Uncontrolled cell proliferation is one of the hallmarks of cancer and its inhibition is desired in cancer therapy. The thymidine analogue 3’-deoxy-3’-[18F]-fluorothymidine ([18F]FLT) is used to image tumour proliferation by positron emission tomography (PET). To be retained in cells, [18F]FLT is phosphorylated by thymidine kinase 1 (TK1), the first enzyme in the salvage pathway for DNA synthesis, and for this reason the cellular incorporation of [18F]FLT is dependent on TK1 activity. Of the mechanisms regulating TK1 activity, TK1 transcription is well recognised while post-translational enzyme modifications are less well understood.
TK1 protein phosphorylation was investigated in cancer cells, hypothesising that, throughout the cell cycle, but particularly during G2/M phase, TK1 is subjected to different types of phosphorylation, which are responsible for regulating its activity, and therefore potentially modulating [18F]FLT uptake.
An acrylamide phos-tag™ gel method was validated to enable discrimination of phosphorylated TK1. Three different phosphorylated forms of TK1 were detected during progression of cells within the cell cycle, one of which was specifically produced upon G2/M arrest. There were significant changes in [18F]FLT uptake subsequent to cell cycle arrest by biological means and following treatment with anti-cancer drugs and pharmacological modulators. Changes in TK1 enzyme activity were detected as variations in [18F]FLT retention, with significantly reduced uptake upon serum starvation-induced G1 arrest, and marked decreased uptake during S-phase arrest and after nocodazole- or paclitaxel-induced G2/M arrest. Phosphorylation of serine-13 and serine-231 of TK1 were implicated in regulating [18F]FLT uptake.
To assess whether or not [18F]FLT-PET highlighted changes in proliferation in vivo, HCT116 tumour-bearing mice were treated with paclitaxel. Although long term treatment resulted in tumour growth delay, in the model and at drug doses and early time points considered for imaging, no significant changes in tumour [18F]FLT retention were observed in treated animals compared to controls. This correlated with similarly unremarkable changes in Ki67 and TK1 expression in excised tumour samples.
Investigation of whether or not alternative nucleoside analogue scaffolds were incorporated into the DNA and if they were providing greater sensitivity for detecting cell proliferation, compared to [18F]FLT, was carried out. Two new radiotracers ([18F]FTT and [18F]FOT) were developed and tested for a) phosphorylation by TK1, b) accumulation into cells and c) metabolism and biodistribution. Both radiotracers proved not to be useful, being minimally phosphorylated by TK1 and therefore not retained in cells, and showing catabolism in vivo.
Finally, specific mitotic inhibitors were characterised in vitro to determine their effects on TK1 phosphorylation and proteins involved in [18F]FLT uptake, in order to verify if [18F]FLT is a suitable biomarker to highlight mitotic arrest with these modern drugs.
The key novel finding to evolve from this thesis is that measurement of tumour proliferation by [18F]FLT-PET is modulated by TK1 phosphorylation. This changes the notion that [18F]FLT uptake simply reflects changes in S-phase arrest/DNA synthesis to one that represents broad sensitivity to proliferation including G2/M arrest. New radiotracers that are specifically incorporated into DNA and whose uptake is TK1-independent could provide additional selectivity for imaging DNA synthesis.Open Acces
