Gail S. Duncan

Inhibition of lymphocyte-mediated cytolysis by adenosine analogs: Biochemical studies concerning mechanism of action

Abstract A number of adenosine (Ado) analogs have been found to inhibit lymphocyte-mediated cytolysis (LMC) in vitro at μM concentrations. Those analogs which are substrates for adenosine deaminase were more inhibitory to LMC in the presence of erythro-9-(2-hydroxy-3-nonyl)adenine, an inhibitor of the deaminase. The inhibitory activity of most of these analogs (the exceptions being 2′-deoxyadenosine, 9-β- d -arabinofuranosyladenine and 7-deazaadenosine) was markedly enchanced by an inhibitor (Ro 20-1724) of cyclic AMP phosphodiesterase. With the exceptions of 2-fluoroadenosine, 7-deazaadenosine and formycin A, the inhibition of LMC caused by the Ado analogs was fully reversible upon removal of the analogs from the medium. In general, the Ado analogs did not cause a reduction in the pool sizes of endogenous ribonucleoside 5′-triphosphates in the lymphocytes. Some inhibitory and non-inhibitory analogs were metabolized to their corresponding 5′-triphosphates. Lymphocytes pretreated with a reversible inhibitor of LMC, 2-aminoadenosine, retained most of the resultant 2-amino-ATP during subsequent incubation in analog-free medium. Most of the Ado analogs which were inhibitory to LMC caused a substantial elevation of lymphocyte cyclic AMP; the magnitude of this elevation was enhanced by Ro 20-1724. Collectively, these results suggest that Ado and many of its structural analogs inhibit LMC by reason of their ability to stimulate the formation and consequent build-up of cyclic AMP in the cytotoxic lymphocytes. This stimulation of adenylate cyclase appears to result from the binding of an appropriate nucleoside to an adenosine receptor located on the membrane of the lymphocytes.

Metabolic formation of nucleoside-modified analogues of S-adenosylmethionine.

Abstract Evidence is presented for the metabolism of six different adenosine analogues (formycin, 7-deazaadenosine, 8-azaadenosine, 2-fluoroadenosine, purine ribonucleoside and 3′-deoxyadenosine) to their corresponding S-nucleosidylmethionine derivatives in human erythrocytes and mouse lymphocytes. The identification of these novel metabolites was based upon chromatographic and enzymatic characterization of the unique radioactive substance found in cells incubated with each of these adenosine analogues plus L-[methyl-3H]methionine. The metabolic formation of analogues of S-adenosylmethionine may contribute to the inhibition of cellular methylation reactions caused by various purine analogues.

Potentiation by homocysteine of adenosine-stimulated elevation of cellular adenosine 3′,5′-monophosphate

Abstract The novel ability of l -homocysteine (Hcy) to potentiate the cellular elevation of adenosine 3′,5′-monophosphate (cAMP) caused by adenosine (Ado) is described. This effect of Hcy is highly selective in that it is not mimicked by l -cysteine, and Hcy does not potentiate the elevation of cellular cAMP caused by either 2-chloroadenosine or prostaglandin E1. Hcy also augments the Ado-stimulated increase in 2-fluoroadenosine 3′,5′-monophosphate in cells preloaded with nucleotides of 2-fluoroadenosine. Addition of Hcy to cells during their incubation with radioactive Ado results in a decrease in the cellular content of radioactive Ado and a concomitant buildup of S-adenosylhomocysteine. The enhancive effect of Hcy on the Ado-stimulated elevation of cAMP may be due to this associated reduction in the intracellular pool of Ado (due to condensation of Ado with Hcy via S-adenosylhomocysteinase) and to a resultant reduction in inhibition of adenylate cyclase by intracellular Ado, thereby allowing greater net stimulation of the cyclase by extracellular Ado.

Inhibition of lymphocyte-mediated cytolysis by 2-fluoroadenosine--evidence for two discrete mechanisms of drug action.

