Hypoxanthine transport in human glioblastoma cells and effect on cell susceptibility to methotrexate.
OBJECTIVE
Cancer cells may circumvent the cytotoxic effect of antimetabolite drugs that inhibit de novo nucleotide synthesis via the uptake of extracellular preformed nucleobases or nucleosides. The goal of this study was to investigate the nucleobase transport mechanism in human U-118 glioblastoma cells and to determine whether the purine nucleobase hypoxanthine affects cell susceptibility to methotrexate.


METHODS
Uptake experiments were performed using 3H-labeled hypoxanthine. RT-PCR was used to determine the expression of nucleoside transporters. Methotrexate-induced apoptosis was analyzed using annexin V staining and FACScan analysis.


RESULTS
Hypoxanthine transport in U-118 cells involved both carrier-mediated (Km = 10.5 +/- 6.3 microM, Vmax = 1.45 +/- 0.69 pmol/10(5) cells/60 s) and simple diffusion processes (Kd = 0.36 +/- 0.009 microm/10(5) cells/60 s). Uptake was sensitive to Na+ and inhibited by nucleobases but not nucleosides or nucleoside transport inhibitors. In contrast, uptake of a nucleoside, uridine, was inhibited by nucleosides but not nucleobases. RT-PCR analysis suggested the presence of hENT1, hENT2, and hCNTI nucleoside transporters in U-118 cells. In the absence of hypoxanthine, methotrexate inhibited U-118 cell proliferation and induced apoptosis. These toxic effects were diminished when hypoxanthine was present at physiologically relevant concentrations.


CONCLUSIONS
Hypoxanthine transport in U-118 cells involves a Na+-dependent, high-affinity nucleobase transport system functionally distinct from nucleoside transporters. At physiologic concentrations, hypoxanthine protects glioblastoma cells from the cytotoxicity of methotrexate.
Kong
Wei
W
Department of Pharmaceutics, School of Pharmacy, University of Washington, Seattle, WA 98195-7610, USA.
Wang
Joanne
J
D001692:Biological Transport / Q000187:drug effects / Q000502:physiology; D045744:Cell Line, Tumor; D004305:Dose-Response Relationship, Drug; D005909:Glioblastoma / Q000378:metabolism*; D006801:Humans; D019271:Hypoxanthine / Q000493:pharmacokinetics*; D008727:Methotrexate / Q000494:pharmacology*; D033703:Nucleoside Transport Proteins / Q000096:biosynthesis / Q000378:metabolism
D033703:Nucleoside Transport Proteins; D019271:Hypoxanthine; D008727:Methotrexate