Cellular localization and functional characterization of the equilibrative nucleoside transporters of antitumor nucleosides

Abstract

Nucleoside transporters play an important role in the disposition of nucleosides and their analogs. To elucidate the relationship between chemosensitivity to antitumor nucleosides and the functional expression of equilibrative nucleoside transporters (ENT), we established stable cell lines of human fibrosarcoma HT-1080 and gastric carcinoma TMK-1 that constitutively overexpressed green fluorescent protein-tagged hENT1, hENT2, hENT3 and hENT4. Both hENT1 and hENT2 were predictably localized to the plasma membrane, whereas hENT3 and hENT4 were localized to the intracellular organelles. The chemosensitivity of TMK-1 cells expressing hENT1 and hENT2 to cytarabine and 1-(3-C-ethynyl-beta-D-ribopentofuranosyl) cytosine increased markedly in comparison to that of mock cells. However, no remarkable changes in sensitivity to antitumor nucleosides were observed in cell lines that expressed both hENT3 and hENT4. These data suggest that hENT3 and hENT4, which are mainly located in the intracellular organelles, are not prominent nucleoside transporters like hENT1 and hENT2, which are responsible for antitumor nucleoside uptake.

Figure 1

The mRNA expression of nucleoside transporters was determined by reverse transcription–polymerase chain reaction analysis. Total RNA from TMK‐1, HT‐1080 and HT‐29 cells was used to analyze the mRNA expression of nucleoside transporters.

Figure 2

The localization of human equilibrative nucleoside transporters (hENT) (green fluorescent protein [GFP]‐hENT1–4) in HT‐1080 cells that stably overexpressed the GFP‐hENT fusion protein (green). (a) Lysosomes (red), (b) mitochondria (red) and (f) nuclei (red) were stained by organelle‐selective dyes. (c) Golgi complexes (red), (d) early endosomes (red) and (e) late endosomes (red) were identified immunologically by specific antibodies.

Figure 3

The transport function of human equilibrative nucleoside transporters (hENT) analyzed as the intracellular uptake of cytarabine (Ara‐C). The uptake of [³H]‐Ara‐C into HT‐1080 and TMK‐1 cells that stably overexpressed the green fluorescent protein (GFP)‐hENT fusion protein. Total cellular accumulation of [³H]‐Ara‐C was measured at 37°C for 15 min (open column). To evaluate the functional transporter for [³H]‐Ara‐C, nucleoside transporter inhibitors such as nitrobenzylthioinosine (NBMPR) (2 µM, hatched column) or dipyridamole (DPM) (20 µM, closed column) were added to each well. Statistical analysis against the mock control was carried out using Student's t‐test. *P < 0.05, **P < 0.01.