As a consequence of the inhibition of de novo purine synthesis by methotrexate (MTX) there is an increase in 5-phosphoribosyl-1-pyrophosphate (PRPP) concentration. In cells where 5-fluorouracil (FUra) is activated via orotate phosphoribosyltransferase (OPRtase), increased PRPP results in greater conversion of FUra to nucleotides. In the murine CD8F1 breast tumor system, MTX markedly enhances the antitumor activity of FUra, increasing both the activation of FUra to FUMP and the incorporation of FUTP into RNA. However, in contrast to reported tumor tissue culture studies, MTX pretreatment in vivo prevents the stable incorporation of FUra into CD8F1 bone marrow DNA. Pretreatment with MTX (300 mg/kg) 2.5 hr prior to [3H]FUra (100 mg/kg), with a 2-hr labeling, reduced the level of FUra in DNA from 921 pmol to 66 pmol/mg of DNA. Without MTX pretreatment, 59% of the total incorporation of FUra into nucleic acids was into DNA when FUra was administered. After MTX the percentage of incorporation into DNA was reduced to 9%. Additionally, the ratio of [3H]FUra to 32P in DNA when both were given simultaneously was reduced by greater than 90%, suggesting that MTX must be specifically blocking the incorporation of FUra rather than nonspecifically preventing its incorporation by inhibiting DNA synthesis. In contrast, MTX failed to reduce the formation of DNA containing fluorouracil residues from FdUrd. To test whether MTX prevents the initial incorporation of FUra into DNA, or acts to enhance removal by the DNA glycosylase repair system, DNA was prelabeled in vivo with [3H]FUra, and MTX or MTX plus dThd was then administered. The level of FUra in bone marrow DNA was not reduced by subsequent treatment with MTX, or MTX plus dThd, indicating that MTX does not enhance the removal of FUra from DNA. The level of total free fluorodeoxynucleotides formed from FUra was reduced by two-thirds following MTX pretreatment, suggesting that the action of MTX in preventing the stable incorporation of FUra into DNA was to reduce the availability of FdUTP.

The purpose of the present investigation was to determine whether a single bolus intravenous injection (2000 mg/kg) of uridine diphosphoglucose (UDPG) could affect levels of PRPP in a transplanted mammary adenocarcinoma and in liver of CD8FI mice. Six hours following a single intravenous injection of UDPG, 2000 mg/kg, tumor PRPP was lowered to 80 pmol/mg protein, a 53% decrease compared to saline control tumors. Liver was more sensitive than tumor to the 5-phosphoribosyl pyrophosphate (PRPP)-depleting effects of a single bolus intravenous injection of UDPG, since significantly lower levels of PRPP were found in liver, but not in tumor, at doses of 500–1000 mg/kg of UDPG. Maximal depression (30% of saline control) or PRPP occurred in liver 6 hr after intravenous UDPG at 1000–2000 mg/kg. Enhanced levels of UDPG in plasma (half-life less than 10 min) and tumor was detected at 30 min after intravenous UDPG at 2000 mg/kg. There was no detectable increase in endogenous levels of UDPG in liver at this time, probably as a result of rapid metabolism of UDPG by liver. At this same time, a twofold increase in uridine triphosphate (UTP) was measured in liver after intravenously administered UDPG. In contrast, the level of UTP was not increased significantly above control values in tumor. These data suggest the potential use of UDPG to elevate UTP pools in normal tissues in the delayed rescue of cancer chemotherapeutic drugs such as 5-fluorouracil which function as a uridine analogue in these tissues.

5-Phosphoribosyl pyrophosphate (PRPP) is a necessary metabolic intermediate in the synthesis of purine and pyrimidine nucleotides as well as other important molecules. Perturbation of the intracellular level of PRPP has been found to be relevant to the origin of certain metabolic diseases [1, 2]. Too little PRPP leads to a condition that to a limited degree mimics Lesch-Nyhan disease. Too much leads to overproduction of purines and the catabolic end product uric acid, and hence gout.

