Characterization of a Novel Na+-dependent, Guanosine-specific, Nitrobenzylthioinosine-sensitive Transporter in Acute Promyelocytic Leukemia Cells

Abstract

NB4 cells are the only bona fide in vitro model of human acute promyelocytic leukemia. We have examined cytidine and guanosine transport in this cell line and characterized a novel guanosine-specific transporter. Cytidine transport occurred predominately by equilibrative nitrobenzylthioinosine (NBMPR)-sensitive (es) transport. In the presence of Na ؉ , guanosine at various concentrations accumulated at least 6-fold above equilibrium. The initial rate of guanosine transport in Na ؉ buffer decreased by 75% with the addition of 1 M NBMPR and the IC 50 for NBMPR inhibition was 0.7 ؎ 0.1 nM. Replacement of Na ؉ with choline also resulted in a 75% decrease in total guanosine transport. The potent inhibition of guanosine transport by NBMPR and the loss of transport in choline suggested that a Na ؉ -dependent NBMPR-sensitive transporter was responsible for the majority of guanosine uptake. This concentrative, sensitive transporter is Na ؉ dependent with a stoichiometric coupling ratio of 1:1. This novel transporter, referred to as csg, is guanosine-specific with total guanosine transport inhibited by only 50% in the presence of 1 mM competing nucleosides. HL-60, acute myelocytic leukemia cells, do not exhibit csg activity while L1210, murine acute lymphocytic leukemia cells, exhibit csg transport. The presence of the csg transporter suggests an important role for guanosine in particular forms of leukemia and may provide a new target for cytotoxic therapy.Purine and pyrimidine nucleotides, and their related metabolic products, participate in numerous biological processes.

Nucleoside anticancer drugs: the role of nucleoside transporters in resistance to cancer chemotherapy

“…The transporter proteins that are responsible for these activities are cit (hCNT1), cif (hCNT2) and cib (hCNT3). However, the proteins responsible for cs (concentrative and sensitive to NBMPR) (Paterson et al, 1993), csg (concentrative, sensitive to NBMPR and accepts guanosine as permeant) (Flanagan and Meckling-Gill, 1997) and cit with specificity towards guanosine have not been identified (Gutierrez et al, 1992;Giacomini, 1993, 1994) and are not discussed further in this review. The mammalian transporter responsible for the cit process (now known to be CNT1) was first described in freshly isolated mouse intestinal cells (Vijayalakshmi and Belt, 1988) and exhibited a preference for pyrimidine nucleosides.…”

“…Six functional subtypes of CNT processes (Gutierrez et al, 1992;Belt et al, 1993;Giacomini, 1993, 1994;Paterson et al, 1993;Cass, 1995;Griffith and Jarvis, 1996;Flanagan and Meckling-Gill, 1997;Ritzel et al, 1997Ritzel et al, , 1998Ritzel et al, , 2001aCass et al, 1999) have been described in human cells or tissues. The three best characterized CNTs are Na þ -nucleoside cotransporters and are termed cit, cif and cib to indicate that they are concentrative, insensitive to NBMPR and accept as permeants, respectively, pyrimidine nucleosides, purine nucleosides or both pyrimidine and purine nucleosides.…”

Molecular Identification and Characterization of Novel Human and Mouse Concentrative Na+-Nucleoside Cotransporter Proteins (hCNT3 and mCNT3) Broadly Selective for Purine and Pyrimidine Nucleosides (System cib)

“…Although their permeant preferences have not been well defined, the csg process (34) accepts guanosine, and the cs process (35) accepts adenosine analogs as permeants. In contrast to cit, cif, and cib, both are inhibited by nanomolar concentrations of NBMPR (34,35). hCNT3 was unaffected by NBMPR or other equilibrative nucleoside transport inhibitors, dipyridamole and dilazep, at concentrations up to 10 M (100 M for dilazep, which is more soluble), eliminating hCNT3 as a possible contributor to csg or cs transport activity (Fig.…”

“…In addition to the three major mammalian concentrative nucleoside transport systems cit, cif, and cib, there are two minor Na ϩ -dependent nucleoside transport processes, csg and cs, which have been described only in leukemic cells (34,35). Although their permeant preferences have not been well defined, the csg process (34) accepts guanosine, and the cs process (35) accepts adenosine analogs as permeants.…”

Equilibrative and Concentrative Nucleoside Transporters Mediate Influx of Extracellular Cyclic ADP-Ribose into 3T3 Murine Fibroblasts

“…Two additional concentrative nucleoside transport processes have been reported in leukemic cells and shown to be inhibited by nanomolar concentrations of NBMPR and by micromolar concentrations of dipyridamole, but not yet molecularly identified: cs (concentrative, sensitive, Ref. 47) and csg (concentrative, sensitive, and guanosine-preferring) (48,49).…”