Robert C. Jackson

Biochemical characterization of a factor released by macrophages

Abstract Peritoneal macrophages from the mouse are able to release a factor, which inhibits the incorporation of [ 3 H]thymidine into the DNA of lymphocytes. The biochemical characterization of this factor reveals that this factor is thymidine, a degradation product of cells dying in culture.

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

Abstract 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.

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

Abstract 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.

Guanosine-5′-phosphate synthetase and guanosine-5′-phosphate kinase in rat hepatomas and kidney tumors

The behavior of the activities of GMP synthetase (xanthosine-5-phosphate: L-glutamine amino-ligase(AMP-forming),EC 6.3.5.2) and GMP kinase (ATP: (d)GMP phosphotransferase,EC 2.7.4.8) was elucidated in cytosol preparations of rat tissues, including fetal, neonatal and regenerating liver, in a transplantable kidney tumor, and in a spectrum of 11 hepatomas of different growth rates. GMP kinase activity was 60-fold or more higher than GMP synthetase activity in all of the examined tissues. GMP synthetase activity was increased in all hepatomas and in the kidney tumor, compared to control tissues, reaching 5.5-fold the normal liver values in the most rapidly growing hepatoma. This increase correlated with the tumor growth rates. GMP kinase activity showed no consistent pattern of alteration in the tumors. In both fetal and neonatal rat liver the activity of GMP synthetase was 2.5-times higher than in livers of adult rats, but GMP kinase activity did not change markedly during liver development. After partial hepatectomy GMP synthetase activity was elevated, reaching a peak of 155% of the sham-operated control values by 36 h after the operation. GMP kinase activity was not affected by partial hepatectomy. After 3 days starvation hepatic GMP kinase activity decreased slightly faster than total cytosol protein, while GMP synthetase activity was preferentially maintained. These results indicate that GMP synthetase activity was linked with cellular proliferation in differentiating, regenerating and neoplastic tissues.

Enzyme pattern directed chemotheraphy: The effects of combinations of methotrexate, 5-fluorodeoxyuridine and thymidine on rat hepatoma cells in vitro☆

Abstract The effects of methotrexate (MTX), 5-fluorodeoxyuridine (FUdR) and thymidine on the growth of four rat hepatoma lines were examined in vitro. In the rapidly growing 3924A and Novikoff lines, MTX exerted a marked anti-purine effect, as indicated by its continued toxicity in the presence of thymidine. Addition to the slower growing hepatoma lines 8999R and 8999S of 200 μM thymidine was able to sustain growth for 48 hr in the presence of MTX. However, at a lower thymidine concentration (20 μM) the MTX toxicity toward the 8999R and 8999S lines was not prevented, even in presence of hypoxanthine. Similarly, thymidine at 20 μM was able to protect the 3924A and Novikoff cell lines completely from the toxic effect of 10 μM FUdR, but this thymidine concentration did not protect the 8999R and 8999S lines. The failure of thymidine to protect the latter hepatoma lines was attributed to the rapid breakdown of thymidine in these cells. This interpretation was supported by experiments where addition of 5-diazouracil, an inhibitor of the rate-limiting enzyme of thymidine catabolism (dihydrothymine dehydrogenase), to the cultures resulted in an increased degree of rescue from FUdR by 20 μM thymidine. The combination of MTX and FUdR treatment in the slow growing hepatomas showed summation, but the two agents were less than additive in the rapidly growing lines. The results of these drug combination studies are interpreted in the context of the patterns of competing enzymes characteristic of the various tumor cell lines.

Enzyme pattern-directed chemotherapy: Effects of antipyrimidine combinations on the ribonucleotide content of hepatomas

Abstract Using combinations of three antipyrimidine drugs, chosen on the basis of expected antihepatoma selectivity, we examined the effects on pyrimidine ribonucleotide pools in rat hepatomas (Morris 8999 and 3924A) and host livers. d -Galactosamine gave a marked decrease in UTP in hepatomas, 3-deazauridine caused decreases in CTP concentration, and these two agents together gave very pronounced (up to 86 per cent) decreases in hepatoma CTP content. Pyrazofurin caused decreases in UTP in both hepatoma and host liver, but in combination with d -galactosamine, pyrazofurin decreased hepatoma CTP by up to 79 per cent without decreasing the host liver CTP. The combination of pyrazofurin and d -galactosamine also gave a significantly greater depression of UTP levels in hepatoma 8999 than in host liver. Uridine and orotic acid both restored the d -galactosamine-induced depletion of UTP in liver and hepatoma 8999.