Uridine diphosphoglucose (UDPG) has been shown to have tissue-specific effects that have proved to be of clinical value in the treatment of some liver ailments. In an effort to determine something about the mechanism of action, we investigated the effect of UDPG on the levels of 5-phosphoribosyl pyrophosphate (PRPP) and PRPP synthetase in mouse liver, spleen and transplanted tumors. Three strains of mice were studied with and without tumors under various experimental conditions. Balb/c mice were infused with UDPG intraperitoneally at levels of 0.16 g/kg/day (0.28 mmole) to 1.6 g/kg/day (2.8 mmoles) for 5 days. At the low dose rate the PRPP level in the liver was found to increase 3-fold. A slight increase was noted in the activity of PRPP synthetase. However, when the UDPG was infused at a level of 2.8 mmoles/kg/day, the increases in both the synthetase and PRPP were inhibited. Both CRF1 and CD8 mice were less sensitive to the effects of UDPG per se. However, the high level of PRPP in the tumors they carried was greatly affected by the UDPG infusion. The tumor-specific inhibition of PRPP suggests that this action might prove to be useful combination therapy with inhibitors of purine and pyrimidine nucleotide synthesis in various rescue regimens. UDPG was found to enter cells intact before it was cleaved into glucose phosphate and UMP. The fact that UDPG was also found in the membrane fraction suggests that either there is a specific transport mechanism or UDPG exerts its action via interaction with the cell membrane.

Two major factors have contributed to a widely held disenchantment with murine tumor models for drug screening in cancer research: (a) the higher costs of these models in comparison to studies performed with tumor cells in vitro; and (b) the perception that these models have failed to demonstrate satisfactory correlation of chemosensitivity with analogous human tumor types; i.e., murine tumors generally have proved to be sensitive to many more agents than are found to be active in the clinic. The perceived failure of the murine models is discussed with particular reference to the difference in criteria used for evaluating drug sensitivity in murine tumor models versus clinical trials, and we conclude that the perception about murine models is not tenable in light of present information. The very important role of murine tumor models in optimizing dosage and administration schedules and, most importantly, in the development of a new drug to its most useful potential in combination chemotherapy is discussed. The value of this in vivo methodology is stressed.

Annals of the New York Academy of SciencesVolume 451, Issue 1 p. 142-149 Adenosine Deaminase and Malignant Cells M. EARL BALIS, M. EARL BALIS Sloan-Kettering Institute New York, New York 10021Search for more papers by this author M. EARL BALIS, M. EARL BALIS Sloan-Kettering Institute New York, New York 10021Search for more papers by this author First published: October 1985 https://doi.org/10.1111/j.1749-6632.1985.tb27105.xCitations: 24AboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onEmailFacebookTwitterLinkedInRedditWechat References 1 Balis, M. E. 1976. Adv. Clin. Chem. 18: 213. 2 Stutman, O. 1983. In Basic and Clinical Tumor Immunology, vol. 1. R. B. Herberman, Ed. Martinus Nijhoff. Boston . 3 Trotta, P. P. & M. E. Balis. 1977. Cancer Res. 37: 2297. 4 Troita, P. P. & M. E. Balis 1978. Biochemistry 17: 270. 5 Nishihara, H., S. Ishikawa, K. Shinkai & H. Akedo. 1973. Biochim. Biophys. Acta 302: 429. 6 Schrader, W. P. & A. R. Stacy. 1977. J. Biol. Chem. 252: 6409. 7 Trotta, P. P. & M. E. Balis. 1980. In Purine Metabolism in Man, part B. A. Rapado, R. W. E. Watts & C. H. M. M. DeBruyn, Eds.: 163. Plenum. New York . 8 Trowa, P. P., R. A. Peterfreund, R. Schonberg & M. E. Balis. 1979. Biochemistry 18: 2953. 9 Herbschleb, V. E., K. J. Ten & K. P. Meera. 1983. Anticancer Res. 3: 95. 10 Smyth, J. F. & K. R. Harrap. 1975. Br. J. Cancer 31: 544. 11 Meier, J., M. S. Coleman & J. J. Hutton. 1976. Br. J. Cancer 33: 312. 12 Chechik, B. E. & J. Minowada. 1979. JNCI 63: 609. 13 Chechik, B. E., W. P. Schrader & P. E. Daddona. 1980. JNCI 64: 1077. 14 Rutzky, L-P. & M. J. Siciliano. 1982. JNCI 68: 81. 15 Hall, J. G., L. Gyure, J. Peppard & E. Orlans. 1979. Br. J. Cancer 40: 751. 16 Grinevich, Y., V. Umansky, L. Kamenets & I. S. Nikolsky. 1984. Neoplasma 31: 21. 17 Umansky, V. & L. Kamenets. 1980. Eksp. Oncol. 2: 47. 18 Belyanchikova, N. I., G. I. Vornovitskaya, S. N. Khramtsova & V. S. Shapot. 1981. Byull. Eksp. Biol. Med. 92: 476. 19 Russo, M., R. Giancane, G. Apice & B. Galanti. 1981. Br. J. Cancer 43: 196. 20 Dinescu-Romalo, G., C. Mihaj & L. Vlad. 1977. Rev. Roum. Biochim. 14: 161. 21 Trotta, P. P., M. P. Ahl, G. B. Brown & M. E. Balis. 1978. Molec. Prarmacol. 14: 199. 22 Tedde, A., M. E. Balis, R. Schonberg & P. P. Trotta. 1979. Cancer Res. 39: 3044. Citing Literature Volume451, Issue1Adenosine Deaminase in Disorders of Purine Metabolism and in Immune DeficiencyOctober 1985Pages 142-149 ReferencesRelatedInformation

