File S1 - Endothelin-1 Promotes Survival and Chemoresistance in Chronic Lymphocytic Leukemia B Cells through ETA Receptor

Contains the files: Figure S1. ET-1 expression in mutated vs. unmutated IGHV CLL subsets. (A) ET-1 expression levels were evaluated by quantitative reverse-transcription PCR on mutated IGHV CLL (n = 3) and unmutated IGHV CLL (n = 7) cells purified from peripheral blood. Histograms depict mean±SEM of ET-1 relative expression. Results were normalized to the expression of GAPDH housekeeping gene. No differential expression of ET-1 mRNA is evident between the two subsets. (B) Big ET-1, the 38-aa precursor of ET-1, was quantified by ELISA in conditioned media obtained after 72 h-culture from 4 mutated IGHV CLL and 9 unmutated ones. Histograms depict mean±SEM of big-ET-1 levels in pg/mL. Unmutated CLL cells secrete higher levels of big-ET-1 as compared to mutated CLL (*pFigure S2. ET-1 signaling improves CLL survival and promotes fludarabine resistance. (A) CLL cells (n = 6), pre-treated or not with 0.1 µM or 1 µM BQ-123, were stimulated with 100 nM ET-1. Viability was inspected by flow cytometry using Annexin-PI staining. Histograms represent mean±SEM of the percentage of viable cells (Annexin V-/PI-) in 3 independent time course experiments from 48 h to 96 h. ET-1 stimulation improves CLL viability at 96 h when leukemic cells decrease their spontaneous apoptosis resistance in vitro. (B) CLL cells (n = 11), pre-treated or not with 0.1 µM or 1 µM BQ-123, were cultured in contact with endothelial layers. Viability was inspected by flow cytometry using Annexin-PI staining. Histograms represent mean±SEM of the percentage of viable cells (Annexin V-/PI-) in 4 independent time course experiments from 48 h to 96 h. The blockade of ETAR by BQ-123 affects EC-mediated survival advantage at 72 h and 96 h. CLL cells (n = 8) were cultured (panel C) alone in complete medium or (panel D) in contact with HUVEC layer (HC). Fludarabine was added at 1 µM. Cells were also treated with 100 nM ET-1 and, as indicated, pretreated with 0.1 µM BQ-123 (20 min). Histograms summarize data at 24 h and 48 h, showing ET-1 mediated fludarabine-resistance at 48 hours. Control is defined as viability of CLL cells cultured alone in complete medium in panels A, B and C or in co-culture in panel D. (*pFigure S3. The blockade of ETAR by BQ-123 induces apoptosis on both mutated IGHV and unmutated IGHV CLL subsets. (A) CLL cells (n = 6, 3 mutated IGHV and 3 unmutated IGHV CLL), pre-treated or not with 0.1 µM or 1 µM BQ-123, were stimulated with 100 nM ET-1. (B) CLL cells (n = 11, 4 mutated IGHV and 7 unmutated IGHV CLL), pre-treated or not with 0.1 µM or 1 µM BQ-123, were cultured in contact with endothelial layers. Viability was inspected by flow cytometry using Annexin-PI staining. Histograms represent mean±SEM of the percentage of viable cells (Annexin V-/PI-) at 96 h of CLL divided into mutated vs. unmutated IGHV subsets. Control is defined as viability of CLL cells cultured alone in complete medium. (*pTable S1. Patients' characteristics (n = 151). (DOCX)