Interaction of 2′,2′-Difluorodeoxycytidine (Gemcitabine) and Formycin B with the Na+-Dependent and -Independent Nucleoside Transporters of Ehrlich Ascites Tumor Cells (original) (raw)
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Solubilization and reconstitution of a nucleoside-transport system from Ehrlich ascites-tumour cells
Biochemical Journal, 1989
Uptake of [3H]uridine by Ehrlich cells was mediated by both nitrobenzylthioinosine (NBMPR)-sensitive (75%) and NBMPR-insensitive (25%) mechanisms. Each cell contained approx. 26,000 high-affinity (KD = 0.19 nM) recognition sites for [3H]NBMPR, and binding was inhibited by dipyridamole and adenosine at concentrations similar to those required for inhibition of [3H]uridine uptake. Calculations show that each cell contains a total of about 35,000 nucleoside transporters. Photoaffinity labelling of a partially purified preparation of plasma membranes with [3H]NBMPR resulted in a single broad 3H-labelled band on SDS/polyacrylamide gels, with an apparent molecular-mass peak of 42 kDa. This is in contrast with human erythrocyte membranes, where [3H]NBMPR photolabelled two broad bands with peaks at 55 and 80 kDa. Treatment of photoaffinity-labelled membranes with endoglycosidase F decreased the apparent molecular masses of both the Ehrlich-cell and erythrocyte [3H]NBMPR-labelled proteins to...
Cancer research, 1998
Gemcitabine (2',2'-difluorodeoxycytidine) is a novel pyrimidine nucleoside drug with clinical efficacy in several common epithelial cancers. We have proposed that gemcitabine requires nucleoside transporter (NT) proteins to permeate the plasma membrane and to exhibit pharmacological activity. In humans, there are seven reported distinct NT activities varying in substrate specificity, sodium dependence, and sensitivity to inhibition by nitrobenzylthioinosine (NBMPR) and dipyridamole. To determine which NTs are required for gemcitabine-dependent growth inhibition, cultures from a panel of 12 cell lines with defined plasma membrane NT activities were incubated with different concentrations of gemcitabine. Cell proliferation was assessed by the sulforhodamine B assay and cell enumeration to identify the concentrations of gemcitabine that inhibited cell replication by 50% (IC50s). NT activity was a prerequisite for growth inhibition in vitro because: (a) the nucleoside transport-...
Biochimica et Biophysica Acta (BBA) - Biomembranes, 1990
Cr.1 cells, LK35.2 mouse B-cell hybridoma cells and cultured mouse peritoneal macrophages express both Na+-depe ndent, active and nonconcentrative, facilitated nucleoside transport systems. In the mouse cell lines, active transport represented only a minor nucleoside transport component and was detected only by measuring formycin B uptake in the presence of dipyridamole or nitrobenzylthioinosine, strong inhibitors of facilitated, but not of active, nucleoside transport. Inhibition of facilitated transport resulted in the concentrative accumulation of formycin B in cells expressing active nucleoside transport. Concentrative formycin B accumulation was abolished by treatment of the cells with gramicidin or absence of Na + in the extracellular medium and strongly inhibited by ATP depletion or ouabain treatment. Mouse macrophages accumulated formycin B to 70-times the extracellnlar concentration in the absence of dipyridamole during 90 min of incubation at 37 ° C. Thus active transport represents a major nucleoside transport system of these cells, similarly as previously reported for mouse spleen lymphocytes. In contrast to the various types of mouse cells, active formycin B transport was not detected in human HeLa cells, human H9, Jurkat and CEM T lymphoidal cells and pig spleen lymphocytes. These cells expressed only facilitated nucleoside transport with kinetic properties similar to those of the facilitated transporters of other mammalian cells.
Journal of Biological Chemistry
Nucleoside transport was examined in freshly isolated mouse intestinal epithelial cells. The uptake of formycin By the C nucleoside analog of inosine, was concentrative and required extracellular sodium. The initial rate of sodium-dependent formycin B transport was saturable with a K,,, of 45 f 3 p~. The purine nucleosides adenosine, inosine, guanosine, and deoxyadenosine were all good inhibitors of sodium-dependent formycin B transport with 50% inhibition (ICSO) observed at concentrations less than 30 pM. Of the pyrimidine nucleosides examined, only uridine ( G O , 4 1 f 9 p~) was a good inhibitor. Thymidine and cytidine were poor inhibitors with ICao values greater than 300 p~. Direct measurements of ['Hlthymidine transport revealed, however, that the uptake of this nucleoside was also mediated by a sodium-dependent mechanism. Thymidine transport was inhibited by low concentrations of cytidine, uridine, adenosine, and deoxyadenosine (ICao values less than 25 pM), but not by formycin B, inosine, or guanosine (ICs0 values greater than 600 p~) .
