Methylthioadenosine deaminase in an alternative quorum sensing pathway in Pseudomonas aeruginosa.

Pseudomonas aeruginosa possesses an unusual pathway for 5'-methylthioadenosine (MTA) metabolism involving deamination to 5'-methylthioinosine (MTI) followed by N-ribosyl phosphorolysis to hypoxanthine and 5-methylthio-α-d-ribose 1-phosphate. The specific MTI phosphorylase of P. aeruginosa has been reported [Guan, R., Ho, M. C., Almo, S. C., and Schramm, V. L. (2011) Biochemistry 50, 1247-1254], and here we characterize MTA deaminase from P. aeruginosa (PaMTADA). Genomic analysis indicated the PA3170 locus to be a candidate for MTA deaminase (MTADA). Protein encoded by PA3170 was expressed and shown to deaminate MTA with 40-fold greater catalytic efficiency for MTA than for adenosine. The k(cat)/K(m) value of 1.6 × 10(7) M(-1) s(-1) for MTA is the highest catalytic efficiency known for an MTA deaminase. 5'-Methylthiocoformycin (MTCF) is a 4.8 pM transition state analogue for PaMTADA but causes no significant inhibition of human adenosine deaminase or MTA phosphorylase. MTCF is permeable to P. aeruginosa and exhibits an IC(50) of 3 nM on cellular PaMTADA activity. PaMTADA is the only activity in P. aeruginosa extracts to act on MTA. MTA and 5-methylthio-α-d-ribose are involved in quorum sensing pathways; thus, PaMTADA is a potential target for quorum sensing. The crystal structure of PaMTADA in complex with MTCF shows the transition state mimic 8(R)-hydroxyl group in contact with a catalytic site Zn(2+), the 5'-methylthio group in a hydrophobic pocket, and the transition state mimic of the diazepine ring in contact with a catalytic site Glu.

Structural basis for the growth factor activity of human adenosine deaminase ADA2.

Two distinct adenosine deaminases, ADA1 and ADA2, are found in humans. ADA1 has an important role in lymphocyte function and inherited mutations in ADA1 result in severe combined immunodeficiency. The recently isolated ADA2 belongs to the novel family of adenosine deaminase growth factors (ADGFs), which play an important role in tissue development. The crystal structures of ADA2 and ADA2 bound to a transition state analogue presented here reveal the structural basis of the catalytic/signaling activity of ADGF/ADA2 proteins. In addition to the catalytic domain, the structures discovered two ADGF/ADA2-specific domains of novel folds that mediate the protein dimerization and binding to the cell surface receptors. This complex architecture is in sharp contrast with that of monomeric single domain ADA1. An extensive glycosylation and the presence of a conserved disulfide bond and a signal peptide in ADA2 strongly suggest that ADA2, in contrast to ADA1, is specifically designed to act in the extracellular environment. The comparison of catalytic sites of ADA2 and ADA1 demonstrates large differences in the arrangement of the substrate-binding pockets. These structural differences explain the substrate and inhibitor specificity of adenosine deaminases and provide the basis for a rational design of ADA2-targeting drugs to modulate the immune system responses in pathophysiological conditions.

Adenosine in the tuberomammillary nucleus inhibits the histaminergic system via A1 receptors and promotes non-rapid eye movement sleep.

