Figure 1.
Expression of HS1 protein in CLL B lymphocytes.
The lysates obtained from normal B lymphocytes and leukemic B cells from CLL patients were analyzed by immunostaining with antibody against HS1. Blots were reprobed with anti-β-actin antibody as loading control. Figure 1A is representative of four CLL and four healthy subjects with the respective densitometry of HS1/β-actin ratio. Figure 1B shows HS1/β-actin ratio of 71 CLL patients and 26 normal controls. Data has been normalized putting equal to 1 the ratio calculated in Jurkat cell line. Data obtained were evaluated for their statistical significance with the Student’s t-test (* p<0.01 between normal controls and CLL patients, B) or ANOVA (** p<0.01 between normal vs mutated CLL vs unmutated CLL, C; normal vs CD38 neg CLL vs CD38 pos CLL, D; normal vs 13q- and normal karyotype CLL vs 17p- and 11q- CLL, E; normal vs treated CLL vs untreated CLL, F; normal vs still alive patients vs dead patients, G). Medians are represented by solid lines. Figure 1H represents the overall survival comparison between patients (n = 16) presenting high levels of HS1 (HS1>0.93, dotted line) and patients (n = 54) presenting low levels of HS1 (HS1<0.93, solid line); the difference between curves is statistically significant (p<0.05, Kaplan Meier). 0.93 is the median of HS1 levels in normal controls.
Figure 2.
Expression of HS1 mRNA in CLL B lymphocytes.
RNA extracted from normal B lymphocytes and leukemic B cells from CLL patients were analyzed for HS1 expression and normalized on GAPDH. Figure 2A shows HS1 mRNA expression of 30 CLL patients and 8 normal controls. Data obtained were evaluated for their statistical significance with the Student’s t-test (* p<0.05 between normal controls and CLL patients, A) or ANOVA (** p<0.05 between normal vs mutated CLL vs unmutated CLL, B; normal vs CD38 neg CLL vs CD38 pos CLL, C; normal vs 13q- and normal karyotype CLL vs 17p- and 11q- CLL, D; normal vs still alive patients vs dead patients, E). Medians are represented by solid lines. Figure 2F represents the overall survival comparison between patients (n = 8) presenting high levels of HS1 mRNA (HS1>7.99, dotted line) and patients (n = 26) presenting low levels of HS1 mRNA (HS1<7.99, solid line); 7.99 is the median of HS1 levels in normal controls.
Figure 3.
Effect of in vivo therapy with FLU and Cy on HS1 protein.
The lysates obtained from leukemic B cells (5×105 for sample) from 32 patients subject to FLU-Cy therapy were analyzed by immunostaining with antibodies against HS1 and β-actin before (-) and after (+) the administration of FLU and Cy according to FLU-Cy protocol. The same cells were processed for RNA extraction, reverse transcription in cDNA and amplification of HS1 and GAPDH by Real-Time PCR. (A) Histograms represent HS1 percentage of variation measured by western blotting analysis. (B) Histograms represent HS1 percentage of variation measured by using Real-Time PCR. (C) Graphics reports WBC count before and after therapy in responsive (left) and unresponsive (right) patients; the reduction of WBC count underlines the responsiveness to therapy. (D) The western blot in the left panel is representative of 26 patients who responded to therapy; right panel is representative of 6 patients not responding to therapy. Cy, cyclophosphamide; FLU, fludarabine.
Figure 4.
Effect of in vitro treatment with FLU and Cy on HS1 protein.
(A) CLL cells were cultured for 24 hours alone or in presence of Cy (5 mM), FLU (20 µM) and Cy and FLU together (left panel), with the same drugs but in co-culture with the HS-5 stromal cell line (panel in the middle) or with PP2 (20 µM) (right panel). Subsequently, cells were processed for SDS/PAGE, transferred on nitrocellulose membrane and treated with polyclonal anti-PARP antibody, to put in evidence cell apoptosis, anti-HS1 polyclonal and anti-β-actin. Histograms represents full length HS1/β-actin ratio densitometric analysis. (B) Cell viability under the same conditions of point A was assessed by Annexin/PI test; histograms represent the mean±SD of percentage of cell viability of 8 CLL patients samples cultured alone (histograms on the left) or in co-culture with stromal cells (histograms on the right). (C) Confocal microscopy analysis of HS1 (FITC, green) in leukemic B lymphocytes cultured 24 hours alone or in presence of Cy, FLU and Cy and FLU together; nuclei were stained with DAPI (blue). UltraView LCI confocal system, UltraView LCI 5.0 acquisition software; original magnification, ×60. Cy, cyclophosphamide; FLU, fludarabine.
Figure 5.
Subcellular localization of HS1 in normal and CLL B cells.
(A) Analysis by differential ultracentrifugation. B cells were sonicated in isotonic buffer and nuclei (I), cytosol (II) and microsomes (III) were separated by ultracentrifugation. Comparable aliquots of the different fractions were loaded on SDS/PAGE and the separated proteins were immunostained with anti-HS1 antibody, anti-LDH (cytosolic marker), anti-calnexin (endoplasmic reticulum marker), anti-PMCA (plasma membrane marker), anti-lamin-B (nuclear marker) and anti-aconitase (mitochondrial marker) antibodies. Figure is representative of different experiments performed on 10 CLL patients and 5 normal controls. The diagram shows the mean ± SD of HS1 of all the samples examined. (B) Confocal microscopy analysis of HS1 (FITC, green) in normal and leukemic B lymphocytes obtained from peripheral blood; nuclei were stained with DAPI (blue). The analysis of HS1 immunolocalization was performed in 6 different normal and 15 CLL samples. (C) Confocal microscopy analysis of HS1 (FITC, green) in leukemic cells from spleen and lymph node of a CLL patient. (D) Confocal microscopy analysis of normal and leukemic B lymphocytes after incubation alone or with anti-human IgM (10 µg/ml) for 10 minutes at 37°C for BCR activation. Cells were immunostained with anti-HS1 antibody (FITC, green). (E) The same experiment of point D was performed in CLL lymphocytes pre-incubating cells with the Lyn kinase inhibitor PP2 (10 µM). The figure is representative of the experiments performed in 4 CLL patients. UltraView LCI confocal system, UltraView LCI 5.0 acquisition software; original magnification, ×60.
Table 1.
Biological and clinical characteristic of patients.