Nucleoside transport and its significance for anticancer drug resistance
Drug Resistance Updates, 1998
AbstractThis article discusses the role of nucleoside transport processes in the cytotoxicity of clinically important anticancer nucleosides.This article summarizes recent advances in the molecular biology of nucleoside transport proteins, review the current state of knowledge of the transportability of therapeutically useful anticancer nucleosides, and provide an overview of the role of nucleoside transport deficiency as a mechanism of resistance to nucleoside cytotoxicity are summarized. Several strategies for utilization of nucleoside transport processes to improve the therapeutic index of anticancer therapies, including the use of nucleoside-transport inhibitors to modulate toxicity of both nucleoside and nonnucleoside antimetabolite drugs are also presented.
Biochimica et Biophysica Acta (BBA) - Biomembranes, 1990
Na+-dependent, active and Na+-independent facilitated nucleoside transport were characterized in mouse spleen cells using rapid kinetic techniques and formycin B, a metabolically inert analog of inosine, as substrate. The Michaelis-Menten constants for formycin B transport by the two transporters were about 30 and 400 pM, respectively. The firstorder rate constant for Na+-dependent transport was about 4-times higher than that for facilitated formycin B transport. The Na+-dependent carrier is specific for uridine and purine nucleosides and accumulates formycin B concentratively in an unmodified form.
Nucleoside uptake in rat liver parenchymal cells
Biochemical Journal, 1996
Rat liver parenchymal cells express Na+-dependent and Na+-independent nucleoside transport activity. The Na+-dependent component shows kinetic properties and substrate specificity similar to those reported for plasma membrane vesicles [Ruiz-Montasell, Casado, Felipe and Pastor-Anglada (1992) J. Membr. Biol. 128, 227–233]. This transport activity shows apparent Km values for uridine in the range 8–13 μM and a Vmax of 246 pmol of uridine per 3 min per 106 cells. Most nucleosides, including the analogue formycin B, cis-inhibit Na+-dependent uridine transport, although thymidine and cytidine are poor inhibitors. Inosine and adenosine inhibit Na+-dependent uridine uptake in a dose-dependent manner, reaching total inhibition. Guanosine also inhibits Na+-dependent uridine uptake, although there is some residual transport activity (35% of the control values) that is resistant to high concentrations of guanosine but may be inhibited by low concentrations of adenosine. The transport activity ...
Biochimica et Biophysica Acta (BBA) - Biomembranes, 1968
Transport of cytosine arabinoside and deoxycytidine was examined in a subline of the LI2IO Inurine leukemia unable to metabolize either nucleoside. At 37 °, uptake of these nucleosides was unaffected by metabolic inhibitors, but apparently saturable. A cell/mediuin distribution ratio of I was achieved, within I rain of incubation, except at the highest nucleoside level tested (25 raM). Kinetic studies were mainly carried out at o °, at which temperature nucleoside uptake was slowed and both saturability and structural specificity of nucleoside transport could readily be demonstrated.
Cancer Chemotherapy and Pharmacology, 2008
Gemcitabine is a hydrophilic clinical anticancer drug that requires nucleoside transporters to cross plasma membranes and enter cells. Pancreatic adenocarcinomas with low levels of nucleoside transporters are generally resistant to gemcitabine and are currently a clinical problem. We tested whether transfection of human concentrative nucleoside transporter 3 (hCNT3) using ultrasound and lipid stabilized microbubbles could increase gemcitabine uptake and sensitivity in HEK293 cells made nucleoside transport deficient by pharmacologic treatment with dilazep. To our knowledge, no published data exists regarding the utility of using hCNT3 as a therapeutic gene to reverse gemcitabine resistance. Our ultrasound transfection system-capable of transfection of cell cultures, mouse muscle and xenograft CEM/araC tumors-increased hCNT3 mRNA and 3 H-gemcitabine uptake by .2,000and 3,400-fold, respectively, in dilazep-treated HEK293 cells. Interestingly, HEK293 cells with both functional human equilibrative nucleoside transporters and hCNT3 displayed 5% of 3 Hgemcitabine uptake observed in cells with only functional hCNT3, suggesting that equilibrative nucleoside transporters caused significant efflux of 3 H-gemcitabine. Efflux assays confirmed that dilazep could inhibit the majority of 3 Hgemcitabine efflux from HEK293 cells, suggesting that hENTs were responsible for the majority of efflux from the tested cells. Oocyte uptake transport assays were also performed and provided support for our hypothesis. Gemcitabine uptake and efflux assays were also performed on pancreatic cancer AsPC-1 and MIA PaCa-2 cells with similar results to that of HEK293 cells. Using the MTS proliferation assay, dilazep-treated HEK293 cells demonstrated 13-fold greater resistance to gemcitabine compared to dilazep-untreated HEK293 cells and this resistance could be reversed by transfection of hCNT3 cDNA. We propose that transfection of hCNT3 cDNA using ultrasound and microbubbles may be a method to reverse gemcitabine resistance in pancreatic tumors that have little nucleoside transport activity which are resistant to almost all current anticancer therapies.