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Hypoxanthine Transport in Human Glioblastoma Cells and Effect on Cell Susceptibility to Methotrexate

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Abstract

Purpose. 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 μM, Vmax= 1.45 ± 0.69 pmol/105 cells/60 s) and simple diffusion processes (Kd= 0.36 ± 0.009 μm/105 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 hCNT1 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.

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  1. Department of Pharmaceutics, School of Pharmacy, University of Washington, Seattle, WA, 98195-7610

    Wei Kong & Joanne Wang

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  1. Wei Kong
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  2. Joanne Wang
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Correspondence to Joanne Wang.

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Kong, W., Wang, J. Hypoxanthine Transport in Human Glioblastoma Cells and Effect on Cell Susceptibility to Methotrexate. Pharm Res 20, 1804–1811 (2003). https://doi.org/10.1023/B:PHAM.0000003378.16802.97

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  • DOI: https://doi.org/10.1023/B:PHAM.0000003378.16802.97

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