ET-1 mediates survival signal on CLL cells.

<p>(A) Serum-starved CLL cells were stimulated with 100 nM ET-1 for 1 hour, pretreating or not cells with 0.1 µM BQ-123 (20 min). Western blot analysis of CLL cells was performed with anti-phospho-Akt, anti-Akt, anti-phospho-ERK, anti-ERK and anti-β-actin antibodies. ET-1 stimulates Akt and ERK phosphorylation through ET_AR. The immunoblots depict Akt and ERK activation in a representative case. Histograms represent densitometric quantification (pAkt/total Akt ratio and pERK/total ERK ratio) of bands relative to phospho-Akt, total Akt, phospho-ERK and total ERK normalized on β-actin. Data are presented as mean±SEM of 6 CLL patients relative to unstimulated control (*p<0.05, ***p<0.001). (B) CLL cells (n = 6) were cultured with the addition of recombinant ET-1 peptide at 100 nM for 96 hours. CLL cell viability was assessed by flow cytometry using Annexin V and PI staining. A lymphocyte gate was set according to the different relative size and granularity (forward scatter and side scatter) and viable cells were defined as Annexin V-/PI-. Histograms represent mean±SEM at 96 h of 6 CLL patients evaluated in 3 independent experiments. Two representative cases are depicted on the right, showing cell viability at 48 h, 72 h and 96 h. Control is defined as viability of CLL cells cultured alone in complete medium. ET-1 stimulation improves CLL survival. The effect is inhibited by pre-treating CLL cells with BQ-123 (0.1–1 µM) (**p<0.01, ***p<0.001). (C) CLL cells (n = 11), pretreated or not with BQ-123 (0.1–1 µM), were cultured on HUVEC cell layer for 96 hours. CLL cell viability was assessed by flow cytometry, as described for panel B. Histograms represent data as mean±SEM of 11 CLL patients evaluated in 4 independent experiments. Two representative cases are depicted on the right, showing cell viability at 48 h, 72 h and 96 h. Control is defined as viability of CLL cells cultured alone in complete medium. Note that BQ-123 significantly reduces the CLL pro-survival effect mediated by HUVEC contact (***p<0.001). (D) CLL cells were stimulated with 10 µg/mL of anti-IgM for 48 hours with or without 0.1 µM BQ-123. Dot plots represent cell viability of 6 CLL patients evaluated in 3 independent experiments. The blockade of ET_AR reduces the pro-survival effect of BCR triggering in CLL cells (*p<0.05). (E) Serum-starved CLL cells were pretreated or not with 0.1 µM BQ-123 for 20 minutes before stimulation with anti-IgM for 10 minutes. The immunoblots depict ERK phosphorylation in a representative case.