Adenosine has been proposed to promote sleep through A(1) receptors (A(1)R's) and/or A(2A) receptors in the brain. We previously reported that A(2A) receptors mediate the sleep-promoting effect of prostaglandin D(2), an endogenous sleep-inducing substance, and that activation of these receptors induces sleep and blockade of them by caffeine results in wakefulness. On the other hand, A(1)R has been suggested to increase sleep by inhibition of the cholinergic region of the basal forebrain. However, the role and target sites of A(1)R in sleep-wake regulation remained controversial. In this study, immunohistochemistry revealed that A(1)R was expressed in histaminergic neurons of the rat tuberomammillary nucleus (TMN). In vivo microdialysis showed that the histamine release in the frontal cortex was decreased by microinjection into the TMN of N(6)-cyclopentyladenosine (CPA), an A(1)R agonist, adenosine or coformycin, an inhibitor of adenosine deaminase, which catabolizes adenosine to inosine. Bilateral injection of CPA into the rat TMN significantly increased the amount and the delta power density of non-rapid eye movement (non-REM; NREM) sleep but did not affect REM sleep. CPA-promoted sleep was observed in WT mice but not in KO mice for A(1)R or histamine H(1) receptor, indicating that the NREM sleep promoted by A(1)R-specific agonist depended on the histaminergic system. Furthermore, the bilateral injection of adenosine or coformycin into the rat TMN increased NREM sleep, which was completely abolished by coadministration of 1,3-dimethyl-8-cyclopenthylxanthine, a selective A(1)R antagonist. These results indicate that endogenous adenosine in the TMN suppresses the histaminergic system via A(1)R to promote NREM sleep.

Adenosine: a physiological modulator of superoxide anion generation by human neutrophils.

The effects of adenosine were studied on human neutrophils with respect to their generation of superoxide anion, degranulation, and aggregation in response to soluble stimuli. Adenosine markedly inhibited superoxide anion generation by neutrophils stimulated with N-formyl methionyl leucyl phenylalanine (FMLP), concanavalin A (Con A), calcium ionophore A23187, and zymosan-treated serum; it inhibited this response to PMA to a far lesser extent. The effects of adenosine were evident at concentrations ranging from 1 to 1,000 microM with maximal inhibition at 100 microM. Cellular uptake of adenosine was not required for adenosine-induced inhibition since inhibition was maintained despite the addition of dipyridamole, which blocks nucleoside uptake. Nor was metabolism of adenosine required, since both deoxycoformycin (DCF) and erythro-9-(2-hydroxy-3-nonyl) adenine did not interfere with adenosine inhibition of superoxide anion generation. The finding that 2-chloroadenosine, which is not metabolized, resembled adenosine in its ability to inhibit superoxide anion generation added further evidence that adenosine metabolism was not required for inhibition of superoxide anion generation by neutrophils. Unexpectedly, endogenously generated adenosine was present in supernatants of neutrophil suspensions at 0.14-0.28 microM. Removal of endogenous adenosine by incubation of neutrophils with exogenous adenosine deaminase (ADA) led to marked enhancement of superoxide anion generation in response to FMLP. Inactivation of ADA with DCF abrogated the enhancement of superoxide anion generation. Thus, the enhancement was not due to a nonspecific effect of added protein. Nor was the enhancement due to the generation of hypoxanthine or inosine by deamination of adenosine, since addition of these compounds did not affect neutrophil function. Adenosine did not significantly affect either aggregation or lysozyme release and only modestly affected beta-glucuronidase release by neutrophils stimulated with FMLP. These data indicate that adenosine (at concentrations that are present in plasma) acting via cell surface receptors is a specific modulator of superoxide anion generation by neutrophils.

Structural and metabolic specificity of methylthiocoformycin for malarial adenosine deaminases.

