Purpose: Adenosine is a ubiquitous nucleoside that accumulates at high levels in hypoxic regions of solid tumors, and A 3 adenosine receptors have been recently demonstrated to play a pivotal role in the adenosine-mediated inhibition of tumor cell proliferation. In the present work, we addressed the question of the putative relevance of A 3 subtypes in colorectal adenocarcinomas.Experimental Design: Seventy-three paired samples of tumor and surrounding peritumoral normal mucosa at a distance of 2 and 10 cm from the tumor and blood samples obtained from a cohort of 30 patients with colorectal cancer were investigated to determine the presence of A 3 receptors by means of binding, immunocytochemistry, and real-time reverse transcription-polymerase chain reaction studies.Results: As measured by receptor binding assays, the density of A 3 receptor was higher in colon carcinomas as compared with normal mucosa originating from the same individuals (P < 0.05). Overexpression of A 3 receptors at the protein level was confirmed by immunohistochemical studies, whereas no changes in A 3 mRNA accumulation in tumors as compared with the corresponding normal tissue were revealed. The overexpression of A 3 receptors in tumors was reflected in peripheral blood cells, where the density was approximately 3-fold higher compared with healthy subjects (P < 0.01). In a cohort of 10 patients studied longitudinally, expression of A 3 receptors in circulating blood cells returned to normal after surgical resection for colorectal cancer.Conclusions: This study provides the first evidence that A 3 receptor plays a role in colon tumorigenesis and, more importantly, can potentially be used as a diagnostic marker or a therapeutic target for colon cancer.

1 The present study describes the e ect of low frequency, low energy, pulsing electromagnetic ®elds (PEMFs) on A 2A adenosine receptors in human neutrophils.2 Saturation experiments performed using a high a nity adenosine antagonist [ 3 H]-ZM 241385 revealed a single class of binding sites in control and in PEMF-treated human neutrophils with similar a nity (K D =1.05+0.10 and 1.08+0.12 nM, respectively). Furthermore, after 1 h of exposure to PEMFs the receptor density was statistically increased (P50.01) (B max =126+10 and 215+15 fmol mg 71 protein, respectively).3 The e ect of PEMFs was speci®c to the A 2A adenosine receptors. This e ect was also intensity, time and temperature dependent. 4 In the adenylyl cyclase assays the A 2A receptor agonists, HE-NECA and NECA, increased cyclic AMP accumulation in untreated human neutrophils with an EC 50 value of 43 (40 ± 47) and 255 (228 ± 284) nM, respectively. The capability of HE-NECA and NECA to stimulate cyclic AMP levels in human neutrophils was increased (P50.01) after exposure to PEMFs with an EC 50 value of 10(8 ± 13) and 61(52 ± 71) nM, respectively. 5 In the superoxide anion (O 2 7 ) production assays HE-NECA and NECA inhibited the generation of O 2 7 in untreated human neutrophils, with an EC 50 value of 3.6(3.1 ± 4.2) and of 23(20 ± 27) nM, respectively. Moreover, in PEMF-treated human neutrophils, the same compounds show an EC 50 value of 1.6(1.2 ± 2.1) and of 6.0(4.7 ± 7.5) nM respectively. 6 These results indicate the presence of signi®cant alterations in the expression and in the functionality of adenosine A 2A receptors in human neutrophils treated with PEMFs.

