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Validation of FLT Uptake as a Measure of Thymidine Kinase-1 Activity in A549 Carcinoma Cells

¹ Department of Radiation Oncology, University of Washington School of Medicine, Seattle, Washington
² Department of Radiology, University of Washington School of Medicine, Seattle, Washington

View larger version (18K): [in a new window]   FIGURE 1. (A) Changes in S phase fraction as function of time after subculturing of growth-arrested A549 cells into fresh culture medium. (B) Uptake of 3H-FLT in A549 cells as function of time after 8-d-old growth-arrested cells were subcultured into fresh growth medium. FLT uptake is expressed relative to activity levels in culture medium. Uptake equal to 100% represents equilibration between cells and medium, whereas higher values mean that cells have concentrated tracer. (C) Uptake of 3H-FLT in A549 cells versus percentage in S phase.

View larger version (20K): [in a new window]   FIGURE 2. (A) TK1 activity in A549 cells as function of time. TK1 activity units are picomoles of phosphorylated TdR/30 min/mg of protein, as determined in cell-free enzymatic assay using A549 cell lysate as source of enzyme. (B) Uptake of 3H-FLT in A549 cells versus TK1 activity.

View larger version (19K): [in a new window]   FIGURE 3. (A) Changes in S phase fraction versus time after subculturing of growth-arrested A549-LXSN and A549-E6 cells. (B) Uptake of 3H-FLT in A549-E6 and A549-LXSN cells as function of time after 8-d-old growth-arrested cells were subcultured into fresh growth medium. FLT uptake is expressed relative to activity levels in culture medium. Uptake equal to 100% represents equilibration between cells and medium, whereas higher values mean that cells have concentrated tracer.

View larger version (18K): [in a new window]   FIGURE 4. (A) Effect of 10-Gy -radiation on movement of growth-arrested A549-E6 and A549-LXSN cells into S phase of cell cycle when subcultured into fresh growth medium. Irradiated LXSN cells do not enter cell cycle during first 24 h after being placed into fresh medium. E6-transfected cells, lacking functional p53, begin to enter S phase by 12 h. (B) Uptake of 3H-FLT into irradiated A549-E6 and A549-LXSN cells as function of time after 8-d-old growth-arrested cells were subcultured into fresh growth medium. Cells were irradiated with 10-Gy 137Cs -rays immediately before being placed into fresh medium. FLT uptake is expressed relative to activity levels in culture medium, with 100% uptake representing equilibration between cells and medium. Irradiated LXSN cells do not show increased uptake of FLT during first 24 h, consistent with their failure to move into cell cycle (A). (C) TK1 activity in irradiated A549-E6 and A549-LXSN cells as function of time after growth-arrested cells were subcultured into fresh growth medium. Cells were irradiated with 10-Gy 137Cs -rays immediately before being placed into fresh medium. TK1 activity does not increase in growth-arrested, irradiated LXSN cells.