pentostatin and 4-nitrobenzylthioinosine

The combination of 2'-deoxyadenosine and 2'-deoxycoformycin is toxic for the human colon carcinoma cell line LoVo. In this study we investigated the mode of action of the two compounds and have found that they promote apoptosis. The examination by fluorescence microscopy of the cells treated with the combination revealed the characteristic morphology associated with apoptosis, such as chromatin condensation and nuclear fragmentation. The occurrence of apoptosis was also confirmed by the release of cytochrome c and the proteolytic processing of procaspase-3 in cells subjected to the treatment. To exert its triggering action on the apoptotic process, 2'-deoxyadenosine enters the cells through an equilibrative nitrobenzyl-thioinosine-insensitive carrier, and must be phosphorylated by intracellular kinases. Indeed, in the present work we demonstrate by analysis of the intracellular metabolic derivatives of 2'-deoxyadenosine that, as suggested by our previous findings, in the incubation performed with 2'-deoxyadenosine and 2'-deoxycoformycin, an appreciable amount of dATP was formed. Conversely, when also an inhibitor of adenosine kinase was added to the incubation mixture, dATP was not formed, and the toxic and apoptotic effect of the combination was completely reverted.

Topics: Antineoplastic Agents; Apoptosis; Caspases; Cell Adhesion; Cell Count; Cell Line, Tumor; Colonic Neoplasms; Cytochromes c; Deoxyadenosines; Enzyme Precursors; Flow Cytometry; Humans; Microscopy, Fluorescence; Pentostatin; Thioinosine

Previous studies indicated that the peptide neurotensin (NT) stimulates Cl(-) secretion in animal small intestinal mucosa in vitro. In this study, we investigated whether NT causes Cl(-) secretion in human colonic mucosa and examined the mechanism of this response.. Human mucosal preparations mounted in Ussing chambers were exposed to NT. Drugs for pharmacologic characterization of NT-induced responses were applied 30 minutes before NT.. Serosal, but not luminal, administration of NT (10(-8) to 10(-6) mol/L) induced a rapid, monophasic, concentration- and chloride-dependent, bumetanide-sensitive short-circuit current (Isc) increase that was inhibited by the specific nonpeptide NT receptor antagonists SR 48692 and SR 142948A, the neuronal blocker tetrodotoxin, and the prostaglandin synthesis inhibitor indomethacin. The mast cell stabilizer lodoxamide and the histamine 1 and 2 receptor antagonists pyrilamine and ranitidine, respectively, did not significantly alter NT-induced Isc increase. In contrast, the adenosine receptor 1 and 2 antagonists inhibited this secretory response, whereas the adenosine uptake inhibitors S-(4-nitrobenzyl)-6-thioguanosine and S-(4-nitrobenzyl)-6-thioinosine and the adenosine deaminase inhibitor deoxycoformycin potentiated NT-induced Isc increase. Serosal adenosine induced a rapid, monophasic, concentration- and chloride-dependent, bumetanide-sensitive Isc increase.. NT stimulates chloride secretion in human colon by a pathway(s) involving mucosal nerves, adenosine, and prostaglandins.

Topics: Adamantane; Adenosine; Adenosine Deaminase Inhibitors; Affinity Labels; Anti-Inflammatory Agents, Non-Steroidal; Cells, Cultured; Chlorides; Colon; Electrophysiology; Enteric Nervous System; Enzyme Inhibitors; Guanosine; Histamine; Humans; Imidazoles; In Vitro Techniques; Indomethacin; Intestinal Mucosa; Mast Cells; Membrane Potentials; Neurotensin; Pentostatin; Pyrazoles; Quinolines; Receptors, Neurotensin; Tetrodotoxin; Thioinosine; Thionucleosides

A1 adenosine receptors in the rat prepiriform cortex play an important role in the inhibition of bicuculline methiodide-induced convulsions. In the present study we evaluated manipulation of endogenous adenosine in this brain area as a strategy to effect seizure suppression. All compounds evaluated were unilaterally microinjected into the rat prepiriform cortex. Administration of exogenous adenosine afforded a dose-dependent protection (ED50 = 48.1 +/- 8.4 nmol) against bicuculline methiodide-induced seizures, and these anticonvulsant effects were significantly potentiated by treatment with an adenosine kinase inhibitor, 5'-amino-5'-deoxyadenosine; by the adenosine transport blockers, dilazep or nitrobenzylthioinosine 5'-monophosphate; and by an adenosine deaminase inhibitor, 2'-deoxycoformycin. When administered alone, 5'-amino-5'-deoxyadenosine, 5'-iodotubercidin and dilazep were found to be highly efficacious as anticonvulsants with respective ED50 values of 2.6 +/- 0.8, 4.0 +/- 2.7 and 5.6 +/- 1.5 nmol. In contrast, 2'-deoxycoformycin was both less potent and less efficacious. These results suggest that accumulation of endogenous adenosine may contribute to seizure suppression, and that adenosine kinase and adenosine transport may play a pivotal role in the regulation of extracellular levels of adenosine in the central nervous system. The adenosine antagonist, 8-(p-sulfophenyl)theophylline, increased markedly the severity of bicuculline methiodide-induced seizures. Moreover, reduction of extracellular adenosine formation by a focal injection of an ecto-5'-nucleotidase inhibitor, alpha, beta-methyleneadenosine diphosphate, produced generalized seizures (ED50 = 37.3 +/- 22.7 nmol). Together the proconvulsant effect of an adenosine receptor antagonist and the convulsant action of an ecto-5'-nucleotidase inhibitor further support the role of endogenous adenosine as a tonically active antiepileptogenic substance in the rat prepiriform cortex.