This work compares the pharmacological and biochemical properties of A 3 adenosine receptors in human polymorphonuclear neutrophil granulocytes (PMNs) and promyelocytic HL60 cells. The gene expression of A 3 receptors was examined by reverse transcription-polymerase chain reaction experiments, whereas the amount of A 3 subtype on the plasma membrane was quantified by using the high-affinity and selective A 3 antagonist -(3-iodo-benzyl)-2-chloro-adenosine-5Ј-N-methyluronamide (Cl-IB-MECA) for inhibition of cAMP levels via A 3 receptors are in good agreement with the binding data; furthermore, the response is potently inhibited by MRE 3008F20. In contrast, the high micromolar concentrations of Cl-IB-MECA and MRE 3008F20 in stimulating and blocking Ca 2ϩ mobilization, respectively, are not completely consistent with the involvement of an A 3 receptor. Furthermore, an important finding of this work is that the inhibition of PMNs oxidative burst is predominantly A 2A -mediated, even though an effect of A 3 subtype could not be excluded. This conclusion is based on potent blockade of Cl-IB-MECA-mediated inhibition of oxidative burst by SCH 58261 and a minor but significant blockade by MRE 3008F20. In conclusion, HL60 cells express A 3 receptors similar to those in PMNs, thus providing a useful model for investigation of biochemical pathways leading to A 3 receptor activation.Neutrophils play major roles in host defense against the invasion of microorganisms and in acute inflammation and have been implicated in the pathogenesis of a number of human diseases. Adenosine, which has been identified as an important regulator of neutrophil function, particularly with regard to neutrophil superoxide production and neutrophil adherence, may therefore be protective against neutrophilmediated tissue injury (Firestein et al., 1995;Cronstein, 1997). So far, four adenosine receptors have been classified (A 1 , A 2A , A 2B , and A 3 ), all of which are coupled to G proteins (Fredholm et al., 2000;Linden, 2001). Human neutrophils contain both A 2A and A 1 subtypes, which produce opposite effects on several cell functions (Fredholm et al., 1996): activation of A 1 by adenosine at low concentrations is associated with augmentation of chemotaxis and phagocytosis (Sullivan and Linden, 1998), whereas occupation of A 2A by adenosine

1 The present work characterizes, from a pharmacological and biochemical point of view, adenosine receptors in the human malignant melanoma A375 cell line. 3 The pharmacological pro®le of radioligand binding on A375 cells was established using typical adenosine ligands which displayed a rank order of potency typical of the di erent adenosine receptor subtype. 4 Thermodynamic data indicated that radioligand binding to adenosine receptor subtypes in A375 cells was entropy-and enthalpy-driven. 5 In functional assays the high a nity A 2A agonists HE-NECA, CGS 21680 and A 2A ± A 2B agonist NECA were able to increase cyclic AMP accumulation in A375 cells whereas A 3 agonists Cl-IB-MECA, IB-MECA and NECA were able to stimulate Ca 2+ mobilization. 6 In conclusion, all these data indicate, for the ®rst time, that adenosine receptors with a pharmacological and biochemical pro®le typical of the A 1 , A 2A , A 2B and A 3 receptor subtype are present on A375 melanoma cell line.

Adenosine may affect several pathophysiological processes, including cellular proliferation, through interaction with A(1), A(2A), A(2B), and A(3) receptors. In this study we characterized adenosine receptors in human colon cancer tissues and in colon cancer cell lines Caco2, DLD1, HT29. mRNA of all adenosine subtypes was detected in cancer tissues and cell lines. At a protein levels low amount of A(1), A(2A), and A(2B) receptors were detected, whilst the A(3) was the most abundant subtype in both cancer tissues and cells, with a pharmacological profile typical of the A(3) subtype. All the receptors were coupled to stimulation/inhibition of adenylyl-cyclase in cancer cells, with the exception of A(1) subtype. Adenosine increased cell proliferation with an EC(50) of 3-12 microM in cancer cells. This effect was not essentially reduced by adenosine receptor antagonists. However dypiridamol, an adenosine transport inhibitor, increased the stimulatory effect induced by adenosine, suggesting an action at the cell surface. Addition of adenosine deaminase makes the A(3) agonist 2-chloro-N6-(3-iodobenzyl)-N-methyl-5'-carbamoyladenosine (Cl-IB-MECA) able to stimulate cell proliferation with an EC(50) of 0.5-0.9 nM in cancer cells, suggesting a tonic proliferative effect induced by endogenous adenosine. This effect was antagonized by 5-N-(4-methoxyphenyl-carbamoyl)amino-8-propyl-2(2furyl)-pyrazolo-[4,3e]-1,2,4-triazolo [1,5-c] pyrimidine (MRE 3008F20) 10 nM. Cl-IB-MECA-stimulated cell proliferation involved extracellular-signal-regulated-kinases (ERK1/2) pathway, as demonstrated by reduction of proliferation with 1,4-diamino-2,3-dicyano-1,4-bis-[2-amino-phenylthio]-butadiene (U0126) and by ERK1/2 phosphorylation. In conclusion this study indicates for the first time that in colon cancer cell lines endogenous adenosine, through the interaction with A(3) receptors, mediates a tonic proliferative effect.