Sealed and unsealed plasma membrane vesicles were prepared from human erythrocytes and lymphocytes. Phosphoribosylpyrophosphate synthetase (PRibPP synthetase), hypoxanthine phosphoribosyltransferase (HPRTase), and adenine phosphoribosyltransferase (APRTase) activities are detectable on both inside-out and right-side-out sealed vesicles. Ghost preparations were about 0.2%, 1%, and 1.2% of the total erythrocyte and 0.5%, 5.3%, and 9.7% of the lymphocyte APRTase, HPRTase, and PRibPP synthetase activities. The rapid decrease in these enzyme activities, upon further purification of the membranes, seemed to suggest that they might be loosely bound extrinsic proteins. Evidence confirming the localization of these enzymes on the cell surface was obtained by measuring production of [14C]AMP by intact cells in medium containing [14C]adenine, ribose 5-phosphate, and Mg2+ATP. The formation of AMP was linear with time and number of cells present. Magnesium and phosphate exerted different effects on the production of extracellular AMP than on intracellular, which involves transport as well as phosphoribosylation. Cytosoluble and membrane-bound APRTase and PRibPP synthetase exhibited different catalytic properties and sensitivities to effectors. Membranes of erythrocytes of HPRTase-deficient patients contain little or no HPRTase activity when assayed in the absence of Triton. Reisolation of these membranes from admixture with normal hemolysates did not result in any bound activity; thus, the membrane-bound activity is not an artifact of the isolation procedure. Lysis with Triton released activity equal to about half that of control membranes. This is further evidence that the enzyme is firmly bound to the membrane.

The effect of continuous infusion into C57BL/6J mice of 2'-deoxycoformycin (DCF), a tight-binding inhibitor of adenosine deaminase, on the biological function of bone marrow stem cells and T- and B-lymphocytes was evaluated. Greater than 85% inhibition of adenosine deaminase in erythrocytes, thymus, and bone marrow was noted after DCF infusion at 0.4 mg per kg body weight per day, while lesser extents of inhibition were characteristic of spleen and lymph nodes. The reconstitution of lethally irradiated C57BL/6J mice with bone marrow cells from DCF- and 0.9% NaCl infused mice of the same strain was compared. The two groups of animals were virtually identical with respect to (a) the number of spleen colony-forming units, (b) the response of splenic lymphocytes to both B- and T-cell mitogens, (c) hematological analysis of peripheral blood elements, and (d) survival time, thus strongly supporting the lack of effect of DCF infusion on the capacity of stem cells to differentiate. In contradistinction, DCF infusion was highly lymphocytotoxic as noted by the severe necrosis in both B- and T-cell regions in lymph nodes and spleen and by the dramatic weight reduction in spleen and thymus. Histopathology of other tissues including bone marrow was normal except for the occurrence of hepatitis. A striking decrease in blastogenesis induced by the mitogens concanavalin A, phytohemagglutinin, and Escherichia coli lipopolysaccharides was also observed after DCF infusion. Consistent with these data, in vitro incubation of bone marrow cells with DCF did not impair the number of spleen colony-forming units produced in lethally irradiated mice. These data suggest a potential use for adenosine deaminase inhibitors in the prevention of graft-versus-host disease in hematopoietic transplantation.

5-Phosphoribosyl 1-pyrophosphate synthetase (PRibPP synthetase EC 2.7.6.1) isolated from rat intestinal mucosa was found to be membrane associated. The subcellular distribution of PRibPP synthetase activity seems to parallel that of gamma-glutamyl transpeptidase, indicating it to be in the brush border. The tip cells of rat intestinal mucosa were richer in PRibPP synthetase than the crypt cells. Chromatography of a Triton-solubilized particulate fraction unmasked a peak of hypoxanthine phosphoribosyltransferase activity that was not detectable before. The activity, too, was concentrated in the brush border. The coexistence of these two activities in the fraction of the bowl involved in absorption has led to the suggestin that the synthetase and phosphoribosyl-transferase are part of a coupled transport system.