Topics: Adenosine; Adenosine Deaminase Inhibitors; Adenosine Diphosphate; Adenosine Kinase; Animals; Bicuculline; Cerebral Cortex; Male; Pentostatin; Rats; Rats, Sprague-Dawley; Receptors, Purinergic; Seizures; Theophylline; Thioinosine

Topics: Biological Transport; Carrier Proteins; Humans; Leukemia; Lymphocytes; Lymphoma; Membrane Proteins; Nucleoside Transport Proteins; Pentostatin; Thioinosine; Tumor Cells, Cultured

The uptake of 2',3'-dideoxyadenosine was examined in a human immunodeficiency virus (HIV) infected and uninfected T cell line (H9 cells), a B cell line (Namalwa), and in normal peripheral blood mononuclear cells. After a 10-minute incubation at ambient temperature, the intracellular 2',3'-dideoxyadenosine-derived radioactivity was 8- to 16-fold higher than the extracellular radioactivity. In metabolically inactive cells (0 degrees C), the intracellular and extracellular 2',3'-dideoxyadenosine-derived radioactivities were nearly equal. In infected and noninfected H9 cells, a large excess of p-nitrobenzylmercaptopurine riboside or pyrimidine nucleosides weakly inhibited the uptake of 2',3'-dideoxyadenosine (7-30%), whereas deoxycoformycin was a stronger inhibitor (50-80%). Purine nucleosides minimally enhanced the uptake (10-20%). The cellular uptake was not associated with the accumulation of dideoxyadenosine triphosphate. In normal peripheral blood mononuclear cells, the uptake of 2',3'-dideoxyadenosine was inhibited by all agents except 2'-deoxyadenosine (15% enhancement). In contrast to H9 cells, the formation and accumulation of dideoxyadenosine triphosphate paralleled the uptake of dideoxyadenosine. The results of these studies suggest that the major route of transport of 2',3'-dideoxyadenosine into cells is by simple diffusion and that different metabolic patterns exist among cell lines and normal peripheral blood mononuclear cells. An understanding of these cellular differences could aid in the development of therapeutic strategies directed against HIV.

Topics: Acquired Immunodeficiency Syndrome; Antiviral Agents; Cell Line; Deoxyadenosines; Dideoxyadenosine; Diffusion; HIV; Humans; Nucleosides; Osmolar Concentration; Pentostatin; Temperature; Thioinosine

8-Azidoadenosine was employed as a possible covalent probe of the erythrocyte nucleoside transporter. 8-Azidoadenosine was shown to enter human erythrocytes by a saturable mechanism (apparent Km for influx 80 microM) that was inhibited by nitrobenzylthioinosine (NBMPR), a potent inhibitor of nucleoside transport, and competitively inhibit uridine influx and NBMPR binding. Irradiation with UV light of human erythrocyte membranes or a partially purified preparation of the nucleoside transporter in the presence of [3H]8-azidoadenosine and dithiothreitol (as a free radical scavenger) resulted in selective covalent incorporation into the band 4.5 region of sodium dodecyl sulfate-polyacrylamide gels (Mr 66,000-45,000). Covalent labeling of band 4.5 was inhibited by adenosine, uridine, and inosine, but NBMPR had no effect. Surprisingly, D-glucose and cytochalasin B, but not L-glucose and cytochalasin E, blocked covalent attachment of the ligand. No incorporation of radioactivity into membranes from rabbit and pig erythrocytes was observed, cells which transport nucleosides rapidly, but have little or no functional glucose carrier. Limited treatment with trypsin of unsealed human erythrocyte membranes photolabeled with [3H]8-azidoadenosine yielded a single radioactive fragment of Mr 19,000, a pattern identical to that obtained with [3H]cytochalasin B-labeled membranes. These results suggest that, despite 8-azidoadenosine being a permeant for the nucleoside transporter, under photoactivation 8-azidoadenosine preferentially labeled the glucose carrier.

