Extracellular ATP is known to trigger apoptosis of thymocytes and lymphocytes through a P2Z receptor at which ATP is a partial agonist, giving only 70% of the maximum response of 3'-O-(4-benzoyl)benzoyl-adenosine 5'-triphosphate (BzATP), a full agonist. This cytolytic receptor and its associated ion channel are Ca2+ (and Ba2+) selective but also pass molecules up to the size of ethidium cation (314 Da). RT-PCR showed identity between lymphocyte P2Z and the hP2X7 gene recently cloned from human monocytes. When human leukemic B lymphocytes were incubated with ATP and 133Ba2+, an immediate influx of isotope occurred. It was augmented by 45% when ATP was added 10 min before isotope. Time-resolved flow cytometry was used to examine kinetics of ethidium uptake in cells incubated with BzATP or the partial agonists ATP, 2-methylthioadenosine 5'-triphosphate, or adenosine 5'-O-(3-thiotriphosphate). Maximally effective concentrations of BzATP (50 microM) induced immediate uptake of ethidium at a rate linear with time. In contrast, a delay was observed (30 s) before ethidium uptake commenced after addition of maximally effective ATP concentrations (500 microM) at 37 degreesC, and the delay was longer at 24 degreesC. ATP addition 2-10 min before ethidium abolished the delay. The delay was longer with other partial agonists and inversely related to maximal flux produced by agonist. A delay was also observed for submaximal BzATP concentrations (10-20 microM). P2Z/P2X7 inhibitors, KN-62 and 5-(N, N-hexamethylene)-amiloride, reduced the rate of agonist-induced ethidium uptake and lengthened the delay. The results support a model in which agonists for P2Z/P2X7 receptor mediate an immediate channel opening allowing passage of small inorganic cations, followed by a slow further permeability increase allowing passage of larger permeant cations like ethidium. The rate of the second step depends on time and temperature and the efficacy and concentration of agonist and is slowed by antagonists, suggesting it depends on the fraction of P2Z/P2X7 channels held in the initial open state.

1 Extracellular adenosine triphosphate (ATP) is known to open a receptor-operated ion channel (P-2Z class) in human lymphocytes which conducts a range of cationic permeants. The activity of a range of different agonists and inhibitors towards the P-2Z-purinoceptor was investigated by measuring the agonist-induced influx of Ba2+ into fura-2 loaded lymphocytes.2 The most potent agonist was 2' and 3'-0-(4-benzoylbenzoyl)-ATP (benzoylbenzoic ATP) which gave 2 fold greater maximum Ba2+ influx and had a 10 fold lower EC(50) than for ATP. The rank order of agonist potency in K+-media was benzoylbenzoic ATP >> ATP = 2-methylthio ATP = 2-chloro ATP > ATP-gamma-S. ADP, UTP and alpha,beta-methylene ATP were unable to stimulate Ba2+ influx.3 Extracellular Na+ inhibited the increment of Ba2+ influx induced by all concentrations of ATP, 2-methylthio ATP, 2-chloroATP and ATP-gamma-S, This inhibitory effect of extracellular Na+ is also reflected in the different EC(50)s for benzoylbenzoic ATP (8 mu M in K+-media, 18 mu M in Na+-media) but the maximal response to this agonist was the same in the presence or absence of Na+.4 Treatment of lymphocytes with 2,3 dialdehyde ATP (oxidized ATP) at 300 mu M for 60 min gave total and irreversible inhibition of ATP-induced Ba2+ influx. 5'-p-Fluorosulphonyl benzoyiadenosine (FSBA) also was an irreversible inhibitor but the maximal inhibition achieved was 90%.5 It is concluded that the P-2Z-purinoceptor of human lymphocytes has a rank order of agonist potency which clearly distinguishes it from other P-2-receptors and that oxidized ATP is a convenient irreversible inhibitor for the P-2Z-purinoceptor.

