Analysis of Primary Chronic Lymphocytic Leukemia Cells' Signaling Pathways

Abstract

Chronic lymphocytic leukemia (CLL) is a lymphoproliferative disorder characterized by a specific expansion of mature B-cell clones. We hypothesized that the disease has a heterogeneous clinical outcome that depends on the genes and signaling pathways active in the malignant clone of the individual patient. It was found that several signaling pathways are active in CLL, namely, NOTCH1, the Ikaros family genes, BCL2, and NF-κB, all of which contribute to cell survival and the proliferation of the leukemic clone. Therefore, we analyzed primary CLL cells for the gene and protein expression of NOTCH1, DELTEX1, HES1, and AIOLOS in both peripheral blood lymphocytes (PBLs) and the bone marrow (BM) of patients, as well as the expression of BCL2 and miRNAs to see if they correlate with any of these genes. BCL2 and AIOLOS were highly expressed in all CLL samples as previously described, but we show here for the first time that AIOLOS expression was higher in the PBLs than in the BM. On the other hand, NOTCH1 activation was higher in the BM. In addition, miR-15a, miR-181, and miR-146 were decreased and miR-155 had increased expression in most samples. The activation of the NOTCH pathway in vitro increases the susceptibility of primary CLL cells to apoptosis despite high BCL2 expression.

Keywords: AIOLOS; BCL-2; CLL; NOTCH; leukemia; survival.

Figure 1

Multiparameter flow cytometry analysis of AIOLOS, HES1, and cleaved NOCTH1 in the malignant CLL clone and in the CD19⁺ peripheral blood lymphocytes. Dead cells were excluded from the analysis. A total of 18 CLL samples and control cells were incubated with the corresponding antibodies and analyzed using multiparametric flow cytometry. Light lines: control samples; dark lines: CLL clones.

Figure 2

qPCR analysis of CLL samples for the mRNA expression of NOTCH1, DELTEX1, HES1, and AIOLOS. cDNA was prepared from RNA isolated from blood samples of CLL patients and compared with normal CD19⁺ peripheral B cells. Relative expression levels were normalized with GUS gene expression. Data are expressed as 2^−ΔCT. Mean values and SEM of replicates are shown. The values of the control samples are shown as a solid line.

Figure 3

Correlation plot of data from gene expression (top) and protein analysis (bottom) of CLL samples for NOTCH1, HES1, DELTEX, and AIOLOS. Correlation coefficients and p-values are indicated.

Figure 4

(A) Analysis of BCL2 expression in a representative CLL blood sample (top) and in a peripheral blood sample from healthy control (bottom) using flow cytometry. The cell subpopulations according to the expression of CD5 and CD19 were analyzed for BCL2 expression. (B) Mean fluorescence intensity for each population calculated by normalizing the MFI of a gene with a corresponding isotype control. The bars represent the mean and SEM of 10 patients’ samples. The p-value is indicated in the figure. BCL2 expression (black line) and the according isotype control (gray line).

Figure 5

miRNA expression detected with TaqMan Advanced miRNA Assays. Relative gene expression for miR-7, miR-34a, miR-15, miR-155, miR-181, miR29a, and miR-146 for seven independent B-CLL samples (patients no 1, 3, 4, 5, 10, 13, and 16) is shown as individual values with mean ± SD indicated. The values of the control samples are shown as a solid line at value 1.

Figure 6

Gene and protein expression profile of primary leukemia samples in peripheral blood and bone marrow. (A) Ten B-CLL cells, collected from peripheral blood (PBL) and bone marrow (BM), were analyzed for NOTCH1, DELTEX1, HES1, and AIOLOS gene expression using qRT-PCR. Ct values were normalized to the expression of the GUSB housekeeping gene, and expressed as relative gene expression. (B) The protein expression of NOTCH1, HES1, and AIOLOS was analyzed using flow cytometry after the labelling of cells with the corresponding antibodies.

Figure 7

(A–C) Representative results of the flow cytometric analysis of cell death of a CLL sample cultured on an OP9-DL cell layer (A), an OP9-GFP cell layer (B), or in medium alone (C). To exclude the detached OP9 cells from the analysis, the samples were gated for CD45-positive cells. (D) Comparison of the percentage of dead (apoptotic and necrotic) cells recovered from the cultures described in A and B. The bars represent the mean ± SEM of four experiments. (E) Comparison of the absolute number of cells recovered from cultures described in (A,B). The bars represent the mean ± SEM of four experiments.