Background:  3’-Azido-2’,3’-dideoxythymidine (AZT) is a nucleoside reverse transcriptase inhibitor (NRTI) frequently used during HAART. It has been suggested that a reduced influx of NRTI could compromise optimal intracellular levels of a specific drug, favoring the selection of resistant viruses. Some proteins of the gene families SLC28, SLC29, and SLC22 have been extensively studied regarding the uptake mechanisms of nucleoside analogs used in antiviral treatment. Therefore, we aimed to study the contribution of human nucleoside transporters (hENT and hCNT) and human organic anion transporters (hOAT) in the uptake of NRTI, focusing on entry mechanisms of AZT in T-lymphocytes, main targets of HIV.

Methods:  The mRNA expression of hENT and hCNT was studied in 6 different T-cell lines, peripheral blood mononuclear cells (PBMC), purified CD4⁺ T lymphocytes, monocytes, monocyte-derived macrophages (MDM), and monocyte-derived dendritic cells (MDDC) by quantitative real-time polymerase chain reaction (RT-PCR). Similarly, the presence of hOAT in T lymphocytes and macrophages was assessed by RT-PCR. To determine the uptake of NRTI by influx transporters, [³H]-AZT cis-inhibition experiments were performed in Molt-4 cell line and PBMC. Moreover, uptake at different temperatures and in phytohemagglutinin (PHA) -stimulated PBMC was studied. To assess statistical significance we used an unpaired t-test and a Mann-Whitney test.

Results:  In T-cell lines and primary lymphocytes, hCNT1 and hCNT3 were minimally expressed, whereas hCNT2 and hENT showed high mRNA expression, being hENT1 the most expressed transporter (4.5 to 6.5 log₁₀ copies of mRNA/µg RNA). By contrast, macrophages and immature DC (iDC) showed a higher expression of hCNT3 than PBMC or CD4⁺ T cells (4.0 vs 1.5 log₁₀; p <0.05). Cis-inhibition experiments demonstrated that hNT play a negligible role in NRTI uptake, whereas hOAT were not detected in T cells. Uptake of [³H]-AZT in PHA-stimulated PBMC was 2-fold higher than in non-stimulated PBMC. AZT showed a temperature-sensitive entry route as the uptake at 4ºC was diminished to 20 to 25% (p <0.01).

Conclusions:  These results demonstrate that hNT and hOAT are not involved in AZT uptake in T lymphocytes, but that hCNT3 could play a role in the entry of these drugs in macrophages and iDC. Nevertheless, AZT uptake into T cells involves a carrier-mediated transport mechanism. The identification of specific NRTI transporters could be a novel target to optimize chemotherapeutic responses.