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A Novel In Vitro Assay to Assess Phosphorylation of 3′-[18F]fluoro-3′-Deoxythymidine

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Abstract

Purpose

3′-[18F]fluoro-3'-deoxythymidine ([18F]FLT) is phosphorylated by thymidine kinase 1 (TK-1), a cell cycle regulated enzyme. Appropriate use of [18F]FLT tracer requires validation of the TK-1 activity. Here, we report development of a novel phosphoryl-transfer assay to assess phosphorylation of [18F]FLT both in tumor cell lysates and tumor cells.

Procedures

The intrinsic F-18 radioactivity was used to quantify both substrate and phosphorylated products using a rapid thin layer chromatography method. Phosphorylation kinetics of [18F]FLT in SW480 and DiFi tumor cell lysates and cellular uptake were measured.

Results

The apparent Michaelis–Menten kinetic parameters for [18F]FLT are \( {K_{\rm{m}}} = {4}.{8}\pm 0.{3}\;{{\mu M}} \) and V max = 7.4 pmol min−1 per 1 × 106 cells with ∼2-fold higher TK-1 activity in DiFi versus SW480 lysates.

Conclusions

The apparent K m of [18F]FLT was comparable to the value reported with purified recombinant TK-1. The uptake of [18F]FLT by SW480 cells is inhibited by nitrobenzylthioinosine or dipyridamole indicating that uptake is mediated predominantly by the equilibrative nucleoside transporters in these tumor cells.

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Acknowledgements

The authors thank Dr. Roger Colbran for advice on the use of NaF as a general phosphatase inhibitor in the phosphoryl-transfer assay and both Jeffrey Clanton and Jarrod Driskill for cyclotron-produced [18F]fluoride. This work was supported by grants from National Institutes of Health, NIH/NCI R25T-CA092043, NIH 5R21MH073800-02, the GI Special Program of Research Excellence (P50 95103), ICMIC (P50CA128323), the NCI-funded South-Eastern Center for Small-Animal Imaging, (U24CA126588), Vanderbilt Department of Radiology & Radiological Sciences and Department of Cancer Biology. H. Charles Manning acknowledges support from a Career Development Award from the National Cancer Institute (K25CA 127349).

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Authors and Affiliations

  1. Department of Radiology and Radiological Sciences, Vanderbilt University School of Medicine, 1161 21st Avenue South, Nashville, TN, 37232, USA

    Ning Guo, Jingping Xie, H. Charles Manning, Natasha G. Deane, M. Sib Ansari, John Gore, Ronald R. Price & Ronald M. Baldwin

  2. Vanderbilt University Institute of Imaging Science (VUIIS), Vanderbilt University School of Medicine, 1161 21st Avenue South, Nashville, TN, 37232, USA

    Ning Guo, Jingping Xie, H. Charles Manning, M. Sib Ansari, John Gore, Ronald M. Baldwin & J. Oliver McIntyre

  3. Department of Cancer Biology, Vanderbilt University School of Medicine, 1161 21st Avenue South, Nashville, TN, 37232, USA

    J. Oliver McIntyre

  4. Department of Neurosurgery, Vanderbilt University School of Medicine, 1161 21st Avenue South, Nashville, TN, 37232, USA

    H. Charles Manning

  5. Department of Biomedical Engineering, Vanderbilt University School of Medicine, 1161 21st Avenue South, Nashville, TN, 37232, USA

    H. Charles Manning & John Gore

  6. Department of Medicine, Cell and Developmental Biology, Vanderbilt University School of Medicine, 1161 21st Avenue South, Nashville, TN, 37232, USA

    Robert J. Coffey

  7. The Vanderbilt-Ingram Cancer Center, Vanderbilt University School of Medicine, 1161 21st Avenue South, Nashville, TN, 37232, USA

    H. Charles Manning, Robert J. Coffey, John Gore, Ronald M. Baldwin & J. Oliver McIntyre

  8. Program in Chemical and Physical Biology, Vanderbilt University, 1161 21st Avenue South, Nashville, TN, 37232, USA

    H. Charles Manning

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  1. Ning Guo
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  2. Jingping Xie
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Correspondence to Ning Guo.

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Guo, N., Xie, J., Charles Manning, H. et al. A Novel In Vitro Assay to Assess Phosphorylation of 3′-[18F]fluoro-3′-Deoxythymidine. Mol Imaging Biol 13, 257–264 (2011). https://doi.org/10.1007/s11307-010-0351-8

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