Gemcitabine Transport in Xenopus Oocytes Expressing Recombinant Plasma Membrane Mammalian Nucleoside Transporters

Abstract

Background: Gemcitabine, a pyrimidine analogue of deoxycytidine, is an anticancer nucleoside drug that requires functional plasma membrane nucleoside transporter proteins to reach its intracellular targets and cause cytotoxicity. Because of technical difficulties inherent in studying nucleoside transport in human cells, we rigorously defined gemcitabine membrane transportability by producing each of the available human (h) and rat (r) recombinant nucleoside transporters (NTs) individually in Xenopus laevis oocytes. Methods: Oocytes were microinjected with in vitro-transcribed RNAs derived from complementary DNAs encoding (C = concentrative) rCNT1, rCNT2, hCNT1, hCNT2, (E = equilibrative) rENT1, rENT2, hENT1, and hENT2. Uptake of [ 3 H]gemcitabine and [ 14 C] uridine was measured 3 days after microinjection to determine kinetic constants. We also used the two-electrode, voltage-clamp technique to investigate the electrophysiology of hCNT1-mediated gemcitabine transport. Results: Gemcitabine was transported by most of the tested proteins (the exceptions being the purine-selective rCNT2 and hCNT2), with the greatest uptake occurring in oocytes producing recombinant rCNT1 and hCNT1. Influxes of gemcitabine mediated by hCNT1, hENT1, and hENT2 were saturable and conformed to Michaelis-Menten kinetics with apparent K m values of 24, 160, and 740 µM, respectively. Gemcitabine had a limited ability to cross the lipid bilayer of oocyte membranes by simple diffusion. External application of gemcitabine to oocytes producing recombinant hCNT1 induced an inward current, which demonstrated that hCNT1 functions as a Na

Gemcitabine chemoresistance and molecular markers associated with gemcitabine transport and metabolism in human pancreatic cancer cells

“…It has been shown that modulation of cellular enzymes of gemcitabine transport and metabolism influences drug activity in vitro (Mackey et al, 1998(Mackey et al, , 1999Goan et al, 1999;Ritzel et al, 2001). Cellular enzymes of gemcitabine transport and metabolism, that is, hENT1, dCK, RRM1, and RRM2, are well documented.…”

“…In recent studies performed on human cancer cell lines, human equilibrative nucleoside transporter-1 (hENT1) was found to be the major gemcitabine transporter (Garcia-Manteiga et al, 2003). If gemcitabine is not transported into the cell via hENT1 it cannot inhibit cell growth (Mackey et al, 1998;Rauchwerger et al, 2000), but increased hENT1 abundance facilitates efficient cellular entry of gemcitabine and confers increased cytotoxicity (Mackey et al, 1999;Ritzel et al, 2001). Inside the cell, gemcitabine is phosphorylated by deoxycytidine kinase (dCK) in a rate-limiting step.…”

Nucleoside transporters in chronic lymphocytic leukaemia

“…The latter show significantly lower affinities for substrates than CNTs and may be reversible depending on how the substrate concentration gradients change across the plasma membrane. Substrate selectivity and their pharmacological profiles, [35][36][37][40][41][42][43] still incomplete, are summarized in Table 2.…”

“…Substrate selectivity in the CNT gene family is narrower than for ENTs. For instance, gemcitabine is a high-affinity substrate for hCNT1 but it is not recognized by hCNT2 42 and appears to be less effectively taken up by the hCNT3 isoform. 45 In contrast, hCNT3 takes up fludarabine with high affinity, while hCNT1 does not recognize it and hCNT2, although inhibited by it, does not take it up.…”

Functional characterization of a H+/nucleoside co‐transporter (CaCNT) from Candida albicans, a fungal member of the concentrative nucleoside transporter (CNT) family of membrane proteins

“…CNT1/2/3 proteins transport a broad spectrum of antiviral and anticancer nucleoside analogues (Huang et al, 1995;Yao et al, 1996b;Ritzel et al, 1997Ritzel et al, , 1998Ritzel et al, , 2001Mackey et al, 1999;unpublished observation). hCNT1 and rCNT1, for instance, transport the antiviral drugs 3 -azido-3 -deoxythymidine (AZT) and 2 3 -dideoxycytidine (ddC), but not 2 3 -dideoxyinosine (ddI).…”