Uncontrolled cell proliferation is a hallmark of cancer and its inhibition is desired in cancer therapy. The most widely used radiotracer for proliferation is 18F-fluorothymidine (18F-FLT). To be retained in cells, 18F-FLT is phosphorylated to thymidylate (dTMP) by thymidine kinase 1 (TK1), the rate limiting step in the salvage pathway for DNA synthesis. We studied TK1 protein phosphorylation in mammalian cells, hypothesising that TK1 is subject to different phosphorylations through the cell cycle, which are responsible for the regulation of its activity, therefore modulating 18F-FLT uptake, particularly during G2/M. Using phos-tag™ acrylamide gels we isolated three forms of phosphorylated TK1, one of which was present throughout the cell cycle, but increased during S and G2/M phases, and two phosphorylated forms that appeared upon G2/M arrest. The implications of these phosphorylations on 18F-FLT uptake were tested by conducting FLT cell uptake studies after treating HCT116 human colon cancer cells with cell cycle-arresting drugs. A marked reduction of radiotracer uptake was observed in cells arrested in S phase (aphidicolin) or G2/M phase (nocodazole and paclitaxel), confirming previous findings regarding TK1 phosphorylation during G2/M being responsible of reduced enzymatic activity. Transient transfection of Ost TK1- cells with FLAG-pCMV vectors encoding WT, S13A, S13D or S231A TK1 suggested that S13 phosphorylation occurs throughout the cell cycle and increases upon G2/M arrest after treatment with nocodazole or paclitaxel. Substitution of S231 with alanine (S231A) was linked to G2/M-specific phosphorylation of TK1. Further experiments were performed to assess the role of the two serine phosphorylations on 18F-FLT cell uptake. There was a significant reduction in 18F-FLT uptake in S13A transfected cells compared to WT. S13D and S231A plasmids rescued 18F-FLT uptake almost to levels seen in WT cells, supporting the relevance of S13 and S231 phosphorylation for TK1 enzymatic activity. Nocodazole induced G2/M arrest further reduced 18F-FLT uptake, even in S231A transfection, implicating phosphorylation sites other than those studied here in the reduced TK1 activity. 18F-FLT-PET imaging of HCT116 tumour-bearing nude mice 24h after treatment with 40 mg/kg paclitaxel indicated a significant reduction in tumour 18F-FLT uptake, which correlates with the G2/M-induced reduction in TK1 activity as a result of TK1 phosphorylation. Our present study demonstrates the presence of at least three phosphorylated forms of TK1, which are responsible for the post-translational regulation of TK1 activity, and demonstrates the effect of these modifications on the uptake of 18F-FLT.

Citation Format: Roberta Sala, Quang-De Nguyen, Chirag Patel, David Mann, Joachim H. G. Steinke, Ramon Vilar, Eric O. Aboagye. Regulation of 18F-fluorothymidine uptake by thymidine kinase 1 protein phosphorylation. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2660. doi:10.1158/1538-7445.AM2013-2660