thioinosine and Neuroblastoma

Nucleoside transport regulation in undifferentiated Neuro-2A cells has been studied and found to include Na+-dependent adenosine transport and facilitated diffusion adenosine transport. The latter corresponded to nitrobenzylthioinosine-sensitive nucleoside transport. Short-term treatment of Neuro-2A cells with physiologically relevant signals only modulated the facilitated diffusion component. The stimulation of undifferentiated cells with forskolin or other activators of the protein kinase A pathway, decreased NBTI-sensitive adenosine transport. Treatment of cells with an inactive analogue of forskolin, 1,9-dideoxi-forskolin, had no effect on NBTI-sensitive nucleoside transport. Therefore, the inhibition of protein kinase A activity by pre-incubation with H-89 or the cAMP antagonist, Rp-8-Br-cAMPS, completely prevented the inhibitory effect of forskolin. Similarly, the activation of protein kinase C with phorbol 12,13-dibutyrate (PDBu) and the calcium ionophore A-23187 decreased NBTI-sensitive adenosine transport. The effect of PDBu was reversed by pre-incubation of cells with staurosporine. Maximal transport inhibition was obtained by the simultaneous stimulation of cells with a phorbol ester and A-23187 or a phorbol ester and forskolin. The modulation of NBTI-sensitive nucleoside transport corresponded to changes in specific [3H]NBTI binding to Neuro-2A cells. Maximal inhibition correlated well with a maximal enhancement of cAMP production. However, the Na+-dependent adenosine transport in Neuro-2A cells was not modulated by any of these signals.

Topics: Adenosine; Animals; Binding Sites; Biological Transport; Colforsin; Cyclic AMP; Cyclic AMP-Dependent Protein Kinases; Enzyme Activation; Mice; Neuroblastoma; Protein Kinase C; Sodium; Thioinosine; Tritium; Tumor Cells, Cultured

The nucleoside transport characteristics of undifferentiated and differentiated LA-N-2 human neuroblastoma cells were compared through measurement of the cellular accumulation of [3H]formycin B in the absence and presence of specific nucleoside transport blockers such as dipyridamole and nitrobenzylthioinosine (NBMPR). [3H]NBMPR was also used as a high affinity probe to obtain an estimate of the number of NBMPR-sensitive nucleoside transport proteins. Undifferentiated LA-N-2 cells accumulated [3H]formycin B (25 microM) via a NBMPR/dipyridamole sensitive, Na(+)-independent, nucleoside transport system (Vi = 1.52 pmol/microliters/s; maximum intracellular concentration = 45 pmol/microliters cell water). The undifferentiated cells also had a high density of site-specific [3H]NBMPR binding sites (135,000 sites/cell; KD = 0.4 nM). When cell differentiation was induced by exposure to a serum-free defined medium, the initial rate of transporter-mediated [3H]formycin B uptake increased to 1.92 pmol/microliters/s, and the steady-state intracellular concentration of [3H]formycin B also increased significantly to 73 pmol/microliters. However, there was no concomitant change in the number of [3H]NBMPR binding sites, and the additional uptake was not Na(+)-dependent. This enhanced uptake in the differentiated cells appeared to be due, in part, to an increased functional expression of a NBMPR-resistant form of facilitated nucleoside transporter. Approximately 18% of the transporter-mediated uptake in the differentiated cells was resistant to inhibition by NBMPR at concentrations that blocked transport completely in the undifferentiated cells. This cell model may prove useful for basic studies on regulation of nucleoside transporter subtype expression in neural tissues, and for evaluation of the efficacy and potential host toxicity of cytotoxic nucleoside analogues (+/- specific transport blockers) in the treatment of neuroblastoma.

Topics: Biological Transport; Cell Differentiation; Formycins; Humans; Neuroblastoma; Nucleosides; Thioinosine; Tumor Cells, Cultured

The activities of an endogenous nucleoside, 5'-deoxy-5'-methylthioadenosine (MTA), on adenosine sensitive sites such as adenosine A1 and A2 receptors and the P-site, as well as on purine nucleoside transport, have been studied. This nucleoside competitively antagonized the A2 receptor-mediated stimulation of neuroblastoma adenylate cyclase, produced a GTP-dependent and 8-p-sulfophenyltheophylline-sensitive inhibition of adenylate cyclase activity in rat cerebellar membranes, and decreased the spontaneous contractile activity of isolated segments of rabbit jejunum. MTA was neither active at the P-site nor did it diminish the binding of [3H]nitrobenzylthioinosine, a nucleoside transport inhibitor. We conclude that (a) MTA is an agonist at the adenosine A1 receptor but an antagonist at the A2 receptor, and (b) the adenosine receptor which causes relaxation of rabbit jejunum is not a neuroblastoma-type A2 receptor which activates adenylate cyclase.

Topics: Adenosine; Adenosine-5'-(N-ethylcarboxamide); Adenylyl Cyclase Inhibitors; Animals; Cerebellum; Deoxyadenosines; In Vitro Techniques; Jejunum; Mice; Muscle Contraction; Neuroblastoma; Rabbits; Rats; Receptors, Purinergic; Thioinosine; Thionucleosides

Tubercidin, an adenosine analogue, is toxic to human neuroblastoma cell lines, to peripheral blood mononuclear cells (PBMCs), and to myeloid colony-forming cells (CFU-C) as tested by a short-term labeled precursor uptake and by a clonogenic assay. When it was co-administered with a potent purine transport inhibitor, nitrobenzyl thioinosine (NBTI), the cytotoxic effect of tubercidin was abolished in PBMCs but not in neuroblastoma cells. Studies of nucleoside transport in neuroblastoma cells demonstrate that although [3H]NBTI binds to the plasma membrane of these cells, the transport of thymidine into the cells is only partially inhibited in the presence of excess NBTI. These data imply that neuroblastoma cells contain a nucleoside transport mechanism which is insensitive to NBTI. "Host protection" with a nucleoside transport inhibitor such as NBTI, may allow effective therapy with otherwise toxic dosages of tubercidin and other cytotoxic nucleosides in patients with neuroblastoma.

Topics: Biological Transport; Cell Line; Colony-Forming Units Assay; Humans; Inosine; Lymphocytes; Neuroblastoma; Ribonucleosides; Thioinosine; Thymidine; Tubercidin; Tumor Stem Cell Assay