Plasmodium falciparum is a purine auxotroph requiring hypoxanthine as a key metabolic precursor. Erythrocyte adenine nucleotides are the source of the purine precursors, making adenosine deaminase (ADA) a key enzyme in the pathway of hypoxanthine formation. Methylthioadenosine (MTA) is a substrate for most malarial ADAs, but not for human ADA. The catalytic site specificity of malarial ADAs permits methylthiocoformycin (MT-coformycin) to act as a Plasmodium-specific transition state analogue with low affinity for human ADA [Tyler, P. C., Taylor, E. A., Frohlich, R. G. G., and Schramm, V. L. (2007) J. Am. Chem. Soc. 129, 6872-6879]. The structural basis for MTA and MT-coformycin specificity in malarial ADAs is the subject of speculation [Larson, E. T., et al. (2008) J. Mol. Biol. 381, 975-988]. Here, the crystal structure of ADA from Plasmodium vivax (PvADA) in a complex with MT-coformycin reveals an unprecedented binding geometry for 5'-methylthioribosyl groups in the malarial ADAs. Compared to malarial ADA complexes with adenosine or deoxycoformycin, 5'-methylthioribosyl groups are rotated 130 degrees . A hydrogen bonding network between Asp172 and the 3'-hydroxyl of MT-coformycin is essential for recognition of the 5'-methylthioribosyl group. Water occupies the 5'-hydroxyl binding site when MT-coformycin is bound. Mutagenesis of Asp172 destroys the substrate specificity for MTA and MT-coformycin. Kinetic, mutagenic, and structural analyses of PvADA and kinetic analysis of five other Plasmodium ADAs establish the unique structural basis for its specificity for MTA and MT-coformycin. Plasmodium gallinaceum ADA does not use MTA as a substrate, is not inhibited by MT-coformycin, and is missing Asp172. Treatment of P. falciparum cultures with coformycin or MT-coformycin in the presence of MTA is effective in inhibiting parasite growth.

Metabolic and functional consequences of inhibiting adenosine deaminase during renal ischemia in rats.

The concentrations of renal ATP have been measured by 31P-nuclear magnetic resonance (NMR) before, during, and after bilateral renal artery occlusion. Using in vivo NMR, the initial postischemic recovery of ATP increased with the magnitude of the residual nucleotide pool at the end of ischemia. ATP levels after 120 min of reflow correlated with functional recovery at 24 h. In the present study the effect of blocking the degradation of ATP during ischemia upon the postischemic restoration of ATP was investigated. Inhibition of adenosine deaminase by 80% with the tight-binding inhibitor 2'-deoxycoformycin led to a 20% increase in the residual adenine nucleotide pool. This increased the ATP initial recovery after 45 min of ischemia from 52% (in controls) to 62% (in the treated animals), as compared to the basal levels. The inhibition also caused an accelerated postischemic restoration of cellular ATP so that at 120 min it was 83% in treated rats vs. 63% in untreated animals. There was a corresponding improvement in the functional recovery from the insult (increase of 33% in inulin clearance 24 h after the injury). Inhibition of adenosine deaminase during ischemia results in a injury similar to that seen after a shorter period of insult.

The erythrocyte as instigator of inflammation. Generation of amidated C3 by erythrocyte adenosine deaminase.

Myocardial ischemia is characterized by the liberation of adenosine and by complement-mediated inflammation. We have reported that amidated C3, formed when ammonia (NH3) disrupts the thiolester bond of C3, serves as an alternative pathway convertase, generates C5b-9, and stimulates phagocytic oxidative metabolism. We investigated whether the deamination of adenosine by adenosine deaminase in hematopoietic cells might liberate sufficient ammonia to form amidated C3 and thereby trigger complement-mediated inflammation at ischemic sites. In the presence of 4 mM adenosine, NH3 production per erythrocyte (RBC) was equal to that per neutrophil (PMN) (3.3 X 10(-15) mol/cell per h). Because RBC outnumber PMN in normal blood by a thousandfold, RBC are the major source of NH3 production in the presence of adenosine. NH3 production derived only from the deamination of adenosine by the enzyme adenosine deaminase and was abolished by 0.4 microM 2'-deoxycoformycin, a specific inhibitor of adenosine deaminase. When purified human C3 was incubated with 5 X 10(8) human RBC in the presence of adenosine, disruption of the C3 thiolester increased more than twofold over that measured in C3 incubated with buffer, or in C3 incubated with RBC (P less than 0.05). The formation of amidated C3 was abolished by the preincubation of RBC with 2'-deoxycoformycin (P less than 0.001). Amidated C3 elicited statistically significant release of superoxide, myeloperoxidase, and lactoferrin from PMN. Thus, the formation of amidated C3 by RBC deamination of adenosine triggers a cascade of complement-mediated inflammatory reactions.

Bone marrow transplantation only partially restores purine metabolites to normal in adenosine deaminase-deficient patients.

