Chronic lymphocytic leukemia B cells contain anomalous Lyn tyrosine kinase, a putative contribution to defective apoptosis

Abstract

B cell chronic lymphocytic leukemia (B-CLL) is a neoplastic disorder characterized by accumulation of B lymphocytes due to uncontrolled growth and resistance to apoptosis. Analysis of B cells freshly isolated from 40 patients with chronic lymphocytic leukemia demonstrated that the Src kinase Lyn, the switch molecule that couples the B cell receptor to downstream signaling, displays anomalous properties. Lyn is remarkably overexpressed at the protein level in leukemic cells as compared with normal B lymphocytes, with a substantial aliquot of the kinase anomalously present in the cytosol. Whereas in normal B lymphocytes Lyn activation is dependent on B cell-receptor stimulation, in resting malignant cells, the constitutive activity of the kinase accounts for high basal protein tyrosine phosphorylation and low responsiveness to IgM ligation. Addition of the Lyn inhibitors PP2 and SU6656 to leukemic cell cultures restores cell apoptosis, and treatment of malignant cells with drugs that induce cell apoptosis decreases both activity and amount of the tyrosine kinase. These findings suggest a direct correlation between high basal Lyn activity and defects in the induction of apoptosis in leukemic cells. They also support a critical role for Lyn in B-CLL pathogenesis and identify this tyrosine kinase as a potential therapeutic target.

Figure 1

Analysis of protein tyrosine phosphorylation in normal and CLL B cells. Immunoblot analysis of cellular tyrosine-phosphorylated proteins in normal B cells, from both tonsil (T) and peripheral blood (P), and in leukemic B-CLL cells. Cells were lysed, and the proteins were analyzed by Western blotting (Wb) and immunostaining with antibody against phosphorylated tyrosine. The filter was reprobed with anti–β-actin antibody as loading control. The molecular mass of protein standards is indicated on the right. The data are representative of experiments performed with 3 different tonsils, 5 peripheral blood samples, and 40 B-CLL samples.

Figure 2

Effect of several inhibitors on CLL cellular tyrosine phosphorylation and tyrosine kinase activities. Freshly isolated B cells obtained from CLL patients no. 19 and no. 40 were cultured for 15 minutes without or with the indicated tyrosine kinase inhibitors. (A) Cells were lysed and analyzed by immunostaining with antibody against phosphorylated tyrosine as in Figure 1. The molecular mass (kDa) of protein standards is indicated in the center. (B and C) Cells were lysed, and the tyrosine kinase activity was detected either on the Src-specific peptide substrate cdc2(6–20) (B) or on the nonspecific random polymer polyGlu₄Tyr (C). The data are representative of experiments performed with 8 different B-CLL samples. Ctrl, control.

Figure 3

Expression of Src kinase proteins and Lyn mRNA in normal and CLL B cells. (A) Immunoblot analysis of the cellular protein level of different Src kinases. The lysates obtained from normal B lymphocytes and leukemic B cells from CLL patients were analyzed by immunostaining with antibodies against the indicated Src kinases. Blots were reprobed with anti–β-actin antibody as loading control (the reprobing of the Lyn blot is shown). The panel is representative of Lyn analysis performed with 3 different tonsils, 5 peripheral blood samples, and 40 B-CLL samples. The analysis of the other Src kinases was performed with 10 CLL samples. (B) The analysis of Lyn immunolocalization was performed by confocal microscopy in 3 different normal and 10 B-CLL samples. Original magnification, ×60. (C) The analysis of Lyn and β-actin mRNA expression was performed by Northern blotting in 16 B-CLL samples and the same normal B cells shown in A. Data from 8 representative B-CLL patients are shown.

Figure 4

Kinase activity of Lyn in normal and CLL B cells before and after IgM ligation. (A) Analysis of Lyn activity in unstimulated B cells. Normal cells refer to tonsil (T), total peripheral blood B cells (P_T), naive peripheral blood B lymphocytes (P_N), and memory peripheral blood B lymphocytes (P_M). Normal and leukemic B cells from CLL patients were lysed and Lyn was immunoprecipitated. The immunocomplexes were then analyzed for both the in vitro kinase activity tested toward the Src-specific peptide substrate cdc2(6–20) and anti-Lyn immunostaining. The data are representative of experiments performed with 3 different tonsils, 5 peripheral blood samples, and 40 B-CLL samples. (B) Analysis of Lyn activity in B cells after IgM ligation. B lymphocytes were isolated from 1 healthy donor and 2 CLL patients, 1 (CLL no. 29) expressing high levels and 1 (CLL no. 18) expressing low levels of IgM, as indicated by flow cytometry analysis (upper panels). B cells were incubated with anti-human IgM (20 μg/ml) at 37–C for the indicated times and the kinase activity of anti-Lyn immunoprecipitates was detected as described in A (lower panels). The data are representative of 3 separate experiments.

Figure 5

Subcellular localization of Lyn in normal and CLL B cells. (A) Analysis by differential ultracentrifugation. B cells were sonicated in isotonic buffer and cytosol, and microsomes (II particulate fraction, II-P) were separated from the cell debris and the other cellular particles (I particulate fraction, I-P) by ultracentrifugation. Comparable aliquots of the different fractions were loaded on SDS/PAGE and the separated proteins were immunostained with either anti-Lyn antibody or the antibodies specific for the indicated distribution control markers: complex II (I particulate, mitochondria), SERCA2 (II particulate, endoplasmic reticulum), and CD79b (II particulate, plasma membrane). Other aliquots were immunoprecipitated with anti-Lyn antibody, and the kinase activity of the immunocomplexes was detected in vitro. The data are representative of experiments performed with 3 different normal and 14 B-CLL samples. (B) Confocal microscopic analysis of the ganglioside GM1 labeled with cholera toxin B subunit (Texas Red) and Lyn (FITC) in normal and leukemic B lymphocytes. The data are representative of experiments performed with 5 normal and 10 different leukemic samples. Original magnification, ×60.

Figure 6

Effect of different compounds on the apoptosis of CLL B cells. Leukemic cells were cultured for 24 hours alone or in the presence of CsA (8 μM), Dex (10 μM), PP2 (10 μM), SU6656 (10 μM), or EGCG (10 μM), and cell apoptosis was analyzed by annexin V–PI flow cytometry. (A) Flow cytometry analysis of B-CLL sample no. 14. (B) Percentage of apoptotic cells obtained in the different conditions with the following B-CLL samples: no. 2 (filled inverted triangles), no. 4 (open inverted triangles), no. 6 (filled triangles), no. 9 (open circles), no. 14 (filled squares), no. 17 (filled circles), no. 28 (filled diamonds), no. 33 (arrows), no. 35 (open triangles), and no. 39 (open squares). (C) The data are reported as median, lower and upper quartile, and minimum and maximum. *P < 0.005 compared with cells cultured in medium alone (Ctrl). Min-max, minimum–maximum.

Figure 7

Effect of different compounds on caspase and Lyn activities of CLL B cells. Leukemic lymphocytes were cultured without or with drugs as described in Figure 6. (A) Caspase activity was analyzed by Western blotting with an antibody against PARP protein, a caspase substrate. The arrows indicate the position of the full-length protein (116 kDa) and its cleaved fragment (89 kDa). (B) Leukemic cells were lysed and Lyn was immunoprecipitated. The immunocomplexes were then analyzed for both in vitro kinase activity and anti-Lyn immunostaining. The data, relative to sample no. 14, are representative of experiments performed with the same B-CLL samples analyzed in Figure 6B.