Nucleoside transporter expression has been linked to proliferation in a variety of haemopoietic cell types. Granulocyte-macrophage colony-stimulating factor (GM-CSF) was given for 72 h before commencing chemotherapy in 15 patients with relapsed or refractory acute myeloid leukaemia (AML) and in 11 patients serial bone marrows were taken for measurement of [3H]thymidine labelling index, Ki-67 positivity and maximal binding of 5-(SAENTA-x8)-fluorescein, a flow cytometry ligand which enumerates nucleoside transporter sites. GM-CSF caused proliferation of marrow myeloblasts in eight of 11 patients, while in three patients there was no change in proliferative indices. The expression of nucleoside transporters increased up to 4-fold in the myeloblasts from the patients showing a proliferative response to GM-CSF but there was no increase in transporters on the myeloblasts from the three non-responding patients. A close correlation was found between the fold increase in nucleoside transporter expression and the fold increase in labelling index of marrow myeloblasts (r = 0.86, n = 9, p < 0.01). In one patient with acute megakaryoblastic leukemia, GM-CSF caused parallel increases in labelling index, Ki-67 positivity and numbers of nucleoside transporters on peripheral blood blast cells. Thus induction of proliferation by cytokine increases the expression of nucleoside transporters on leukaemic myeloblasts studied in serial samples from the same source (bone marrow or blood). The suitability of 5-(SAENTA-x8)-fluorescein for two colour flow cytometric analysis allows the rapid enumeration of nucleoside transporters in the myeloblast compartment of heterogeneous marrow samples.

Quantitation of equilibrative, nitrobenzylthioinosine (NBMPR) sensitive (es) nucleoside transporters on blast cells isolated from patients with acute myeloblastic leukemia is useful in predicting intracellular accumulation of the antileukemic nucleoside drug, cytosine arabinoside. We previously reported the synthesis of a fluorescein-labeled ligand for the es nucleoside transporter, 5-(SAENTA-x2)-fluorescein. This paper reports the synthesis of 5-(SAENTA-x8)-fluorescein in which the linkage between fluorescein and nucleoside ligand has been increased from 2 atoms to 8 atoms. This new ligand had a sixfold increase in affinity (Kd 0.9 +/- 0.1 nM) as well as an 86% increase in the cell associated fluorescence output compared to its prototype 5-(SAENTA-x2)-fluorescein. The fluorescence signal arising from 5-(SAENTA-x8)-fluorescein specifically bound to freshly isolated and cultured leukemic myeloblasts was converted to molecules of equivalent soluble fluorescein (MESF) using standardized fluorescein microbeads and compared with the number of es nucleoside transporter sites assayed concurrently by [3H]NBMPR equilibrium binding analysis. A high correlation between the two assays was observed (r = 0.98), which enabled the cell-bound fluorescence output of 5-(SAENTA-x8)-fluorescein to be expressed in numbers of es nucleoside transporter sites per cell. The improved properties of 5-(SAENTA-x8)-fluorescein over those of its prototype molecule make it a suitable reagent for flow cytometric quantitation of nucleoside transporter expression on leukemic cells isolated from patient samples.

New strategies are needed to improve the outlook for patients with acute myeloid leukemia (AML). The use of granulocyte-macrophage colony stimulating factor (GM-CSF) given prior to and in combination with chemotherapy represents such a new approach and its safety and efficacy have been established [1, 2]. GM-CSF is a growth promoter of leukemic colony-forming cells in vitro [3, 4] and can render such cells more susceptible to the cytotoxic action of cytosine arabinoside (araC). When given in vivo to patients with AML, GMCSF usually increases the proliferative activity of myeloblasts and in a small study is associated with a high remission rate to subsequent chemotherapy incorporating araC.