An in vivo murine model for immunodeficiency of both B and T cells is produced by continuous intraperitoneal infusion of 2'-deoxycoformycin (DCF), a specific tightly binding inhibitor of adenosine deaminase (ADase; adenosine aminohydrolase, EC 3.5.4.4). After DCF infusion, ADase of thymus, spleen, and lymph nodes was inhibited to varying degrees ranging from 57% to 100%. Immunodeficiency under these conditions was indicated by: (i) a striking decrease in lymphocyte response to the T-cell mitogens concanavalin A and phytohemagglutinin; (ii) an impairment of delayed hypersensitivity measured by the footpad reaction; (iii) a decrease in antibody production measured in both in vivo and in vitro plaque-forming cell assay; (iv) a significant prolongation of mouse skin allograft survival after transplantation into the C57BL/6J (H-2b) strain of skin from BALB/c (H-2d) mice; and (v) a marked lymphopenia. Histological examination indicated lymphoid degeneration in the thymus, lymph nodes, and spleen with no alterations in other tissues including bone marrow, kidney, lung, gastrointestinal tract, and liver except for the occurrence of hepatitis. A decrease in the number of Thy-1-positive cells in both spleen and lymph nodes further supported the fact of cytotoxicity of DCF to T cells. Anorexia and weight loss were observed within 5 days of continuous DCF infusion at 0.4 mg/kg body weight per day. These data indicate that this method provides an experimental model for future studies on the biochemical mechanisms responsible for the genetically determined severe combined immunodeficiency disease in man.

ADVERTISEMENT RETURN TO ISSUEPREVArticleNEXTPolyamine-polyphosphate complexes as enzyme inhibitorsLily C. Yip and M. Earl BalisCite this: Biochemistry 1980, 19, 9, 1849–1856Publication Date (Print):April 1, 1980Publication History Published online1 May 2002Published inissue 1 April 1980https://doi.org/10.1021/bi00550a019RIGHTS & PERMISSIONSArticle Views94Altmetric-Citations14LEARN ABOUT THESE METRICSArticle Views are the COUNTER-compliant sum of full text article downloads since November 2008 (both PDF and HTML) across all institutions and individuals. These metrics are regularly updated to reflect usage leading up to the last few days.Citations are the number of other articles citing this article, calculated by Crossref and updated daily. Find more information about Crossref citation counts.The Altmetric Attention Score is a quantitative measure of the attention that a research article has received online. Clicking on the donut icon will load a page at altmetric.com with additional details about the score and the social media presence for the given article. Find more information on the Altmetric Attention Score and how the score is calculated. Share Add toView InAdd Full Text with ReferenceAdd Description ExportRISCitationCitation and abstractCitation and referencesMore Options Share onFacebookTwitterWechatLinked InReddit PDF (946 KB) Get e-Alerts Get e-Alerts

The effect of 2′-deoxycoformycin (DCF) on activity levels and physicochemical properties of mouse adenosine deaminase (ADase) and purine nucleoside phosphorylase from nine tissues was characterized. This tight-binding inhibitor of ADase was continuously infused into C57BL mice for five days. Various levels of ADase which were tissue dependent remained after this period. Thymus activity was the most severely inhibited; jejunum, ileum, and spleen activities were only marginally depressed; and stomach activity was not inhibited. The presence of a transplantable colon tumor resulted in significantly lower ADase after infusion, especially in jejunum. Tumor ADase was depressed to approximately 15% of the control value. The extent of inhibition was found to be dependent on the mouse strain, i.e. , ADase from tissues of CD-1 mice was generally inhibited to a greater extent than was enzyme from the C57BL strain. Levels of purine nucleoside phosphorylase were relatively insensitive to the infusion. With the exception of the thymus, in vitro inhibition of ADase by DCF exceeded that produced in vivo . Residual levels of activity remaining in both cases were generally less sensitive to inhibition by DCF than untreated enzyme. Ackermann-Potter plots establish the inhibition as stoichiometric both before and after DCF infusion and provide evidence for a significant decrease in affinity after infusion. The data suggest the possibility of the induction of a form of ADase less sensitive to inhibition than is native enzyme. These results may be of eventual usefulness in the design of combined chemotherapeutic regimens involving adenosine analogs and tight-binding inhibitors.