Phosphorylation Status of Thymidine Kinase 1 Following Antiproliferative Drug Treatment Mediates 3′-Deoxy-3′-[18F]-Fluorothymidine Cellular Retention
Figure 3
Effect of drug-induced alteration of cell cycle progression on TK1 phosphorylation and [18F]FLT cell uptake.
The following drug concentrations were used in each experiment: 10 µM aphidicolin (APH), 0.5 µg/ml nocodazole (NOC), 250 nM paclitaxel (PAX), 50 µM roscovitine. A) Representative cell cycle distribution profiles of HCT116 cells after 24 h drug-induced cell cycle arrest. Treatment with 50 µM roscovitine was carried out for 4 h. B) Characterization of TK1 phosphorylation bands (using 75 µM MnCl2-phos-tag gels) in cell cycle arrested HCT116 cell lysates by alkaline phosphatase (CIP) treatment (100 U per 100 µg of protein at 37°C for 1 h). HCT116 cells were treated with nocodazole or paclitaxel for 24 h. Roscovitine treatment was carried out for 4 h. C) Representative TK1 phosphorylation profile after treatment with the indicated agents for 24 h (4 h for roscovitine treatment). D) [18F]FLT uptake and phospho-TK1 (P-TK1) expression in HCT116 following treatments for 24 h (4 h for roscovitine treatment). The left y-axis represents mean ± S.E.M. expressed as quantitative counts per µg of protein. Stars highlight significant differences compared to untreated control. The right y-axis represents semiquantitative P-TK1 density (band #2+band #3). An arbitrary value of 1 was assigned to P-TK1 expression in control cells and this value was used as comparison for the other treatments.