FUNCTIONAL NUCLEOSIDE TRANSPORTERS ARE REQUIRED FOR GEMCITABINE INFLUX AND MANIFESTATION OF TOXICITY IN CANCER CELL-LINES

Gemcitabine (2',2'-difluorodeoxycytidine) is a novel pyrimidine nucleo side 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 pharmacologi cal activity. In humans, there are seven reported distinct NT activiti es varying in substrate specificity, sodium dependence, and sensitivit y to inhibition by nitrobenzylthioinosine (NBMPR) and dipyridamole, To determine which NTs are required for gemcitabine-dependent growth inh ibition, cultures from a panel of 12 cell lines with defined plasma me mbrane NT activities were incubated with different concentrations of g emcitabine, Cell proliferation was assessed by the sulforhodamine B as say and cell enumeration to identify the concentrations of gemcitabine that inhibited cell replication by 50% (IC(50)s). NT activity was a p rerequisite for growth inhibition in vitro because: (a) the nucleoside transport-deficient cells were highly resistant to gemcitabine; and ( b) treatment of cells that exhibited only equilibrative NT activity wi th NBMPR or dipyridamole increased resistance to gemcitabine by 39- to 1800-fold. These data suggested that the type of NT activities posses sed by a cell mag be an important determinant of its sensitivity to ge mcitabine and that NT deficiency may confer significant gemcitabine re sistance. We analyzed the uptake kinetics of [H-3]gemcitabine by each of five human NT activities in cell lines that exhibited a single NT a ctivity in isolation; transient transfection of the cDNAs encoding the human concentrative NT proteins (hCNT1 and hCNT2) was used to study t he cit and cif activities, respectively. The efficiency of gemcitabine uptake varied markedly among the cell lines with single NTs: es congr uent to cit > ei > cib > > > cif. The transportability of [H-3]gemcita bine was demonstrated by reconstitution of the human es NT in proteoli posomes, confirming that gemcitabine permeation is a protein-mediated process.