Research ArticleBasic Science Investigation

Different Modes of Transport for 3H-Thymidine, 3H-FLT, and 3H-FMAU in Proliferating and Nonproliferating Human Tumor Cells

David A. Plotnik, Lindsay E. Emerick, Kenneth A. Krohn, Jashvant D. Unadkat and Jeffrey L. Schwartz
Journal of Nuclear Medicine September 2010, 51 (9) 1464-1471; DOI: https://doi.org/10.2967/jnumed.110.076794

Article Figures & Data

Figures

  • FIGURE 1.
    FIGURE 1.

    Time-dependent nucleoside uptake in noncycling, plateau-phase cultures of A549 cells. (A) Intracellular levels of tracer (disintegrations per minute [dpm]/106 cells) in cells incubated in tracer-containing buffer. (B) Intracellular levels of tracer (percentage of activity in cell-equivalent volume of buffer) in cells incubated in tracer-containing buffer. (C) Intracellular levels of tracer (dpm/106 cells) in cells incubated in tracer-containing buffer with 10−4 M NBMPR. (D) Intracellular levels of tracer (percentage of activity in cell-equivalent volume of buffer) in cells incubated in tracer-containing buffer with 10−4 M NBMPR. Results are mean ± SD of at least 3 replicates. ○ = 3H-thymidine; ● = 3H-FLT; ▲ = 3H-FMAU; dotted line = relative activity in buffer.

  • FIGURE 2.
    FIGURE 2.

    Concentration-dependent inhibition of nucleoside uptake in noncycling, plateau-phase cultures of A549 cells. (A) One-minute nucleoside tracer uptake values (disintegrations per minute [dpm]/106 cells) as function of increasing concentrations of NBMPR. (B) Results normalized to no-inhibitor levels. (C) One-minute nucleoside tracer uptake values (dpm/106 cells) as function of increasing concentrations of carrier-added thymidine. (D) Results normalized to no-carrier-added thymidine. Results are mean ± SD of at least 3 replicates. ○ = 3H-thymidine; ● = 3H-FLT; and ▲ = 3H-FMAU; TdR = thymidine.

  • FIGURE 3.
    FIGURE 3.

    Nucleoside efflux from noncycling, plateau-phase cultures of A549 cells. (A) Relationship between nucleoside uptake and efflux in cells preloaded with tracers and then incubated in tracer-free buffer for 15–60 s. (B) NBMPR-sensitive and -resistant nucleoside efflux in cells preloaded with tracers and then incubated in tracer-free buffer for 15–60 s. Results are mean ± SD of at least 3 replicates. ○ = 3H-thymidine; ● = 3H-FLT; ▲ = 3H-FMAU; dpm = disintegrations per minute.

  • FIGURE 4.
    FIGURE 4.

    (A) Western blot probed with rabbit anti-hENT1 antibody. Bands from left to right are whole-cell lysates from noncycling cells, whole-cell lysates from exponentially growing cells, membrane preparations from noncycling cells, and membrane preparations from exponentially growing cells. (B) ENT content measured in membrane preparations by 3H-NBMPR binding assay. White bar = noncycling cells; black bar = exponentially growing cells. (C) Tracer uptake after incubation for 60 s in buffer ± 10−4 M NBMPR in plateau-phase, noncycling, and asynchronous exponentially growing A549 cells.

Tables

  • TABLE 1.

    Metabolism of 3H-Tracers*

    TimeTracerThymineNucleosideMonophosphateDiphosphateTriphosphateDNA†
    60-s incubation3H-thymidine13.6656.354.0910.8714.760.26
     3H-FLT—95.005.000.000.000.00
     3H-FMAU—100.000.000.000.000.00
    60-min incubation3H-thymidine4.758.514.7314.2962.744.97
     3H-FLT—13.0064.808.0014.000.00
     3H-FMAU—33.2126.379.9128.352.16
    60-min washout‡3H-thymidine0.000.004.7814.1666.3514.72
     3H-FLT—0.0041.1011.4047.500.00
     3H-FMAU—7.7824.4115.7748.581.47

    • ↵* Percentage of total activity in cells.

    • ↵† Activity in DNA.

    • ↵‡ Cells were incubated for 60 min in labeled tracer, then washed and cultured in tracer-free medium for 60 min before analysis.

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