To delineate the extent to which bone marrow transplantation provides "enzyme replacement therapy", we have determined metabolite concentrations in two patients with adenosine deaminase (ADA) deficiency treated with bone marrow transplants and rendered immunologically normal. 10 yr after engraftment of lymphoid cells, erythrocyte deoxy ATP was markedly decreased compared to the marked elevations of deoxy ATP observed in untreated patients, but was still significantly elevated (62 and 90 vs. normal of 6.0 +/- 6.0 nmol/ml packed erythrocytes). Similarly, deoxyadenosine and adenosine excretion were both markedly diminished compared to that of untreated patients but deoxyadenosine excretion was still clearly increased (20.1 and 38.6 vs. normal of less than 0.2 nmol/mg creatinine) while adenosine excretion was in the upper range of normal (7.0 and 8.1 vs. normal of 5.6 +/- 3.6 nmol/mg creatinine). Mononuclear cell deoxy ATP content was also elevated compared to normal (5.25 and 14.4 vs. 1.2 +/- 0.3). Separated mononuclear cells of bone marrow transplanted patients contain both donor lymphocytes and recipient monocytes. When mononuclear cells were depleted of the cells enriched for donor lymphocytes (i.e. monocyte depleted) was lower than that of the mixed mononuclear cells (2.2 vs. 5.26). Surprisingly, plasma adenosine was as high as in untreated ADA-deficient patients (3.2 and 1.5 vs. untreated of 0.3-3 microM). Consistent with the elevated plasma adenosine and urinary deoxyadenosine, erythrocyte S-adenosyl homocysteine hydrolase activity was diminished (0.88 and 1.02 vs. normal of 5.64 +/- 0.25). Thus, bone marrow transplantation of ADA-deficient patients not only provides lymphoid stem cells, but also partially, albeit incompletely, clears abnormally increased metabolites from nonlymphoid body compartments.

Mechanism of deoxyadenosine and 2-chlorodeoxyadenosine toxicity to nondividing human lymphocytes.

Deoxyadenosine has been implicated as the toxic metabolite causing profound lymphopenia in immunodeficient children with a genetic deficiency of adenosine deaminase (ADA), and in adults treated with the potent ADA inhibitor deoxycoformycin. However, the biochemical basis for deoxyadenosine toxicity toward lymphocytes remains controversial. The present experiments have examined in detail the sequential metabolic changes induced in nondividing human peripheral blood lymphocytes by incubation with deoxyadenosine plus deoxycoformycin, or with 2-chlorodeoxyadenosine (CdA), an ADA resistant deoxyadenosine congener with anti-leukemic and immunosuppressive properties. The lymphotoxic effect of deoxyadenosine and CdA required their phosphorylation, and was inhibited by deoxycytidine. As early as 4 h after exposure to the deoxynucleosides, strand breaks in lymphocyte DNA began to accumulate, and RNA synthesis decreased. These changes were followed by a significant fall in intracellular NAD levels at 8 h, a drop in ATP pools at 24 h, and cell death by 48 h. Incubation of the lymphocytes with 5 mM nicotinamide, a NAD precursor and an inhibitor of poly(ADP-ribose) synthetase, prevented NAD depletion. The nicotinamide treatment also rendered the lymphocytes highly resistant to deoxyadenosine and CdA toxicity, without altering dATP formation or the accumulation of DNA strand breaks. The poly(ADP-ribose) synthetase inhibitor 3-aminobenzamide exerted a similar although less potent effect. These results suggest that NAD depletion, probably triggered by poly(ADP-ribose) formation, is the principle cause of death in normal resting human lymphocytes exposed to deoxyadenosine plus deoxycoformycin, or to CdA.

Stepwise isolation and properties of unstable Chinese hamster cell variants that overproduce adenylate deaminase.