Neoplastic transformation-linked alterations in adenylosuccinate synthetase activity

Abstract Adenylosuccinate synthetase has been measured in rats in normal, differentiating, and regenerating liver, transplantable hepatomas of different growth rates, kidney cortex, and a transplantable kidney tumor. The activity was increased 1.6 to 3.7-fold in all the tumors. The activity showed no correlation with the degree of histological or biochemical differentiation of the tumors, nor with their growth rate. Adenylosuccinate synthetase activity in regenerating liver was unchanged and in neonatal liver it was much lower than in adult liver. It is concluded that the ubiquitous increase in the tumors of liver and kidney was linked with the neoplastic transformation.

Synergistic and antagonistic interactions of methotrexate and 1-β-D-arabinofuranosylcytosine in hepatoma cells: The modulating effect of purines

Abstract A 4-hr pretreatment with methotrexate antagonized the cytotoxic effect of subsequent arabinosylcytosine treatment in rat hepatoma cells of lines N 1 S 1 and 3924A, but in the hepatoma line 8999R and the fibroblast line BF5, MTX pretreatment was synergistic with the arabinosylcytosine treatment. Measurement of cellular deoxyribonucleoside triphosphate concentrations showed that in those lines in which antagonism was found the dCTP increased, whereas in the lines where the drugs were synergistic the dCTP pool was decreased. Conversely, dATP levels were high when the drugs were synergistic and low when antagonism was obtained. Although methotrexate pretreatment antagonized arabinosylcytosine in N 1 S 1 and 3924A cells, pretreatment of these cells with the combination of methotrexate plus a purine (either hypoxanthine or 2′-deoxyadenosine) resulted in synergism with arabinosylcytosine. Deoxynucleotide pool measurements showed that methotrexate in combination with either hypoxanthine or 2′-deoxyadenosine increased dATP and decreased dCTP in the N 1 S 1 and 3924A hepatoma cells. In N 1 S 1 cells, pretreatment with 2′-deoxyadenosine alone for 4 hr was synergistic with arabinosylcytosine. It was concluded that elevated dATP pools enhanced arabinosylcytosine cytotoxicity by depleting the dCTP pool, through feedback inhibition of ribonucleotide reductase, thus causing greater inhibition of DNA biosynthesis and greater incorporation of AraCTP into nucleic acid. Methotrexate was synergistic in those cell lines where dATP accumulated and dCTP was decreased, but when methotrexate had a potent antipurine effect dCTP pools increased and arabinosylcytosine was antagonized. The synergistic interaction was more marked at cytocidal drug concentrations than it was at growth-inhibitory doses.

Increase in liver and kidney deoxycytidine kinase activity linked to neoplastic transformation

Abstract Deoxycytidine kinase activity in normal rat liver cytosol was low (0.8 nmol/hr/mg protein); it increased 2–26-fold in 12 lines of chemically-induced, transplantable rat hepatomas of different growth rates. The increased kinase activity correlated positively with the hepatoma growth rate. The kinase activity did not change in the regenerating liver and the activity in the differentiating, neonatal rat liver was similar to values in adult liver. Deoxycytidine kinase activity in 2 chemically-induced, transplantable rat kidney tumors was increased to twice the value found in normal renal cortex. Among 15 normal rat tissues examined the highest kinase activities were observed in thymus, bone marrow and spleen. Of the normal and malignant rat tissues tested, only testis had detectable cytidine deaminase activity.

Increased adenylosuccinase activity in hepatomas and kidney tumors

Abstract Adenylosuccinase activity was measured in adult, neonatal and regenerating liver, in nine transplantable hepatomas of different growth rates, and also in kidney cortex and 2 transplantable kidney tumors in the rat. The activity was increased 1.4 to 2.2-fold in all the tumors, irrespective of the growth rate or the degree of histological differentiation of the neoplasms. Adenylosuccinase activity in the average liver cell of one-day-old rats was 21% of the adult value. In the rapidly-growing regenerating adult liver the enzyme activity did not change. It is concluded that the consistent elevation of adenylosuccinase activity in the tumors was linked with the neoplastic transformation and the increase was specific to neoplasia.