Topics: Adenosine; Affinity Labels; Azides; Blood Proteins; Coformycin; Cytochalasin B; Dose-Response Relationship, Drug; Erythrocytes; Humans; Kinetics; Membrane Proteins; Molecular Weight; Monosaccharide Transport Proteins; Nucleoside Transport Proteins; Pentostatin; Phenylisopropyladenosine; Phloretin; Photochemistry; Receptors, Cell Surface; Receptors, Purinergic; Thioinosine; Uridine

The rate of nucleoside transport decreased profoundly in human promyelocytic leukemia HL-60 cells after myeloid differentiation was induced by 5-6 days of exposure to 0.8% N,N-dimethylformamide (DMF). The facilitated diffusion of 100 microM radiolabeled adenosine and 2'-deoxyadenosine, measured by rapid transport assays, decreased 10- to 20-fold. The transport of 2 microM coformycin or 2'-deoxycoformycin, which is mediated by the same mechanism and was monitored by the adenosine deaminase titration assay, decreased 29-fold. The reduction in nucleoside transport capacity after DMF treatment was confirmed by a 19-fold decrease in the number of specific binding sites per cell (from 24-30 X 10(4) to 1.2-1.7 X 10(4)) for [3H]-6-p-nitrobenzylthioinosine, a nucleoside transport inhibitor. The binding affinity of 6-p-nitrobenzylthioinosine was not altered significantly and nucleoside transport remained sensitive to the transport inhibitors, 6-p-nitrobenzylthioinosine, dipyridamole, and dilazep after DMF-induced maturation. Time-dependence studies showed that the rate of 100 microM deoxyadenosine transport was unchanged for the first 24 h of exposure to DMF but fell to about 36% of control rates at 24-26 h and then gradually decreased further to about 4-5% of control rates after 5-6 days. In contrast, transport rates of the purine bases were reduced only 2- to 3-fold in HL-60 cells after 5 days of DMF treatment. The rates of adenosine and deoxyadenosine transport were unchanged or reduced by no more than 2-fold after 5-6 days of exposure to 0.8% DMF in the following human tumor cell lines that are not inducible with DMF: ARH-77 (multiple myeloma), KG-1 (acute myelogenous), and K-562 (chronic myelogenous). Thus, changes in nucleoside transport may serve as an early, membrane-associated marker of differentiation of the HL-60 cell line.

Topics: Adenosine; Adenosine Deaminase Inhibitors; Cell Differentiation; Cell Line; Coformycin; Deoxyadenosines; Dimethylformamide; Humans; Leukemia, Myeloid, Acute; Nucleosides; Pentostatin; Purines; Thioinosine

Rapid kinetic techniques were applied to determine the effect of transport inhibitors on the transport and metabolism of adenosine in human red cells. Dipyridamole inhibited the equilibrium exchange of 500 microM adenosine by deoxycoformycin-treated cells in a similar concentration dependent manner as the equilibrium exchange and zero-trans influx of uridine with 50% inhibition being observed at about 20 nM. Intracellular phosphorylation of adenosine at an extracellular concentration of 5 microM was inhibited only by dipyridamole concentrations greater than or equal to 100 nM, which inhibited transport about 95%. Lower concentrations of dipyridamole actually stimulated adenosine phosphorylation, because the reduced influx of adenosine lessened substrate inhibition of adenosine kinase. When the cells were not treated with deoxycoformycin, greater than 95% of the adenosine entering the cells at a concentration of 100 microM became deaminated. A 95-98% inhibition of adenosine transport by treatment with dipyridamole, dilazep, or nitrobenzylthioinosine inhibited its deamination practically completely, whereas adenosine phosphorylation was inhibited only 50-85%. Whether adenosine entering the cells is phosphorylated or deaminated is strictly based on the kinetic properties of the responsible enzymes, substrate inhibition of adenosine kinase, and the absolute intracellular steady state concentration of adenosine attained. The latter approaches the extracellular concentration of adenosine, since transport is not rate limiting, except when modulated by transport inhibitors. In spite of the extensive adenosine deamination in cells incubated with 100 microM adenosine, little IMP accumulated intracellularly when the medium phosphate concentration was 1 mM, but IMP formation increased progressively with increase in phosphate concentration to 80 mM. The intracellular phosphoribosylation of adenine and hypoxanthine were similarly dependent on phosphate concentration. The results indicate that adenosine is the main purine source for erythrocytes and is very efficiently taken up and converted to nucleotides under physiological conditions, whereas hypoxanthine and adenine are not significantly salvaged. Hypoxanthine resulting from nucleotide turnover in these cells is expected to be primarily released from the cells. Adenosine was also dephosphorylated in human red cells presumably by 5'-methylthioadenosine phosphorylase, but this reaction seems without physiological sign