Addition of coformycin (0.5 microgram/ml) to a culture medium containing adenine causes in Chinese hamster fibroblasts a lethal depletion of IMP. Resistant variants have been recovered, some of which exhibit increased adenylate deaminase activity. (Debatisse et al., J. Cell. Physiol., 106:1-11, 1981). The selective medium was made more specific for the isolation of this class of variants by supplementation with azaserine. The hyperactive variants remained sensitive to coformycin concentrations above that used for their selection and were unstable. Their frequency was not increased by ethyl methane sulfonate mutagenesis. The resistant phenotype and the increased activity of adenylate deaminase behaved as semidominant traits in hybrids. No change was detected in the Km for AMP, the cofactor requirement, or the chromatographic properties of adenylate deaminase in the variants. Through stepwise selection in media supplemented with increasing coformycin concentrations, unstable clones with adenylate deaminase activity up to 150-fold the wild-type level were isolated; from an unstable clone, a stable subclone with reduced resistance and enzyme activity was recovered. Evidence that increased adenylate deaminase activity is the manifestation of overaccumulation of the enzyme protein was supplied by the correlation of enzyme activity with the intensity of a protein band comigrating with purified adenylate deaminase during sodium dodecyl sulfate-polyacrylamide gel electrophoresis of cell extracts. Several unidentified additional bands showed comparable quantitative changes. The striking similarity between the adenylate deaminase-overproducing lines and unstable dihydrofolate reductase-overproducing lines generated by gene amplification strongly suggests that the coformycin-resistant variants also resulted from amplification of an adenylate deaminase gene.

Hemodynamic effects of potentially useful antineoplastic agents.

Adenosine (Ado) and Ado analogues produce multiple hemodynamic effects including coronary vasodilation, bradycardia, alterations in left ventricular contractility, and peripheral vasodilation or vasoconstriction depending on the vascular bed. The intact anesthetized Sprague-Dawley rat was examined in relation to electrocardiogram and blood pressure alterations induced by a series of potentially useful antineoplastic agents that are purine or pyrimidine analogues as part of a preclinical evaluation of these agents. The drugs tested were arabinosyladenine and its 5'-monophosphate derivative arabinosyladenine-5'-monophosphate (ara-AMP), the 2-fluoro derivative of ara-AMP, the pyrazolo[4,3-d]pyrimidines (formycin and formycin B), 8-azaadenosine, 6-methylmercaptopurine riboside, tricyclic nucleoside-5'-monophosphate, 5-fluorouracil, arabinosylcytosine, and 3-deazauridine. Those Ado analogues subject to deamination by adenosine deaminase (ADA) were also studied in the intact Sprague-Dawley rat after pretreatment with the ADA inhibitor 2'-deoxycoformycin. The results indicate that these agents have significant hemodynamic effects and should alert clinicians to potential adverse reactions when infusing these drugs.

Morphologic changes and immunohistochemical localization of adenosine deaminase in tissues of rats given injections of 2'-deoxycoformycin.

2'-Deoxycoformycin (DCF) is a potent inhibitor of adenosine deaminase (ADA) and a potential antineoplastic and immunosuppressive agent. In this study the kinetics of ADA expression was assessed by immunomorphologic and enzymatic methods in tissues of ACI rats given injections of DCF. The rats received a daily ip injection of 10 mg DCF/kg for 3 consecutive days. This treatment destroyed cortical thymocytes, whereas lymphocytes of the thymic medulla were mainly preserved. In control phosphate-buffered saline-injected rats, cortical thymocytes were not affected morphologically and displayed strong ADA staining. It was found unexpectedly that injections of DCF produced activation and, possibly, differentiation of B-cells in the mesenteric lymph nodes and spleen. These activated B-lymphocytes and plasma cells stained strongly for ADA. Transient changes in patterns of ADA expression were also observed in endothelial cells of blood vessels and liver Kupffer's cells, but these changes were not accompanied by degeneration of the cells. The treatment with DCF did not result in any permanent abnormalities in the rat tissues.

Recovery of 2'-deoxycoformycin-inhibited adenosine deaminase of mouse erythrocytes and leukemia L1210 in vivo.

