Elsevier

Biochemical Pharmacology

Volume 80, Issue 10, 15 November 2010, Pages 1528-1536
Biochemical Pharmacology

Induction of thymidine kinase 1 after 5-fluorouracil as a mechanism for 3′-deoxy-3′-[18F]fluorothymidine flare

https://doi.org/10.1016/j.bcp.2010.08.004Get rights and content

Abstract

Imaging the pharmacodynamics of anti-cancer drugs may allow early assessment of anti-cancer effects. Increases in 3′-deoxy-3′-[18F]fluorothymidine ([18F]FLT) uptake early after thymidylate synthase inhibition (TS) inhibition, the so-called flare response, is considered to be largely due to an increase in binding sites for type-1 equilibrative nucleoside transporter. We investigated the induction of thymidine kinase 1 (TK1) after 5-fluorouracil (5-FU) treatment as one of mechanisms for [18F]FLT flare. Exposure of nine cancer cell lines to 5-FU for 24 h induced a 2.5- to 3.5-fold increase in [18F]FLT uptake, significantly higher than the 1.5-fold increase observed 2 h after treatment. The increase of [18F]FLT uptake 24 h after 5-FU exposure accompanied TK1 induction in most cell lines. In representative cell lines (A431 and HT29), 5-FU time-dependently increased [18F]FLT uptake, kinase activity and the levels of protein and mRNA for TK1, sequential cyclin E and A induction, and G1–S phase transition. Cycloheximide treatment and knockdown of TK1 completely inhibited 5-FU-induced [18F]FLT flare. On the other hand, HCT8 cells showed a biphasic [18F]FLT flare with lacked TK1 induction in response to the dosage of 5-FU. Cycloheximide did not inhibit 5-FU-induced [18F]FLT flare in this cells. In vivo dynamic [18F]FLT-PET and ex vivo analysis in HT29 tumor-bearing mice showed significantly increased [18F]FLT flux and TK1 activity of tumor tissue 24 h after 5-FU administration (P < 0.05). Conclusively, 5-FU induced TK1 and TK1-mediated high [18F]FLT flare in most of cell lines. [18F]FLT-PET may be used to assess pharmacodynamics of TS inhibitor by a mechanism involving TK1 induction.

Introduction

Thymidylate synthase (TS), a folate-dependent enzyme that catalyzes a reaction required for the de novo synthesis of thymidylate, is a target for anti-cancer drugs [1], [2], [3]. Unfortunately, the expression levels of TS and folate receptors vary depending on cancer cell type, leading to often unsatisfactory therapeutic efficacy of agents targeting this enzyme. Even 5-FU, a representative TS inhibitor widely used for neo-adjuvant and metastatic chemotherapy in various solid tumors, has clinical response rates of only 10–15% when administered as monotherapy and 20–40% when administered in combination regimens [4], [5], [6].

The characterization of biological factors that correlate with response to TS inhibition may be important in identifying those patients who are most likely to benefit from treatment with TS inhibitors [1]. Alternatively, a method of predicting TS inhibition may be critical in the development of new TS inhibitors. Intratumoral levels of TS, thymidine phosphorylase, dihydropyrimidine dehydrogenase and other biomarkers have been associated with tumor response to 5-FU [1], [7], [8], [9]. These methods, however, are invasive or may not give complete information about tumors.

TS inhibition was reported to increase the thymidine kinase 1 (TK1) activity and nucleoside transporter expression to regulate intracellular thymidine triphosphate pools [10], a mechanism attributed to an increase in salvage kinetics following TS inhibition. In vitro and in vivo positron emission tomography (PET) studies have demonstrated increases in radiolabeled thymidine or 3′-deoxy-3′-[18F]fluorothymidine ([18F]FLT) uptake early after TS inhibition, the so-called flare response [8], [9], [11], [12], [13], [14], [15]. Interestingly, in cancer cells, [18F]FLT flare following TS inhibition was shown to be due largely to an increase in binding sites for type-1 equilibrative nucleoside transporter within hours after drug administration, but without changing TK1 protein levels [9], [13]. In humans, increases in [18F]FLT uptake after oral capecitabine treatment were also attributed to the redistribution of transporters [14]. To date, however, the role of TK1 has not been assessed, and the molecular mechanisms underlying [18F]FLT flare in response to 5-FU have not been fully determined. We therefore investigated whether 5-FU treatment induces TK1, and whether TK1 mediates 5-FU-induced [18F]FLT flare. We also assessed TK1-mediated [18F]FLT flare using PET in mouse tumor model.

Section snippets

Radiopharmaceutical preparation

[18F]FLT was prepared from (5′-O-DMTr-2′-deoxy-3′-O-nosyl-b-d-threopentafuranosyl)-3-N-BOC-thymine by the nucleophilic fluorination of 18F-fluoride in a protic solvent (t-butanol or t-amyl alcohol) [16]. Typically, decay-corrected radiochemical yields ranged from 60% to 70%. The mean ± SD radiochemical purity was 98 ± 1.2%, with a specific activity greater than 60 TBq/mmol.

Cell culture, drug treatment, and preparation of cell lysates

A431, HT29, HeLa, MDA-MB-231, Calu6, A549, HCT116, MCF7, and HCT8 cell lines were obtained from the American Type Culture

5-FU induced changes in [18F]FLT uptake in cancer cell lines

A431, HT29, HeLa, MDA-MB-231, Calu6, A549, HCT116, MCF7, and HCT8 cells were grown for 24 h, and exposed to vehicle or 1, 3, 10, 30, or 100 μM 5-FU for 2 or 24 h; [18F]FLT uptake and cell count were then measured. The tested cells were sensitive to 5-FU, having IC50 within the ranges of tested concentrations (Supplementary Fig. 1). The viable cell number was minimally changed after 2 h treatment, and dose-dependently decreased by 24 h (Supplementary Table 2). In most cell lines, [18F]FLT uptake was

Discussion

To our knowledge, this study is the first to show that TK1 mediates the increase in [18F]FLT uptake induced by 5-FU treatment, both in vivo and in vitro. The high [18F]FLT flare occurring 24 h after 5-FU treatment was accompanied by an increase in TK1 expression in most cell lines. 5-FU-resistant cells, SNU-620-5-FU/1000, showed no changes in [3H]FLT uptake and TK1 expression. TK1 was activated during the G1S cell-cycle transition and TK1 activation was crucial for the [18F]FLT flare, as

Acknowledgments

This study was supported by a grant of the Korea Healthcare Technology R&D Project, Ministry for Health, Welfare & Family Affairs, Republic of Korea (No. A062254) and by the Real Time Molecular Imaging Research Program (No. 2010-002040) of National Research Foundation, which is funded by the Ministry of Education, Science and Technology, Republic of Korea.

We thank Woo Yeon Moon, Haeng Jung Lee, Hye Young Kang, Sang Ju Lee, and Na Young Chung for excellent technical assistance.

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