Abstract
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 pharmacological 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 determine which NTs are required for gemcitabine-dependent growth inhibition, cultures from a panel of 12 cell lines with defined plasma membrane 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 prerequisite 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 with NBMPR or dipyridamole increased resistance to gemcitabine by 39- to 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 (hCNT1 and hCNT2) was used to study the cit and cif activities, respectively. The efficiency of gemcitabine uptake varied markedly among the cell lines with single NTs: es ≅ cit > ei > cib > > > cif. The transportability of [3H]gemcitabine was demonstrated by reconstitution of the human es NT in proteoliposomes, confirming that gemcitabine permeation is a protein-mediated process.
Footnotes
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↵1 Supported by a Canadian Cancer Society operating grant from the National Cancer Institute of Canada and a Pilot Project and New Investigator's Award from the Alberta Cancer Board. C. E. C. is a Terry Fox Cancer Research Scientist of the National Cancer Institute of Canada, and J. D. Y. is a Heritage Medical Scientist of the Alberta Heritage Foundation for Medical Research. Isolation of the L1210/DNC3 and L1210/DU-5 cell lines was also supported by National Cancer Institute (USA) Research Grant CA55056, Cancer Center Support Grant CA21765, and the American Lebanese Syrian Associated Charities of St. Jude Children's Research Hospital (to J. A. B. and C. R. C.).
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↵2 To whom requests for reprints should be addressed, at Cross Cancer Institute, 11560 University Avenue, Edmonton, Alberta, T6G 1Z2 Canada.
- Received March 10, 1998.
- Accepted August 18, 1998.
- ©1998 American Association for Cancer Research.
Volume 58, Issue 19
