Selective loss of nucleoside carrier expression in rat hepatocarcinomas

Abstract

Evidence that hepatoma cell lines show differential expression of concentrative nucleoside transporters (CNT1 and CNT2) prompted us to study the transporter proteins in 2 models of hepatocarcinogenesis, the chemically induced Solt and Farber model and the albumin-SV40 large T antigen (Alb-SV40) transgenic rat. CNT1 expression was lower in tumor biopsy specimens from Alb-SV40 rat livers than in normal tissue. Immunocytochemistry revealed that the CNT1 protein was indeed absent in the tumor lesions. CNT1 was also absent in a cell line, L25, derived from the Alb-SV40 transgenic rat liver tumors, whereas another cell line, L37, derived from the normal-appearing parenchyma, retained the expression of both carrier isoforms. The protein expression correlated with the nucleoside transport properties of these cell lines. Moreover, although CNT2 expression was highly dependent on the growth characteristics of the 2 cell lines, as was CNT1 (albeit to a lower extent) in L37 cells, it was not expressed in L25 cells at any stage of cell growth. In contrast to the transgenic model of hepatocarcinogenesis, in the chemically induced tumors the expression of CNT2 was lower, although still detectable. In summary, these data indicate that hepatocarcinogenesis leads to a selective loss or diminished expression of nucleoside carrier isoforms, a feature that may be relevant to our understanding of the molecular basis of the bioavailability of those drugs that are nucleoside derivatives and may be substrates of these carriers. The transport properties and isoform-expression profile of the L25 and L37 cell lines make them suitable hepatocyte culture models with which to study nucleoside transport processes and drug sensitivity. (HEPATOLOGY 2000;32:239-246.)Nucleoside and nucleobase derivatives, such as fluorouracil, cytarabine, fludarabine, cladribine, and gemcitabine, are currently used in the treatment of a variety of hematopoietic malignancies as well as in the chemotherapy of solid tumors. 1,2 Drug bioavailability may rely on the translocation of these prodrugs across the plasma membrane. Thus, the molecular characterization of the carrier proteins involved in the uptake of nucleosides and nucleoside analogs, a long awaited goal, has recently been achieved by several laboratories. [3][4][5][6][7][8] Four isoforms of nucleoside transporters from rat and human tissues have been identified. [3][4][5][6][7][8] The complementary DNAs (cDNAs) responsible for the concentrative Na ϩ -dependent pyrimidine-(CNT1) and purine-preferring (CNT2 or SPNT) nucleoside transport respectively have been cloned, 3,4,8 as have the broad-specificity nitrobenzylthioinosine-sensitive (ENT1) and -insensitive (ENT2) nucleoside equilibrative transporters. 5-7 These transporters are major routes for the uptake of nucleoside derivatives. 3,5,9,10 Nucleoside transport in liver was initially characterized in plasma membrane vesicles 11-13 and appeared to be up-regulated by insulin and during the early phase of liver growth after partial hepatectomy 14,15 consistent...

Reduced Ribavirin Antiviral Efficacy via Nucleoside Transporter-Mediated Drug Resistance

“…There is evidence that localization of ENT1 and other transporters can change from the plasma membrane to intracellular membrane compartments in response to various stimuli, including adaptation to two-dimensional tissue culture (20) (see below). Similarly, the expression and activity of transporters can change in response to transformation (11). Therefore, although our results with Huh-7 cells may or may not apply to liver cells in vivo, they provide a foundation for future studies of the role of nucleoside transporters in host-based RBV resistance and antiviral efficacy.…”

Role of the Transporter Regulator Protein (RS1) in the Modulation of Concentrative Nucleoside Transporters (CNTs) in Epithelia

“…Changes in CNT-related expression associated with cell density might be cell type-dependent and not necessarily consistent with the reported changes in RS1 nuclear localization and inhibitory function. Although CNT1 protein amounts were shown to increase as cultures became confluent for rat hepatocytederived cell lines (Dragan et al, 2000), CNT-related activities The uptake rates were corrected for uridine uptake measured in noninjected oocytes. B, hCNT1, hCNT2, or hCNT3 cRNA was injected and the oocytes were incubated for 3 days.…”

ATP-Sensitive K+ Channels Regulate the Concentrative Adenosine Transporter CNT2 following Activation by A1 Adenosine Receptors

“…Liver homogenates, basolateral plasma membranes isolated as previously described (14), and crude extracts from FAO cells were used. Samples were treated with 0.5% Triton X-100-10 mM Tris (pH 7.4), and 20-g protein aliquots were run on sodium dodecyl sulfate-10% polyacrylamide gels (10,13). Proteins were transferred to filters (Immobilon-P; 0.45-m-pore size; Millipore, Bedford, Mass.…”