The antibiotic 2'-deoxycoformycin, a potent inhibitor of adenosine deaminase, has potential as a chemotherapeutic agent. Injection of 2'-deoxycoformycin i.v. (0.2 mg/kg) to mice bearing ascites L1210 leukemia cells completely inhibits adenosine deaminase in both erythrocytes and L1210 cells. The recovery of the enzymic activity is markedly different in the two tissues. The recovery is very slow in erythrocytes (13% in 48 hr), whereas 80% recovery occurs during the same time interval in L1210 cells. This marked difference in the recovery of the enzyme in different tissues may play a role in the pharmacological and chemotherapeutic behavior of this drug.

Enhanced cytotoxicity and inhibition of DNA damage repair in irradiated murine L5178Y lymphoblasts and human chronic lymphocytic leukemia cells treated with 2'-deoxycoformycin and deoxyadenosine in vitro.

The effects of irradiation were evaluated in L5178Y lymphoblasts treated with the adenosine deaminase inhibitor, 2'-deoxycoformycin, and deoxyadenosine. A synergistic antitumor effect was observed in resting cells between irradiation and 2'-deoxycoformycin/deoxyadenosine, with the dose required to reduce the surviving cell fraction to 0.1 being 25% lower than predicted for an additive effect. Synergy was enhanced with increasing deoxyadenosine concentration or with increasing radiation dose. When cells were treated with 2'-deoxycoformycin/deoxyadenosine for 1 h prior to irradiation, synergy was increased by prolonging postirradiation drug treatment. With 4-h postirradiation exposure to drug, varying the preirradiation incubation time did not affect synergy. In contrast, only a small enhancement of antitumor activity was observed in irradiated proliferating cells treated with 2'-deoxycoformycin/deoxyadenosine. Incubation of resting cells with 2'-deoxycoformycin/deoxyadenosine resulted in inhibition of the rate and extent of repair of radiation-induced DNA single strand breaks and an increase in dATP, but had no effect on NAD or ATP. With removal of drug, the dATP level fell rapidly and DNA repair resumed. Repair of DNA single strand breaks was more rapid in proliferating cells than in resting cells and was minimally affected by 2'-deoxycoformycin/deoxyadenosine, although the accumulation of dATP in these cells was 2-fold greater than in resting cells. The repair of DNA single strand breaks in chronic lymphocytic leukemia cells was as rapid as for proliferating L5178Y cells, but repair was significantly inhibited by 2'-deoxycoformycin/deoxyadenosine. These results suggest that 2'-deoxycoformycin/deoxyadenosine can function as a radiosensitizer, and this effect is associated with the cellular accumulation of dATP and inhibition of repair of DNA single strand breaks.

Mechanism of adenosine triphosphate catabolism induced by deoxyadenosine and by nucleoside analogues in adenosine deaminase-inhibited human erythrocytes.

The mechanism of the depletion of ATP, recorded in the erythrocytes of adenosine deaminase-deficient children and of leukemia patients treated with deoxycoformycin, was investigated in normal human erythrocytes treated with this inhibitor of adenosine deaminase. Deoxyadenosine, which accumulates in both clinical conditions, provoked a dose-dependent accumulation of dATP, depletion of ATP, and increases in the production of inosine plus hypoxanthine. Concomitantly, there was an increase of AMP and IMP, but not of adenosine, indicating that catabolism proceeded by way of AMP deaminase. A series of nucleoside analogues (9-beta-D-arabinofuranosyladenine, N6-methyladenosine, 6-methylmercaptopurine ribonucleoside, tubercidin, ribavirin, and N-1-ribosyl-5-aminoimidazole-4-carboxamide riboside) also stimulated adenine nucleotide catabolism and increased AMP and IMP to various extents. The effects of deoxyadenosine and of the nucleoside analogues were prevented by 5'-iodotubercidin, an inhibitor of adenosine kinase. Strikingly, they were reversed if the inhibitor was added after the accumulation of nucleotide analogues and initiation of adenine nucleotide catabolism. Further analyses revealed linear relationships between the rate of phosphorylation of deoxyadenosine and nucleoside analogues and the increase in AMP and between the elevation of the latter above a threshold concentration of 10 microM and the rate of adenine nucleotide catabolism. Kinetic studies with purified erythrocytic AMP deaminase, at physiological concentrations of its effectors, showed that the enzyme is nearly inactive up to 10 microM AMP and increases in activity above this threshold. We conclude that the main mechanism whereby deoxyadenosine and nucleoside analogues stimulate catabolism of adenine nucleotides by way of AMP deaminase in erythrocytes is elevation of AMP, secondary to the phosphorylation of the nucleosides.

