Stable transfection of HEK293 cells with SLC43A3_1 and SLC43A3_2. (A) Transfection of HEK293 cells with SLC43A3 was confirmed using PCR with the primers specific for SLC43A3 (top) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (bottom) shown in (Supplemental Table 1). cDNA was prepared from total RNA isolated from untransfected cells (HEK293) or HEK293 cells stably transfected with SLC43A3_1 or SLC43A3_2. (B) Membranes were prepared from HEK293 cells and cells transfected with SLC43A3_1 (ENBT1.1) and SLC43A3_2 (ENBT1.2). Samples were resolved on SDS-PAGE gels, transferred to polyvinyl membranes and probed with anti-SLC43A3 and anti-β-actin antibodies. (C) Parallel immunoblot analyses were performed as described previously, but using anti-myc and anti-β-actin antibodies.

Compensatory changes in gene expression upon transfection of HEK293 cells with SLC43A3. Gene expression, assessed by semiquantitative PCR, is shown relative to the amount detected in untransfected HEK293 cells. Expression was normalized to glyceraldehyde-3-phosphate dehydrogenase transcript levels in each individual experiment. (A) Relative expression of SLC43A3 in HEK293 cells stably transfected with the respective SLC43A3_1, SLC43A3_2, or empty vector (pcDNA3.1) construct (N = 5). (B–D) Relative expression of ENT1 (SLC29A1), ENT2 (SLC29A2), ENT4 (SLC29A4), MRP4 (ABCC4), and MRP5 (ABCC5), and the enzymes TPMT and hypoxanthine-guanine phosphoribosyltransferase (HPRT) in HEK293 cells transfected with either empty pcDNA3.1 (B), SLC43A3_1 (C), or SLC43A3_2 (D). Bars represent the mean ± S.D. of five independent samples; * denotes significant difference in expression between untransfected HEK293 cells and transfected HEK293 cells (Student’s t test, P < 0.05, corrected for multiple comparisons with the Holm-Sidak method).

[³H]Adenine transport by SLC43A3-encoded ENBT1. HEK293 cells and cells transfected with SLC43A3_1 (A), SLC43A3_2 (B), or the empty pcDNA3.1 vector (B) were incubated with 100 µM [³H]adenine at room temperature for the specified times (abscissa) and then centrifuged through oil. Cell pellets were digested overnight in 1 M NaOH and assessed for [³H] content using standard liquid scintillation counting techniques to define picomoles of adenine accumulated per microliter cell pellet (ordinate). ENBT1-mediated uptake was defined as the difference in cellular accumulation by the SLC43A3-transfected cells (SLC43A3-HEK293) and that observed in the untransfected HEK293 cells assessed in parallel. Data points represent the mean ± S.D. of six (A) or five (B) experiments done in duplicate. (C) The kinetics of ENBT1.1- and ENBT1.2-mediated uptake of [³H]adenine were determined by assessing the uptake of a range of concentrations of [³H]adenine (abscissa) as described for (A and B). Initial rates of influx (ordinate) were estimated as the transporter-mediated uptake at 0.5 seconds extrapolated from time course profiles as shown in (A) (ENBT1.1) or directly from the 2-second uptake time point (B) (ENBT1.2). Data shown are the mean ± S.D. of N = five experiments.

[¹⁴C]6-MP transport by SLC43A3-encoded ENBT1. HEK293 cells and cells transfected with SLC43A3_1 (A), SLC43A3_2 (B), or empty pcDNA3.1 (B) were incubated with 100 µM (A) or 30 µM (B) [¹⁴C]6-MP at room temperature, in the presence and absence of 1 mM adenine, for the specified times (abscissa) and then centrifuged through oil. Cell pellets were digested overnight in 1 M NaOH and assessed for [¹⁴C] content using standard liquid scintillation counting techniques to define pmol 6-MP accumulated per microliter cell pellet (ordinate). Data points represent the mean ± S.D. of seven (A) or five (B) experiments done in duplicate. (C) The kinetics of ENBT1.1- and ENBT1.2-mediated [¹⁴C]6-MP uptake were determined by assessing the uptake of a range of concentrations of [¹⁴C]6-MP (abscissa) as described for (A and B). Initial rates of influx (ordinate) were estimated as the transporter-mediated uptake (calculated as the difference in cellular uptake ± adenine) at 0.5 seconds interpolated from time course profiles as shown in (A) (ENBT1.1), or directly from the 2-second uptake time point (ENBT1.2). Data shown are the mean ± S.D. of N = 5 experiments.