Abstract 2-Fluoroadenosine (F-Ado) is a potent, irreversible inhibitor of lymphocyte-mediated cytolysis (LMC) in vitro: the irreversibility of this inhibition has been attributed to the metabolism of F-Ado to 2-fluoroadenosine t′-triphosphate (F-ATP) and 2-fluoroadenosine 3′, 5′-monophosphate (F-cAMP) within the cytotoxic lymphocytes [T. P. Zimmerman, J. L. Rideout, G. Wolberg, G.S. Duncan and G. B. Elion, J. biol. Chem.251, 6757 (1976)]. The present study was undertaken to define better the biochemical events intrinsic to the inhibition of LMC by F-Ado. Several purine ribonucleosides, which are themselves non-inhibitory towar LMC, have been found to inhibit the metabolism of F-Ado to F-ATP and F-cAMP by the cytotoxic lymphocytes. The reduction in F-cAMP formation caused by these ribonucleosides was counterbalanced by their augmentation of the elevation of lymphocytic cyclic AMP (cAMP) caused by F-Ado. While interference with the metabolism of F-Ado had little or no effect on the immediate inhibitory activity of F-Ado toward LMC, prevention of the cellular formation of F-ATP and F-cAMP did allow most of the inhibitory activity of F-Ado to be reversed after washing the lymphocytes free of exogenous F-Ado. The relative efficacy of these ribonucleosides in allowing reversibility of the inhibitory activity of F-Ado toward LMC followed the same order as did their efficacy in preventing the metabolism of F-Ado by the cytotoxic lymphocytes: 8-aza-adenosine > inosine > guanosine. Cytotoxic lymphocytes which had been preloaded with nucleotides of F-Ado (via prior incubation with F-Ado and subsequent washout of residual extracellular drug) exhibited increased inhibition of their cytolytic activity upon subsequent incubation with an inhibitor (Ro 20-1724) of cAMP phosphodiesterase. Under these latter experimental conditions, Ro 20-1724 caused a 2- to 3-fold elevation of F-cAMP in the cytotoxic lymphocytes but did not raise cAMP above control levels. These results suggest that F-Ado can inhibit LMC by either of two distinct mechanisms: (1) an extracellular mechanism, wherein F-Ado binds reversibly to an adenosine receptor present on the plasma membrane of the cytotoxic lymphocytes and reversibly activates a functionally associated adenylate cyclase, thereby causing an elevation of cellular cAMP; and (2) an intracellular mechanism, wherein F-Ado is metabolized irreversibly (during the 1- to 2-hr experimental period) by the cytotoxic lymphocytes to F-cAMP which, by reason of its ability to activate cAMP-dependent protein kinase, mimics the effect of elevated cellular levels of cAMP.

Mechanisms of Adenosine and 2’-Deoxyadenosine Inhibition of Immune Function in Mouse Lymphocytes

Adenosine (Ado) has been recognized as having an inhibitory influence on the immune system since 1970, when it was reported that this purine nucleoside inhibits the mitogenic stimulation of human peripheral blood lymphocytes by phytohemagglutinin [1]. Widespread interest in the possible immune modulatory role of Ado was sparked by the 1972 report of Giblett and her colleagues [2] describing the genetic deficiency of the enzyme Ado deaminase (ADA) in two children with severe combined immunodeficiency. This inherited disorder is characterized by a marked reduction of functional T and B lymphocytes. With the discovery of additional immunodeficient patients lacking ADA, an extensive research effort was begun to elucidate the biochemical mechanisms by which ADA deficiency results in the rather selective demise of the immune system. From 1972 until 1978, it was widely assumed that this immunodeficiency disease results from the buildup of high concentrations of Ado and/or adenine ribonucleotides in ADA-deficient patients. Indeed, in 1976 Mills et al. [3] reported that plasma Ado and adenine, and erythrocyte ATP, were elevated in an ADA-deficient child. Accordingly, on the basis of results obtained with a number of in vitro model systems, several hypotheses were introduced to explain the role of Ado in the pathophysiology of ADA-deficient immunodeficiency, the more prominent of which were Ado-induced pyrimidine starvation, Ado-stimulated increases in cAMP, and Ado-mediated accumulation of S-adenosylhomocysteine (AdoHcy) and consequent inhibition of methylation reactions (for reviews see [4–7]). Subsequently, in 1978, it was reported that 2′-deoxyadenosine (dAdo) was elevated in the plasma and urine of ADA-deficient children and that dATP was elevated in their erythrocytes and lymphocytes. With the further important observation that dAdo is more toxic than Ado to proliferating lymphoid cells, the emphasis in biochemical studies concerning the causal relationship between ADA deficiency and severe combined immunodeficiency shifted away from Ado toward dAdo. The historical development of this interesting field has been the subject of several recent reviews [4–7].

Concerning the nonspecificity of methylation inhibitors

Most of the evidence for the essential participation of S-adenosylmethionine (AdoMet)-mediated methylation reactions in eukaryotic cell functions derives from the use of chemical probes that have been found to be inhibitory to these various cell functions and that are also known to be inhibitory, in a direct or an indirect manner, to some cellular methylation reaction(s). Compounds most often employed experimentally as methylation reaction probes include adenosine (Ado), 3-deazaadenosine (c3Ado), 5’-deoxy-5’-S-isobutylthioadenosine (SIBA) and 5’-deoxy-5’-S-isobutylthio-3-deazaadenosine (c3SIBA). The use of all these compounds as methylation reaction probes is based upon the findings of numerous investigators that S-adenosylhomocysteine (AdoHcy) is a potent inhibitor of many AdoMet-utilizing methyltransferases (see review by Borchardt, 1977). In many of the studies employing Ado and c3Ado, L-homocysteine (Hcy) has been observed to potentiate the physiological effects of these nucleosides (e.g., Ishizaka et al., 1980; Morita et al., 1981; Pike et al., 1978; Rabe et al., 1980; Zimmerman et al., 1978). This potentiation by Hcy has been interpreted as additional evidence that Ado and c3Ado are affecting cell function as the result of an intracellular buildup of AdoHcy or S-3-deazaadenosylhomocysteine (c3AdoHcy), respectively, and consequent inhibition of one or more methyltransferases.