Figure 1.

Human primary CLL cell proliferation is completely dependent on extracellular arginine. (A,B) Primary human CLL cells were isolated from peripheral blood of CLL patients by Ficoll density gradient centrifugation. Cells were activated with a TLR9 agonist (ODN 2006, 7.5 μg/ml) for 48 h or left unstimulated, both either in the presence (+) or absence (–) of 1 mM arginine (Arg). (A) Cell proliferation was determined by the incorporation of [³H]thymidine over 16 h. Values of stimulated cells in the presence of arginine (mean: 5,291 ± 2,668 cpm) were set as 100% (n = 21 from 7 independent CLL patients; P9, 14, 15, 19, 20, 24, and 25). (B) Cell viability: cells were stained with propidium iodide (PI) and then analyzed by flow cytometry. Values are shown as means ± SD (n = 8 independent donors, P9, 14, 15, 19, 20, 24, 25, and 26). Statistical calculations were performed by one way ANOVA with Tukey post-test. (C) ASS and Glycerinaldehyde 3-phosphate dehydrogenase (GAPDH) protein expression was analyzed by Western Blot in PB CLL cells from 18 consecutive patients (P1-18). (D) ASS and GAPDH protein expression were analyzed by Western Blot in PB CLL cells from 3 different patients (P19, 20, 22), cultured as described in (A). EA.hy926 (EA) endothelial cells served as positive control for ASS.