Phosphorylation status of thymidine kinase 1 following antiproliferative drug treatment mediates 3'-deoxy-3'-[18F]-fluorothymidine cellular retention.

BACKGROUND: 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. METHODS: [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. RESULTS: 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. CONCLUSION: 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.

Inhibition of mitogen-activated protein kinase (MAPK) and cyclin-dependent kinase 2 (Cdk2) by platinum(II) phenanthroline complexes.

UNLABELLED: Inhibition of protein kinases in the fight against disease remains a constant challenge for medicinal chemists, who have screened multitudes of predominantly planar organic scaffolds, natural and synthetic, to identify potent-albeit not always selective-kinase inhibitors. Herein, in an effort to investigate the potential biological utility of metal-based compounds as inhibitors against the cancer-relevant targets mitogen-activated protein kinase and cyclin-dependent kinase 2, we explore various parameters in planar platinum(II) complexes with substituted phenanthroline ligands and aliphatic diamine chelate co-ligands, to identify combinations that yield promising inhibitory activity. The individual ligands' steric requirements as well as their pattern of hydrogen bond donors/acceptors appear to alter inhibitory potency when modulated. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1007/s12154-011-0059-5) contains supplementary material, which is available to authorized users.