Inhibition of [¹⁴C]6-MP transport by SLC43A3-transfected HEK293 cells. The uptake of [¹⁴C]6-MP by ENBT1.1 (100 µM) (A) and ENBT1.2 (30 µM) (B) was assessed using a 2-second time point in the presence and absence of the indicated concentration of MTX, 6-TG, MMP, 2-chloroadenosine (Cl-Ado), adenosine (Ado), 2-deoxyadenosine (Deoxy-Ado), hypoxanthine (Hypox), uridine, nitrobenzylthioinosine (NBMPR), or decynium-22 (D22). Data were normalized as percentage of control uptake, where 100% was defined as the uptake of [¹⁴C]6-MP in the absence of inhibitor and 0% was defined as the uptake in the presence of 1 mM adenine. Each bar represents the mean ± S.D. of five experiments done in duplicate; * denotes significantly different from 100% control (Student’s t test, P < 0.05, corrected for multiple comparisons with the Holm-Sidak method). (C) Time courses were constructed at various concentrations of [³H]adenine in the presence of 750 µM 6-TG, as described previously (see Fig. 4C). Initial rates of transport were derived from the rate of uptake at 0.5 seconds as extrapolated from the time course profiles. The dashed line indicates the analogous data obtained in the absence of 6-TG (from Fig. 4C). Data points represent mean ± S.D. of five experiments done in duplicate. (D) A range of concentrations of 6-TG, MMP, and D22 were assessed for their ability to inhibit the 2-second uptake of 100 µM [¹⁴C]6-MP by the ENBT1.1. Data were normalized as the percentage of control uptake with 100% defined as the uptake of 100 µM [¹⁴C]6-MP in the absence of inhibitor and 0% defined as that in the presence of 1 mM adenine. Sigmoid curves were fitted to these data for the determination of IC₅₀ values, which were used to calculate the inhibitor K_i values shown in the text. Each point represents the mean ± S.D. of N = 5 experiments done in duplicate.

6-MP efflux by ENBT1.1. Cells were loaded with 100 µM [¹⁴C]6-MP for 30 seconds (SLC43A3_1 transfected) or 10 minutes (untransfected HEK293), pelleted, and then to initiate efflux suspended in either N-methylglucamine buffer (control) or buffer containing either 1 mM or 100 µM adenine. Aliquots of cell suspension were centrifuged through an oil layer at the specified times (abscissa) and processed to assess intracellular [¹⁴C] content. Data are expressed as percentage of initial [¹⁴C]6-MP load (ordinate) with 100% defined as [¹⁴C]6-MP content at zero time extrapolated from the curve fit (one phase exponential decay) to the 1 mM adenine data. One- or two-phase decay profiles were fitted to the data depending on which fit was determined to be statistically superior (P < 0.05; F test). Each point represents the mean ± S.D. of five (control and 100 µM adenine) or 10 (1 mM adenine) experiments.

Effect of ceefourin-1, zaprinast, and DY on 6-MP efflux. SLC43A3_1-transfected HEK293 cells were loaded with 100 µM [¹⁴C]6-MP for 30 seconds, pelleted, and then to initiate efflux suspended in either N-methylglucamine buffer (control) or buffer containing either 1 mM or 100 µM adenine, as indicated, in the presence and absence of the MRP4 inhibitor ceefourin-1 (50 µM) (A), the MRP5 inhibitor zaprinast (100 µM) (B), a combination of 50 µM ceefourin-1 and 100 µM zaprinast (C), or the ENT2 inhibitor DY (1 µM) (D). Aliquots of cell suspension were centrifuged through an oil layer at the specified times (abscissa) and processed to assess intracellular [¹⁴C] content. Data are expressed as percentage of initial [¹⁴C]6-MP load (ordinate), and either a one- or two-phase decay profile was fitted to the data based on which fit was determined to be statistically superior (P < 0.05; F test). Each point represents the mean ± S.D. of five experiments.

SLC43A3 transfection of HEK293 cells significantly enhances the cytotoxicity of 6-MP. HEK293 cells and cells transfected with empty pcDNA3.1 plasmid or SLC43A3_1 (A) or SLC43A3_2 (B) were plated at a density of 5 × 10⁴ cells/well in 24-well plates and incubated with a range of concentrations of 6-MP (abscissa) for 48 hours at 37°C in a humidified incubator (5% CO₂/95% air). Cell viability was assessed using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay, and expressed as a percentage of the cell viability measured at 48 hours in the absence of 6-MP (percentage of control; ordinate). Biphasic dose-response curves were fitted to these data, and each point represents the mean ± S.D. of five experiments; * denotes significant difference between HEK293 cells and SLC43A3-transfected cells (Student’s t test, P < 0.05, corrected for multiple comparisons with the Holm-Sidak method). (C) Effect of DY on the cytotoxicity of 6-MP. HEK293 cells and cells transfected with SLC43A3_1 were incubated with a range of concentrations of 6-MP in the absence and presence of the ENT1/ENT2 blocker DY (1 µM). Cell viability was assessed and data are presented as described for (A and B) (N = 5). (D) Effect of G418 on the cytotoxicity of 6-MP in SLC43A3_1- and SLC43A3_2-transfected HEK293 cells. Cell viability was assessed upon incubation with the indicated concentrations of 6-MP for 48 hours, as described for (A and B), in the presence (+) and absence (−) of 120 µg/ml G418 (N = 5).