Figure 1.
Relationship between thymidine kinase 1 (TK1) and thymidylate synthase (TS) expression, and doubling time.
A) Representative western blot showing TK1, TS and β actin expression in the analyzed cell lines. Cells were grown in 10 cm plates for 48 h to reach approximately 80% confluency at the time of the analysis. B) Quantification of TK1 and TS expression normalized to β actin. Average of 3 independent experiments. HCT116 are used as reference to calculate statistical significance. C) Cell proliferation rates of HCT116, A549, Hos and Ost TK1− cells. Cells were seeded in 6-well plates in triplicates. Doubling times (DT) were calculated using the macro available on http://www.doubling-time.com/compute.php?lang=en. D) Representative correlation plot between doubling times of each cell line and TK1 protein expression.
Figure 2.
Comparison of thymidine kinase 1 (TK1) expression to [18F]FLT uptake.
A) [18F]FLT uptake in HCT116, A549, Hos and Ost TK1− cells. B) Plot showing the association between [18F]FLT uptake and TK1 protein expression. Values are means ± S.E.M.
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.
Figure 4.
Verification of phosphorylated amino acid residues on TK1 protein.
Ost TK1− cells were transiently transfected with FLAG-pCMV2 plasmids coding for TK1 variants. A) Ost TK1− transient transfections with FLAG-pCMV2 plasmids coding for the indicated TK1 variants were resolved (10 µg) on a 75 µM MnCl2-phos-tag gels and probed for TK1 expression (1∶500). B) [18F]FLT uptake in Ost TK1− cells transiently transfected with the indicated TK1 constructs. The main figure represents the uptake in control and nocodazole (0.5 µg/ml) treated samples. The y-axis represents mean ± S.E.M. expressed as counts per µg of protein. Significant differences following transient transfection compared to non-transfected cells, and significant changes after nocodazole treatment compared to the untreated sample are highlighted by stars. The inset reports the percentage of radiotracer uptake compared to cells transfected with wild type (WT; 100%) TK1. Significant differences are indicated by stars. C) TK1 expression in transfected cells resolved on 12% SDS-PAGE. β actin was used as loading control.
Figure 5.
Determination of the regulation of TK1 phosphorylation by Cdk1 and Cdk2.
A) Cdk1 and Cdk2 knock-down optimization. Representative western blots, with corresponding densitometry (average of 3 independent experiments) and qPCR analysis. Control refers to non-silenced cells; scramble indicates transfection with non-targeting siRNA. B) Determination of the Cdk responsible for TK1 specific phosphorylation. 5 nM of scramble, Cdk1 or Cdk2 siRNA were used to induce Cdk knock-down for 48 h. Cells were treated O/N with 0.5 µg/ml nocodazole (NOC) following Cdk knock-down. Top two panels represent MnCl2-phos-tag gel; bottom three panels report 12% SDS-PAGE.