Phosphorylation Status of Thymidine Kinase 1 Following Antiproliferative Drug Treatment Mediates 3′-Deoxy-3′-[¹⁸F]-Fluorothymidine Cellular Retention

<div><p>Background</p><p>3′-Deoxy-3′-[¹⁸F]-fluorothymidine ([¹⁸F]FLT) is being investigated as a Positron Emission Tomography (PET) proliferation biomarker. The mechanism of cellular [¹⁸F]FLT retention has been assigned primarily to alteration of the strict transcriptionally regulated S-phase expression of thymidine kinase 1 (TK1). This, however, does not explain how anticancer agents acting primarily through G2/M arrest affect [¹⁸F]FLT uptake. We investigated alternative mechanisms of [¹⁸F]FLT cellular retention involving post-translational modification of TK1 during mitosis.</p><p>Methods</p><p>[¹⁸F]FLT cellular retention was assessed in cell lines having different TK1 expression. Drug-induced phosphorylation of TK1 protein was evaluated by MnCl₂-phos-tag gel electrophoresis and correlated with [¹⁸F]FLT cellular retention. We further elaborated the amino acid residues involved in TK1 phosphorylation by transient transfection of FLAG-pCMV2 plasmids encoding wild type or mutant variants of TK1 into TK1 negative cells.</p><p>Results</p><p>Baseline [¹⁸F]FLT cellular retention and TK1 protein expression were associated. S-phase and G2/M phase arrest caused greater than two-fold reduction in [¹⁸F]FLT cellular retention in colon cancer HCT116 cells (p<0.001). G2/M cell cycle arrest increased TK1 phosphorylation as measured by induction of at least one phosphorylated form of the protein on MnCl₂-phos-tag gels. Changes in [¹⁸F]FLT cellular retention reflected TK1 phosphorylation and not expression of total protein, in keeping with the impact of phosphorylation on enzyme catalytic activity. Both Ser13 and Ser231 were shown to be involved in the TK1 phosphorylation-modulated [¹⁸F]FLT cellular retention; although the data suggested involvement of other amino-acid residues.</p><p>Conclusion</p><p>We have defined a regulatory role of TK1 phosphorylation in mediating [¹⁸F]FLT cellular retention and hence reporting of antiproliferative activity, with implications especially for drugs that induce a G2/M cell cycle arrest.</p></div

Verification of phosphorylated amino acid residues on TK1 protein.

<p>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.</p

Comparison of thymidine kinase 1 (TK1) expression to [¹⁸F]FLT uptake.

<p>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.</p

Relationship between thymidine kinase 1 (TK1) and thymidylate synthase (TS) expression, and doubling time.

<p>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 <a href="http://www.doubling-time.com/compute.php?lang=en" target="_blank">http://www.doubling-time.com/compute.php?lang=en</a>. D) Representative correlation plot between doubling times of each cell line and TK1 protein expression.</p

Determination of the regulation of TK1 phosphorylation by Cdk1 and Cdk2.

<p>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.</p