The N6-(4-nitrobenzyl) derivative of adenosine is a tight-binding inhibitor of the equilibrative inhibitor-sensitive nucleoside transporter of mammalian cells. A fluorescent ligand for this transporter has been synthesized by allowing an adenosine analogue. 5'-S-(2-aminoethyl)-N6-(4-nitrobenzyl)-5'-thioadenosine (SAENTA), to react with fluorescein isothiocyanate. The purified adduct had a SAENTA/fluorescein molar ratio of 0.92:1 calculated from its absorption spectrum. The intensity of fluorescent emission from the SAENTA-chi 2-fluorescein adduct was 30% that of fluorescein isothiocyanate (chi 2 is the number of atoms in the linkage between fluorescein and SAENTA). SAENTA-chi 2-fluorescein inhibited the influx of nucleosides into cultured leukaemic cells with an IC50 (total concentration of inhibitor producing 50% inhibition) of 40 nM. The adduct inhibited the binding of [3H]nitrobenzylthioinosine ([3H]NBMPR) with half-maximal inhibition at 50-100 nM. Mass Law analysis of the competitive-binding data suggested the presence of two classes of sites for [3H]NBMPR binding, only one of which was accessible to SAENTA-chi 2-fluorescein. Flow cytometry was used to analyse equilibrium binding of SAENTA-chi 2-fluorescein to leukaemic cells and a Kd of 6 nM was obtained. SAENTA-chi 2-fluorescein is a high-affinity ligand for the equilibrative inhibitor-sensitive nucleoside transporter which allows rapid assessment of transport capacity by flow cytometry.

Accumulation of cytosine arabinoside triphosphate (araCTP) from a range of cytosine arabinoside (araC) concentrations (1–50 μM) was measured during incubations of leukemic cells freshly isolated from patients with acute leukemia. In all but one patient, increments in extracellular araC above 10 μM did not increase intracellular araCTP levels. This maximal level of araCTP accumulation ranged from 254 to 1607 pmol/107 cells attained after 1 h incubation and did not correlate with either the number of nucleoside transporters on the cell membrane or the Vmax of araC phosphorylation in cell free extracts. Extremely low araCTP accumulation (103 pmol/107 cells/h at 50 μM araC) was observed in an AML patient with the unusual finding of micromyeloblasts. These cells also had very low numbers of nucleoside transport sites (< 500 sites/cell) and were mitotically inactive. The unique feature of the myeloblasts from this patient was that intracellular araCTP accumulation showed a linear dependence on extracellular araC up to 50 μM with no evidence of saturation.

Summary The proliferation of mammalian cells requires nucleosides which are provided either by de novo synthesis or by influx of nucleosides via membrane transporters with subsequent metabolic trapping. In this study the density of nucleoside transporters in freshly‐isolated blast cells from patients with leukaemias and lymphomas was quantitated by equilibrium binding of 3H‐nitrobenzylmercaptopurine riboside (NBMPR). In acute myeloid leukaemia (AML) the density of NBMPR binding sites on blast cells ranged from 3800 to 24 200 sites/cell and this value correlated with the 3H‐thymidine labelling index (1–20%) which was used to measure proliferative rate (r=0.80, P < 0.001). Cells from patients with Burkitt's lymphoma, other B‐cell lymphomas, T‐lymphoblastic lymphoma and large cell lymphoma gave a 20‐fold range of NBMPR site densities (from 3700 to 75 300 sites/cell) and site numbers correlated closely with the labelling index (r=0.87, P < 0.001). Non‐proliferating cells from patients with chronic lymphocytic leukaemia expressed the lowest density of NBMPR binding sites (850–2900 sites/cell). Comparison of bone marrow and peripheral blood blasts confirmed the positive correlation between NBMPR binding sites and labelling index for four individual patients. In contrast, the density of NBMPR binding sites on lymphoblasts from non‐T acute lymphoblastic leukaemia (ALL) was low (2300–7400 sites/cell) and showed little dependence on proliferation over a wide range of labelling indices (1–20%). No correlation was observed between NBMPR site density and cell size measured by the intracellular water space. Thus an increased proliferative rate of AML or lymphoma is associated with higher numbers of nucleoside transporters in the cell membrane.

