty acids, and other compounds on strategic enzymes of liver carbohydrate metabolism. The selective action of free fatty acids was characterized in terms of inhibition of key enzymes of glycolysis, and the direct oxidative pathway, and of certain enzymes in the Krebs cycle. Since liver pyruvate kinase is pivotal in the regulation of gluconeogenesis and glycolysis, detailed attention was given to the effects of fructose 1,6-diphosphate, ATP, ADP, NADH, magnesium, free fatty acids, and other regulatory molecules on this enzyme.

Insulin: Inducer of Pyruvate Kinase

The hepatic pyruvate kinase activity markedly decreased when rats were made diabetic by alloxan injection. Insulin treatment induced new synthesis of pyruvate kinase which was prevented by injection of ethionine and actinomycin D. The evidence indicated that the increased pyruvate kinase activity induced by inisulin entails at a certain stage a stimulation of the synthesis of certain RNA species relevant to the production of this enzyme.

Enzymic imbalance in serine metabolism in human colon carcinoma and rat sarcoma

The activities of 3-phosphoglycerate dehydrogenase, an enzyme of serine biosynthesis, and serine hydroxymethyltransferase, serine dehydratase and serine aminotransferase, which are competing enzymes of serine utilization, were assayed in human colon carcinomas from patients and in transplantable rat sarcomas. Serine dehydratase and serine aminotransferase activities were absent, whereas 3-phosphoglycerate dehydrogenase and serine hydroxymethyltransferase activities were markedly increased in both tumour types. Serine hydroxymethyltransferase catalyses the formation of glycine and methylene tetrahydrofolate which are important precursors for nucleotide biosynthesis. The observed enzymic imbalance in these tumours ensures that an increased capacity for the synthesis of serine is coupled to its utilisation for nucleotide biosynthesis as a part of the biochemical commitment to cellular replication in cancer cells. That this pattern is found in sarcomas and carcinomas, and in tumours of human and rodent origin, signifies its universal importance for the biochemistry of the cancer cell and singles it out as a potential target site for anti-cancer chemotherapy.

Control of gene expression in carbohydrate, pyrimidine and DNA metabolism.

Summary The patterns of behavior of key enzymes in carbohydrate and nucleic acid metabolism were investigated under conditions involving modulation of gene expression. 1. The changes in hepatic cellularity and DNA content were examined in the liver during post-natal differentiation. The cellularity was 650 million cells per g wet weight in the liver of the newborn rat and it decreased during differentiation to 220 million in the adult. The total liver cellularity was 168 million in newborn rats and it increased 14-fold to reach a cell population of over 2 billion in the adult. The DNA content per g liver wet weight and per total liver underwent a similar order of magnitude of change. Thus, the DNA content per cell remained unaltered during differentiation. It was suggested that the enzymatic and metabolic changes in differentiation entail sequential alterations in the read-out of nuclear genes and in their selective transcription and translation. These processes then determine the phenotypic pattern of metabolism in liver cells in different age groups. 2. During post-natal differentiation the key enzymes of liver carbohydrate metabolism follow a characteristic pattern of behavior that is in good agreement with predictions that can be deduced from the Functional Genic Unit Concept. After birth, the activities of the key gluconeogenic enzymes are high whereas the key glycolytic ones are low. During subsequent development for a period of about 30 to 40 days the key gluconeogenic enzymes decrease and the key glycolytic ones increase to achieve the activity levels observed in the adult rat. 3. The opposing enzymes of gluconeogenesis and glycolysis reach a steady state balance in the adult; however, as it has been shown it is possible to markedly modulate gene expression by altering the endocrine status of the organism. When rats were made alloxan diabetic, the liver glucose-6-phosphatase activity increased and the pyruvate kinase activity decreased. Administration of insulin caused the activity of the gluconeogenic enzyme to decrease and that of the glycolytic one to increase to the normal levels of the liver of the adult rat. Thus, insulin coordinates hepatic metabolism, in part, through causing an antagonistic behavior for the opposing gluconeogenic and glycolytic enzymes. 4. In neoplasia a characteristic behavior pattern is also seen which agrees with the predictions of the Functional Genic Unit Concept. In the spectrum of hepatomas of different growth rates, in parallel with the rise in tumor proliferation rate, the key glycolytic enzymes increased, whereas the key gluconeogenic ones decreased. The ratios of the opposing key enzymes, e.g., hexokinase/glucose-6-phosphatase, correlate closely with hepatoma growth rate. Thus, the expression of genes for the key enzymes of carbohydrate metabolism is linked with the expression of the potential for cell proliferation rate in the different lines of hepatomas. 5. The examination of the pattern of gene expression and its regulation revealed that the behavior of the key gluconeogenic and glycolytic enzymes exhibits an antagonistic pattern during differentiation, under hormonal regulatory conditions and in neoplasia. The behavior of the activities of the overall pathways of gluconeogenesis and glycolysis is in close accord with that of the key enzymes of these pathways under the same conditions of gene regulation. 6. In normal liver in UMP metabolism the pathway of catabolism is more active than the synthetic pathway that produces UMP. In the spectrum of hepatomas it was observed that the key enzymes involved in the synthesis of UMP increased whereas dihydrouracil dehydrogenase and the overall degradative pathway decreased in parallel with the increase in hepatoma growth rate. Thus, gene expression in pyrimidine metabolism is also coordinated in terms of the antagonistic behavior of opposing pathways and the enzymes involved. 7. The activities of the enzymes of pyrimidine and DNA metabolism were recalculated from the literature and expressed in a uniform fashion for comparison in a Table. The activities of enzymes in the synthetic processes were very low, especially those that play a role at strategic parts of the pathway, e.g., DNA polymerase, ribonucleotide reductase, dTMP synthase and dTMP kinase. In contrast, the activities of enzymes that function in the degradation of uridine and thymidine were several magnitudes higher than those of the synthetic enzymes. 8. In hepatic thymidine metabolism the behavior of the activity of the synthetic utilization (TdR to DNA) contrasts with that of the degradative pathway (TdR to CO 2 ). In the newborn rat the degradative pathway is active and during development the activity increases 2-fold to the high levels of the adult. The activity of TdR to DNA is very high in the liver of the newborn rat and during differentiation it decreases to less than 10% of this value in the adult. In the regenerating liver the synthetic pathway is markedly increased and the catabolic pathway is decreased. In differentiation and in regeneration the antagonistic behavior of the two opposing pathways of thymidine is closely reflected in that of the activities of the enzymes involved. These results suggest the operation of a coordinated behavior pattern of gene expression manifesting opposing changes in the activities of antagonistic pathways and enzymes in thymidine metabolism. 9. In the hepatomas the TdR to DNA pathway increased and concurrently the TdR to CO 2 pathway decreased in parallel with tumor growth rate. Consequently, there was a 140,000-fold increase in the ratio of the two pathways in the rapidly growing hepatomas. In the neoplastic cells the behavior of the opposing pathways of thymidine utilization and the enzymes involved exhibits a mirror picture. 10. The pattern of the activities of the opposing pathways in carbohydrate, pyrimidine and DNA metabolism suggests a close linkage in the expression of the replicative potential of the genome with the extent of the progressive imbalance in these metabolic pathways. These observations provide further support for the validity of the Molecular Correlation Concept in the hepatoma system. The applicability of this Concept has also been shown for kidney tumors, mammary neoplasms and other tumors of different growth rates indicating that the approaches of the Molecular Correlation Concept are valid not only for the hepatomas, but also for various other types of neoplastic cells.