The extracellular fluid of solid carcinomas contains immunosuppressive concentrations of adenosine.

The purine nucleoside adenosine (9-beta-D-ribofuranosyladenine) inhibits a number of lymphocyte functions in vitro, including the ability of activated T lymphocytes and natural killer cells to adhere to and kill tumor targets. Solid tumors, such as adenocarcinomas of the lung and colon, are frequently hypoxic and are, therefore, likely to exhibit increased adenine nucleotide breakdown through the 5'-nucleotidase pathway, yielding adenosine. We examined whether the concentration of adenosine in the extracellular fluid of such tumors is adequate to cause immunosuppression. Murine tumors grown in syngeneic hosts or human tumors grown in immunodeficient nu/nu mice were subjected to microdialysis, and adenosine levels in the microdialysate were measured by high-performance liquid chromatography. Treatment of the tumor microdialysates with adenosine deaminase eliminated the adenosine peak. Recovery of adenosine ranged from 15 to 29%, depending on the microdialysis probe, and concentrations of adenosine in tumors ranged from 0.2 to 2.4 microM with a mean of 0.5 microM. In contrast, the adenosine concentration measured s.c. at the same location was 30 +/- 5 nM (mean +/- SE). Inclusion of the adenosine deaminase inhibitor coformycin (10 microM) and the adenosine kinase inhibitor 5'-iodotubercidin (0.1 microM) in the microdialysis perfusion buffer increased extracellular adenosine concentration in tumors to as high as 13 microM. These data show that extracellular adenosine levels in solid tumors are sufficient to suppress the local antitumor immune response and that interference with pathways of adenosine metabolism causes marked increases in tumor extracellular adenosine concentration.

Combined chemoseparation and immunoseparation of clonogenic T lymphoma cells from human bone marrow using 2'-deoxycoformycin, deoxyadenosine, 3A1 monoclonal antibody, and complement.

Chemoseparation and immunoseparation techniques have been combined to eliminate malignant clonogenic T lymphoma cells from human bone marrow. Incubation with 5 microM 2'-deoxycoformycin and 500 microM deoxyadenosine has eliminated 2 logs of HSB-2 T lymphoma cells from a 20-fold excess of irradiated human bone marrow. Multiple incubations with 3A1 antibody and rabbit complement eliminated approximately 2 logs of HSB-2 cells from similar mixtures. Used in combination, the 2 techniques eliminated up to 4 logs of T lymphoma cells. Incubation of normal human bone marrow under similar conditions failed to affect growth of granulocyte-macrophage colony-forming cell units, burst-forming erythroid units, or multipotential erythroid-granulocyte-megakaryocyte-macrophage colony-forming hematopoietic progenitor cells units.

Clinical pharmacology of 9-beta-D-arabinofuranosyladenine in combination with 2'-deoxycoformycin.

