Figure 3.

CAT-1 is the only arginine importer in primary CLL cells and its expression and arginine transport are coordinately upregulated upon activation. (A–C) mRNA expression of the arginine transporters (A) CAT-1, (B) y⁺LAT1, and (C) y⁺LAT2 was quantified by qRT-PCR in mRNA obtained from PB CLL cells of 18 individual patients (same as in Figure 1C). Depicted is the copy number of the respective mRNAs /ng total mRNA. (D) Protein expression of CAT-1, y⁺LAT1, y⁺LAT2, and GAPDH (as loading control) was determined by Western Blot in lysates from PB CLL cells of 18 individual patients (same as in Figure 1C). (E) The uptake of 100 μM [³H]arginine (10 μCi/ml) into PB CLL cells was assessed over 5, 30, 60, and 120 s. Arginine uptake is depicted in pmol/1 × 10⁶ cells as means ± SD of triplicates of CLL cells from P15 (other donors analyzed: P16, 18, 23). (F–I) PB CLL cells from 4 patients (P19-P22) were incubated for 48 h in cell culture medium in the presence (+) or absence (−) of a TLR9 agonist ODN 2006 (7.5 μg/ml). The mRNA expression of (F) CAT-1, (G) y⁺LAT1, and (H) y⁺LAT2 was quantified by qRT-PCR. mRNA expressions of each transporter are shown in relation to beta 2-Mikroglobulin (B2M). (I) Protein expression of the three transporters and GAPDH was analyzed by Western Blot. (J) The uptake of 100 μM [³H]arginine (10 μCi/ml) was determined over 30 s in the absence or presence of the CAT inhibitor NEM (200 μM) and the presence of 1 mM leucine in 48 h TLR9-stimulated or unstimulated PB CLL cells. Data are shown as means ± SD from PB CLL cells (n = 8) from P9, 19, and 20. The uptake in cells stimulated with TLR9 agonist in the absence of NEM (7.4 ± 1.4 pmol/10⁶ cells) was set to 100% for each patient to address intraindividual variations. Statistical calculations were performed by one way ANOVA with Tukey post-test.