title: Phosphorylation status of thymidine kinase 1 following antiproliferative drug treatment mediates 3'-deoxy-3'-[18F]-fluorothymidine cellular retention.
abstract: 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.
  
  
  CONCLUSIONS
  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.
authors.affiliation: Comprehensive Cancer Imaging Centre, Department of Surgery and Cancer, Imperial College London, London, United Kingdom.
authors.forename: Roberta
authors.initials: R
authors.lastname: Sala
authors.affiliation: Comprehensive Cancer Imaging Centre, Department of Surgery and Cancer, Imperial College London, London, United Kingdom.
authors.forename: Quang-Dé
authors.initials: QD
authors.lastname: Nguyen
authors.affiliation: Institute of Chemical Biology, Department of Chemistry, Imperial College London, London, United Kingdom.
authors.forename: Chirag B K
authors.initials: CB
authors.lastname: Patel
authors.affiliation: Department of Life Sciences, Imperial College London, London, United Kingdom.
authors.forename: David
authors.initials: D
authors.lastname: Mann
authors.affiliation: Institute of Chemical Biology, Department of Chemistry, Imperial College London, London, United Kingdom.
authors.forename: Joachim H G
authors.initials: JH
authors.lastname: Steinke
authors.affiliation: Institute of Chemical Biology, Department of Chemistry, Imperial College London, London, United Kingdom.
authors.forename: Ramon
authors.initials: R
authors.lastname: Vilar
authors.affiliation: Comprehensive Cancer Imaging Centre, Department of Surgery and Cancer, Imperial College London, London, United Kingdom.
authors.forename: Eric O
authors.initials: EO
authors.lastname: Aboagye
mesh_terms: D000963:Antimetabolites / Q000378:metabolism; D000970:Antineoplastic Agents / Q000494:pharmacology*; D015153:Blotting, Western; D001859:Bone Neoplasms / Q000188:drug therapy / Q000201:enzymology / Q000473:pathology; D002453:Cell Cycle; D049109:Cell Proliferation; D003110:Colonic Neoplasms / Q000188:drug therapy / Q000201:enzymology* / Q000473:pathology; D015224:Dideoxynucleosides / Q000378:metabolism*; D019788:Fluorodeoxyglucose F18 / Q000378:metabolism*; D006801:Humans; D012516:Osteosarcoma / Q000188:drug therapy / Q000201:enzymology* / Q000473:pathology; D010766:Phosphorylation; D011499:Protein Processing, Post-Translational; D019275:Radiopharmaceuticals / Q000378:metabolism; D013937:Thymidine Kinase / Q000378:metabolism*; D014407:Tumor Cells, Cultured
chemical_list: D000963:Antimetabolites; D000970:Antineoplastic Agents; D015224:Dideoxynucleosides; D019275:Radiopharmaceuticals; D019788:Fluorodeoxyglucose F18; D013937:Thymidine Kinase; C092717:thymidine kinase 1