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Metabolism of 3′-deoxy-3′-[F-18]fluorothymidine in proliferating A549 cells: Validations for positron emission tomography

https://doi.org/10.1016/j.nucmedbio.2004.06.004Get rights and content

Abstract

3′-Deoxy-3′-[F-18]fluorothymidine (FLT) is under clinical evaluation as a metabolic probe for imaging cell proliferation in vivo using positron emission tomography (PET). As part of our validation effort, we followed the short-term metabolism of FLT in exponentially growing tumor cells to demonstrate the enzyme activities within the DNA salvage pathway that influence retention of radioactivity. In A549 cells, thymidine kinase-1 (TK1) activity produced FLTMP, which dominated the labeled nucleotide pool. Subsequent nucleotide phosphorylations by thymidylate kinase (TMPK) and nucleotide diphosphate kinase (NDPK) led to FLTTP. After 1h, the cellular metabolic pool contained ∼30% FLTTP. A putative deoxynucleotidase (dNT), which degrades FLTMP to FLT, provided the primary mechanism for tracer efflux from cells. In contrast, FLTTP was resistant to degradation and highly retained. The uptake and retention characteristics of FLT were also compared to those of thymidine, FMAU (2′-arabino-fluoro-TdR) and FIAU (2′-arabino-fluoro-5-iodo-2′-dexoyuridine). Despite the fact that FLT lacks the 3′-hydroxy necessary for its incorporation into DNA it out performed both FMAU and FIAU in terms of uptake and retention.

Introduction

Tumor proliferation measures can be used to differentiate benign from malignant lesions and to measure the effect of treatments directed toward actively dividing cells. Non-invasive PET imaging of tumors with fluorine-18 labeled FLT is being evaluated for this purpose [1], [2], [3], [4], [5], [6], [7], [8]. Its mechanism of uptake is based on metabolism within the DNA synthesis pathway as shown in Fig. 1.

FLT can be labeled at high specific activity [9] and the fluorine substitution functions to restrict nucleoside metabolism, similar to the effect on glucose metabolism seen with 2-[F-18]fluoro-2-deoxyglucose (FDG). As a result, [F-18]FLT has become an attractive alternative to [C-11]thymidine for imaging cellular proliferation.

The large pools of thymidine phosphorylase (TP) present in blood, liver and spleen rapidly degrade thymidine in vivo. In contrast, FLT is inert to TP. For thymidine, this limits its incorporation into DNA and produces a large pool of labeled blood metabolites. One consequence of this is that tissues such as proliferative bone marrow exhibit a higher uptake with FLT compared to thymidine [10]. This is despite the fact that FLT has a lower Vmax/Km value for the initial enzyme in the thymidine salvage pathway, thymidine kinase-1 (TK1) [11].

FLT is a selective substrate for TK1, the enzyme used for nuclear DNA replication. In contrast, thymidine also reacts with TK2, the unregulated isozyme, which is used for mitochondrial DNA replication and repair. This situation offers an explanation for why [C-11]thymidine images the heart wheres [F-18]FLT does not.

In proliferating cells, FLT metabolism takes place within the anabolic arm of the DNA salvage pathway. The role of this pathway is to rapidly balance intracellular nucleotide pools used for DNA replication. It operates by using futile metabolic cycles to buffer intracellular nucleotide pools produced by de novo biosynthesis [12]. In these cycles, excess deoxynucleotides are degraded by nucleotidases and ultimately exported as nucleosides. When needed, nucleosides are imported and phosphorylated by kinases to balance the demand for nucleotide triphosphates. The scheme, specific to thymidine and analogs such as FLT, is shown in Fig. 1.

TK1 controls entry into the salvage pathway and converts intracellular thymidine and FLT to their nucleotide monophosphates [11], [12]. This cytosolic enzyme is expressed when cells pass through S-phase of the cell cycle [13], after which it is rapidly degraded. Subsequent phosphorylations by other kinases within the DNA synthesis pathway, thymidylate kinase (TMPK) and nucleotide diphosphate kinase (NDPK), lead to the added presence of FLTDP and FLTTP within cells [14], [15], [16], [17]. As a direct result of TK1 activity, proliferating cells have a select capacity to metabolize FLT during S-phase and to retain its labeled nucleotides.

Intracellular thymidine labels DNA so rapidly that the impact of retrograde synthesis of thymidine from TTP is negligible. However, FLT can only act as a DNA chain terminator [14], [15] because it lacks a 3′-hydroxyl. Consequently, reversible nucleotide metabolism within the salvage pathway is a significant issue. To obtain a better understanding of these processes we studied FLT metabolism using exponentially growing A549 tumor cells. We also compared FLT's cell uptake and washout characteristics to those of alternative fluoronucleoside probes, FMAU (2′-arabino-fluoro-TdR) and FIAU (2′-arabino-fluoro-5-iodo-2′-dexoyuridine), which are inert to TP and have been proposed as PET tracers. In contrast to FLT, both FMAU and FIAU can be incorporated into DNA, which, overall, might lead to better label retention. We also conducted experiments to test this issue.

This report presents a perspective on the cellular metabolism of FLT and the extent to which it has been validated as a radiotracer for cellular proliferation.

Section snippets

Reagents

Reagents were purchased from Aldrich (Milwaukee, WI, USA), or otherwise as indicated. [F-18]FLT was routinely prepared with a specific activity >1 Ci/μmol at the time of use, according to our published method [9]. [F-18]FLT-5′-monophosphate (FLTMP) was prepared enzymatically and purified from [F-18]FLT as described below. [H-3]Thymidine (2.0 Ci/mmol), [H-3]FLT (6.2 Ci/mmol), [H-3]FMAU (2.1 Ci/mmol), and [C-14]FMAU (0.055 Ci/mmol) were purchased from Moravek Biochemicals (Brea, CA, USA) and were

Results

[F-18]FLT metabolism in A549 cell cultures was evaluated after 10 min and after 2 h of exposure. All three FLT-nucleotides were detected in cell extracts using ion exchange-HPLC, Fig. 2. The two times were chosen to separate initial metabolic events from those resulting from prolonged tracer exposure. Initial metabolism was dominated by monophosphate (FLTMP) synthesis, whereas exposure for 2 h led to an additional significant triphosphate (FLTTP) fraction. Table 1 shows the distribution of FLT

Discussion

Several features of the DNA salvage pathway may influence how well FLT reflects cellular proliferation. The following discussion summarizes key data from the literature and our work in further defining the scope and impact metabolism plays in A549 cells.

Conclusions

The metabolism of labeled FLT in A549 cells has been validated by metabolite analyses. Accumulation of intracellular radioactivity results exclusively from formation of FLT-nucleotides (FLTMP, FLTDP, FLTTP) in competition with FLTMP degradation. The kinetics of these processes is dominated by TK1, dNT-1, and TMPK activities. The rate limiting step in the overall conversion of FLT to FLTTP was the phosphorylation of FLTMP by TMPK. This study provides additional validation of FLT metabolism in a

Acknowledgements

This work was supported by Grant Nos. CA42045 and CA34570 from the National Institutes of Health.

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