Functional Nucleoside Transporters Are Required for Gemcitabine Influx and Manifestation of Toxicity in Cancer Cell Lines1

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 pharmacolog ical 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 de termine which NTs are required for gemcitabine-dependent growth inhi bition, cultures from a panel of 12 cell lines with defined plasma mem brane 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 prereq uisite for growth inhibition in vitro because: (a) the nucleoside transportdeficient cells were highly resistant to gemcitabine; and (In treatment of cells that exhibited only equilibrative NT activity with NBMPR or dipyr idamole increased resistance to gemcitabine by 39to 1800-fold. These data suggested that the type of NT activities possessed by a cell may be an important determinant of its sensitivity to gemcitabine and that NT deficiency may confer significant gemcitabine resistance. We analyzed the uptake kinetics of [3H]gemcitabine by each of five human NT activities in cell lines that exhibited a single NT activity in isolation; transient transfection of the cDNAs encoding the human concentrative NT proteins (hCNTl and hCNT2) was used to study the rii and rif activities, respec tively. The efficiency of gemcitabine uptake varied markedly among the cell lines with single NTs: es = dt > ei > db »> df. The transport ability of [3H]gemcitabine was demonstrated by reconstitution of the human es NT in proteoliposomes, confirming that gemcitabine permeation is a protein-mediated process.