Topics: Adenine Phosphoribosyltransferase; Adenosine; Adenosine Kinase; Biological Transport; Coformycin; Dilazep; Dipyridamole; Erythrocytes; Humans; Pentostatin; Phosphates; Phosphorylation; Thioinosine

Incubation of deoxycoformycin-treated L1210 leukemia cells with dipyridamole or nitrobenzylthioinosine, inhibitors of nucleoside transport, enhanced the long-term incorporation of 2'-deoxyadenosine and adenosine into the nucleotide pool and the toxicity of 2'-deoxyadenosine for the cells. In contrast, 2'-deoxyadenosine uptake in deoxycoformycin-treated P388 leukemia cells, which was about 10 times greater than that in L1210 cells, was inhibited by dipyridamole and nitrobenzylthioinosine, and 2'-deoxyadenosine toxicity was not significantly affected by the transport inhibitors. P388 cells also were about 6 times more resistant to 2'-deoxyadenosine than were L1210 cells, in spite of the greater uptake of the nucleoside. We found that purine nucleoside transport in L1210 and P388 cells exhibited similar kinetic properties and sensitivity to dipyridamole and nitrobenzylthioinosine (both influx and efflux) and that the stimulation of 2'-deoxyadenosine uptake by the inhibitors in L1210 cells is not mediated at the level of its transport into the cells but rather reflects an enhanced intracellular net accumulation of deoxyadenosine nucleotides.

Topics: Adenosine; Animals; Biological Transport; Coformycin; Deoxyadenosines; Dipyridamole; Inosine; Leukemia L1210; Leukemia P388; Leukemia, Experimental; Mice; Pentostatin; Thioinosine

The assay of residual adenosine deaminase (ADA) activity was used as a sensitive measure of the transport of deoxycoformycin (dCF) into human erythrocytes. Contrary to prior reports from this laboratory, the inactivation of intraerythrocytic ADA by dCF was linear rather than log-linear, with time. Linear inactivation rates were also seen when erythrocytes were preloaded with a 5-fold excess of calf intestinal ADA. The uptake of tritium-labeled dCF molecules and the rate of inactivation of ADA molecules showed an approximate 1:1 stoichiometry. The nucleoside transport inhibitors, 6-[(4-nitrobenzyl)thio]-9-beta-D-ribofuranosylpurine (NBMPR) and dipyridamole, and the permeant, uridine, inhibited dCF transport with Ki values of 35 nM, 45 nM, and 340 microM respectively. The affinity of dCF for the nucleoside transporter was low with a Ki of approximately 10 mM for the inhibition of adenosine influx.

Topics: Adenosine Deaminase Inhibitors; Biological Transport; Coformycin; Dipyridamole; Erythrocytes; Humans; Kinetics; Nucleoside Deaminases; Nucleosides; Pentostatin; Ribonucleosides; Thioinosine; Tritium; Uridine

Attempts were made to measure adenosine transport in isolated smooth muscle preparations including guinea-pig taenia caeci, beef coronary arteries and longitudinal muscle of rabbit small intestine. Because adenosine-mediated relaxation is potentiated by nucleoside transport inhibitors such as dipyridamole and 6- thiobenzylpurine ribosides in the first two systems but not in rabbit intestinal muscle, possible differences in transport capacities and in the effects of these inhibitors in the three tissues were examined. Transport was to be measured by assessing metabolic products of adenosine including adenine nucleotides and inosine plus hypoxanthine in both tissues and incubation media. Despite extensive rinsing of tissues, adenosine deaminase leaked into the incubation media, requiring its inhibition by 5 nM deoxycoformycin. When measuring apparent transport rates by quantitating metabolic products in the presence of 5 nM deoxycoformycin, no saturation of uptake at 100 to 400 microM adenosine was observed in taenia caeci and rabbit muscle. Comparing these results with literature reports on transport rates in single cell preparations, it appears that the obtained values (20-40 pmol/mg/min) may be at least 100-fold lower, suggesting that rates of diffusion through tissue and intracellular deamination of adenosine were the limiting functions measured by the methodology used in this study, requiring a careful definition for the terms transport and uptake and suggesting that it is practically not possible to measure true transport of adenosine in intact tissues. The uptake of adenosine was inhibited in all three tissues by dipyridamole and 6- thiobenzylpurine ribosides (10 microM) to a similar extent, leaving open the question of why potentiation of the relaxant effects of adenosine is seen in taenia caeci and coronary arteries but not in rabbit intestinal muscle.

Topics: Adenosine; Adenosine Deaminase Inhibitors; Animals; Biological Transport; Cattle; Coformycin; Coronary Vessels; Dipyridamole; Dose-Response Relationship, Drug; Female; Guanosine; Guinea Pigs; In Vitro Techniques; Inosine; Intestinal Mucosa; Male; Muscle, Smooth; Pentostatin; Rabbits; Thioinosine; Thionucleosides