It has been suggested that, by inhibiting the adenosine deaminase (ADA)-mediated catabolism of 9-beta-D-arabinofuranosyladenine (ara-A), 2'-deoxycoformycin (DCF) would increase the half-life (t1/2) of ara-A, a compound with known antileukemic activity. To test this hypothesis, we collected serial plasma samples from five patients with refractory acute lymphoblastic leukemia who participated in a Phase I trial of i.v. DCF 915 mg/sq m) in combination with i.v. single-dose ara-A (120-250 mg/sq m). In four of these patients, of whom three were known to have achieved greater than 98% ADA inhibition, a mean ara-A t1/2 of 227 min was achieved. Extrapolated peak levels (i.e., following infusion of ara-A) ranged from 1.5 to 7.4 micrograms/ml (mean, 4.2 micrograms/ml). Elimination of drug appeared to follow a single-compartment model. In two patients who received ara-A without prior DCF and in a third patient who had significant residual ADA activity despite DCF, ara-A was unmeasurable within 5 min of the end of infusion. These data confirm that the kinetics of ara-A catabolism can be altered by inhibition of ADA and suggest that more than one dose of DCF may be necessary for complete inhibition of the enzyme and optimal pharmacological modulation of ara-A.

Correlation of cytotoxicity with total intracellular exposure to 9-beta-D-arabinofuranosyladenine 5'-triphosphate.

The mechanism by which 9-beta-D-arabinofuranosyladenine produces cell death has been studied extensively, but the details remain controversial. The results presented here describe an evaluation of 9-beta-D-arabinofuranosyladenine-induced cytotoxicity in terms of the product of the total amount of the active 5'-triphosphate metabolite, 9-beta-D-arabinofuranosyladenine 5'-triphosphate (ara-ATP), which accumulated in the cells, and the duration of the exposure expressed in units of ara-ATP microM-hr. It was demonstrated that a strong correlation exists between these parameters which was not affected by the rate of accumulation of ara-ATP. In addition, inhibition of 9-beta-D-arabinofuranosyladenine deamination by 2'-deoxycoformycin did not alter the relationship between cell death and total intracellular exposure to ara-ATP. The consistency of this relationship both within and between experiments indicates that the quantitation of the total cellular exposure to ara-ATP is useful in predicting cytotoxicity.

Reactivation of S-adenosylhomocysteine hydrolase activity in cells exposed to 9-beta-D-arabinofuranosyladenine.

9-beta-D-Arabinofuranosyladenine (ara-A) inactivates isolated S-adenosyl-L-homocysteine (AdoHcy) hydrolase (EC 3.3.1.1) as well as AdoHcy hydrolase in intact cells. Whereas the inactivation in cell-free systems is an irreversible process, the AdoHcy hydrolase activity in rat hepatocytes exposed to ara-A gradually recovered upon prolonged incubation of the cells in a medium devoid of ara-A. This process, tentatively termed reactivation of the enzyme, was nearly totally dependent on a high level of adenosine deaminase in the extracellular medium, which induced a decrease in intracellular content of adenosine as well as ara-A. Reactivation of intracellular enzyme was inhibited by adenosine deaminase inhibitors [2'-deoxycoformycin and erythro-9-(2-hydroxy-3-nonyl)adenine] and the synthetic substrate for AdoHcy hydrolase, 3-deazaadenosine. An inhibitor of protein synthesis (cycloheximide) was without effect. Homocysteine, which protected the intracellular AdoHcy hydrolase against inactivation by ara-A, induced no reactivation of the enzyme. The half-life of the intracellular ara-A-AdoHcy hydrolase complex was about 90 min and was not affected by adenosine deaminase, 3-deazaadenosine, or homocysteine added to the cell suspension. However, the rate of elimination of the complex in the hepatocytes exceeded the rate of reactivation of AdoHcy hydrolase. Thus, the elimination process accounted for the reactivation, but not correlation between these two processes was observed. Reactivation of intracellular AdoHcy hydrolase caused a pronounced fall in cellular content of AdoHcy. The possibility that reduced cellular level of AdoHcy induced the reactivation of AdoHcy hydrolase seemed unlikely. This statement was based on the observation that reactivation was observed also under conditions of high concentrations of AdoHcy (obtained by the addition of homocysteine to the cell suspension). Reactivation of AdoHcy hydrolase with a concomitant decrease in cellular level of AdoHcy could also be demonstrated with mouse plasmacytoma (MPC-11) cells and mouse fibroblasts (L-929) exposed to ara-A, but the reactivation process was far less pronounced than with hepatocytes.