Different Modes of Transport for ³H-Thymidine, ³H-FLT, and ³H-FMAU in Proliferating and Nonproliferating Human Tumor Cells

Abstract

The basis for the use of nucleoside tracers in PET is that activity of the cell-growth–dependent enzyme thymidine kinase 1 is the rate-limiting factor driving tracer retention in tumors. Recent publications suggest that nucleoside transporters might influence uptake and thereby affect the tracer signal in vivo. Understanding transport mechanisms for different nucleoside PET tracers is important for evaluating clinical results. This study examined the relative role of different nucleoside transport mechanisms in uptake and retention of [methyl-3H]-3′-deoxy-3′-fluorothymidine (3H-FLT), [methyl-3H]-thymidine (3H-thymidine), and 3H-1-(2-deoxy-2-fluoro-β-D-arabinofuranosyl)-5-methyluracil (3H-FMAU). Methods Transport of 3H-FLT, 3H-thymidine, and 3H-FMAU was examined in a single human adenocarcinoma cell line, A549, under both nongrowth and exponential-growth conditions. Results 3H-Thymidine transport was dominated by human equilibrative nucleoside transporter 1 (hENT1) under both growth conditions. 3H-FLT was also transported by hENT1, but passive diffusion dominated its transport. 3H-FMAU transport was dominated by human equilibrative nucleoside transporter 2. Cell membrane levels of hENT1 increased in cells under exponential growth, and this increase was associated with a more rapid rate of uptake for both 3H-thymidine and 3H-FLT. 3H-FMAU transport was not affected by changes in growth conditions. All 3 tracers concentrated in the plateau phase, nonproliferating cells at levels many-fold greater than their concentration in buffer, in part because of low levels of nucleoside metabolism, which inhibited tracer efflux. Conclusion Transport mechanisms are not the same for 3H-thymidine, 3H-FLT, and 3H-FMAU. Levels of hENT1, an important transporter of 3H-FLT and 3H-thymidine, increase as proliferating cells enter the cell cycle.

Levels of human equilibrative nucleoside transporter-1 are higher in proliferating regions of A549 tumor cells grown as tumor xenografts in vivo

“…In previous in vitro work by our group, we reported that plateau-phase cultures of A549 cells have lower levels of ENT1 than proliferating cultures of A549 cells [2]. Our observation and those of Cass et al [10] and Coe et al [11] suggest that ENT1 levels are proliferation dependent.…”

“…The use of FLT uptake and retention as a surrogate marker for tumor proliferation is based on the assumption that thymidine kinase-1 (TK1, a cytosolic enzyme) activity up-regulated in S phase is the primary factor driving FLT uptake and retention in tumors [1]. While TK1 is an absolute requirement for radiotracer accumulation [2], variations in levels of the equilibrative nucleoside transporter-1 (ENT1), which facilitate FLT transport into cells, have been shown to modify intracellular peak levels of FLT [2, 3]. …”

[ 18 F]CFA as a clinically translatable probe for PET imaging of deoxycytidine kinase activity

“…1 G and H), further supporting the utility of this probe for imaging dCK activity. (27,28). As shown in Fig.…”

PET Imaging of Proliferation with Pyrimidines

“…Among the nucleosides, thymidine is exclusively used in the DNA structure. After entering the cells by multiple different transporters passively or actively (3,4), thymidine is phosphorylated into a more polarized molecule and cannot freely exit the cells. This mechanism has been used to accurately measure DNA synthesis in cell cultures using 3 H-thymidine.…”