Figure 2.

Human immortalized CLL cells do not express functionally relevant amounts of ASS and are therefore arginine auxotrophic. (A–C) Human HG3 CLL cells were incubated for 48 h in cell culture medium in the presence (+) or absence (–) of 1 mM arginine (Arg). (A) Cell proliferation was determined by the incorporation of [³H]thymidine over 16 h. Data are shown as means ± SD. Proliferation in the presence of arginine (mean: 65,402 ± 52,269 cpm) was set as 100% (n = 8–9). (B,C) Cells were stained with Annexin V and propidium iodide (PI) and were then analyzed by flow cytometry (n = 3). Values are shown as means ± SD. Statistical calculations were performed by t-test. (D,E) HG3 CLL cells were incubated for 24 and 48 h (h) in the presence (+) or absence (–) of 1 mM arginine and citrulline (Cit). ASS and GAPDH protein expression were analyzed by SDS-PAGE and Western Blot in cell lysates. (D) Quantitative analyses of OD values from Western Blots of 3 individual experiments depict ASS normalized to GAPDH expression and calculated as % expression of EA.hy926 cells (EA), used as positive control. Statistical calculations were performed by one way ANOVA with Tukey post-test. (E) A representative Western Blot of 3 independent experiments is shown. (F) HG3 CLL cells were incubated for 48 h in the presence or absence of 1000 μM arginine, and supplemented with different concentrations of citrulline (0–1,000 μM). Cell proliferation was determined by the incorporation of [³H]thymidine for further 16 h (n = 8–9). Data are shown as means ± SD in % of cells incubated in the presence of arginine (mean: 201,625 ± 93,045 cpm). Statistical calculations were performed by one way ANOVA with Tukey post-test.