1-beta-D-Arabinofuranosylcytosine (araC) is an effective drug in the i.p. therapy of ovarian carcinoma but little is known of its transport and metabolism in this tumor. Influx of araC at 1 microM into cultured human ovarian carcinoma cells (CI 80-13S) was largely inhibited by nanomolar concentrations of the nucleoside transport inhibitor, nitrobenzylthioinosine, while the residual influx (approximately 10%) was inhibited only by micromolar concentrations of nitrobenzylthioinosine. There was a two fold greater density of specific [3H]nitrobenzylthioinosine binding to the nucleoside transporters on the ovarian than on cultured human leukemic cells (RC2a). Calculated turnover rates of the nucleoside transporter for 1 microM araC were 5-fold less in ovarian than in leukemic cells. The major metabolic product of araC was 1-beta-D-arabinofuranosylcytosine 5'-triphosphate (araCTP) which accumulated in the ovarian cells to levels half those achieved in the leukemic cells. AraC was the major product of araCTP degradation in ovarian cells consistent with a pathway (araCTP--------araCMP----araC) which is different from that previously found in leukemic cells (araCTP--------araCMP----araUMP----araU). Despite these differences, ovarian carcinoma cells show substantial accumulation of araCTP from extracellular araC.

The intracellular half-life for retention of the active triphosphate metabolite 1-beta-D-arabinofuranosylcytosine 5'-triphosphate (araCTP) of 1-beta-D-arabinofuranosylcytosine was measured in vitro in blast cells from patients with acute myeloblastic leukemia, acute lymphoblastic leukemia, and T-cell lymphoblastic lymphoma. araCTP accumulation from 1 microM 1-beta-D-arabinofuranosylcytosine in leukemic blast cells was closely correlated with the nucleoside transport capacity as measured by equilibrium binding of [3H]nitrobenzylthioinosine. The half-life of araCTP retention was related to araCTP accumulation only when the level of araCTP was expressed as a percentage of total intracellular 1-beta-D-arabinofuranosylcytosine metabolites. Accumulation of 1-beta-D-arabinofuranosyluracil 5'-monophosphate was inversely related to the half-life of araCTP retention and directly related to dCMP deaminase activity in cell free extracts. No conversion of 1-beta-D-arabinofuranosyluracil to 1-beta-D-arabinofuranosyluracil 5'-monophosphate was detectable in intact cells. The end product of araCTP degradation was 1-beta-D-arabinofuranosyluracil and it is proposed that conversion of 1-beta-D-arabinofuranosylcytosine 5'-monophosphate to 1-beta-D-arabinofuranosyluracil 5'-monophosphate is a step in the degradative pathway of araCTP. However, it is the cells' nucleoside transport capacity which primarily determines the level of intracellular araCTP accumulation.

l-/3-D-Arabinofuranosylcytosine(araC) is an effective drug in the ¡.p. therapy of ovarian carcinoma but little is known of its transport and metabolism in this tumor. Influx of araC at l «IM into cultured human ovarian carcinoma cells (CI 80-13S) was largely inhibited by nanomolar concentrations of the nucleoside transport inhibitor, nitrobenzylthioino- sine, while the residual influx (approximately 10%) was inhibited only by micromolar concentrations of nitrobenzylthioinosine. There was a two fold greater density of specific (3H)nitrobenzylthioinosinebinding to the nucleoside transporters on the ovarian than on cultured human leukemic cells (RC2a). Calculated turnover rates of the nucleoside transporter for 1 nM araC were 5-fold less in ovarian than in leukemic cells. The major metabolic product of araC was l-/3-D-arabinofuranosyIcytosine 5'-tri- phosphate (araCTP) which accumulated in the ovarian cells to levels half those achieved in the leukemic cells. AraC was the major product of araCTP degradation in ovarian cells consistent with a pathway (araCTP â€"¿�»â€"» araCMP â€"¿�> araC) which is different from that previously found in leukemic cells (araCTP â€"¿�>â€"» araCMP -* araUMP â€"¿�» araU). Despite these differences, ovarian carcinoma cells show substantial accumulation of araCTP from extracellular araC.