The present dissertation work is devoted to identification of key molecules of the cellular signaling cascades that are altered at a basal level at chronic lymphocytic leukemia (CLL).
In result of our work, several key links of the signaling cascades in CLL were identified, using methods of molecular biology (quantitative polymerase chain reaction (qPCR), qPCR on microarrays), immunology (immunofluorescence analysis), bioinformatics and statistics. We found that several genes, that are induced by activation of the TGFB-SMAD2/3 cellular signaling pathway at the basal level, namely BCL2L1 (BCL-XL), CCND2 (Cyclin D2), ID1 and MYC, are also dependent on activation of the cellular signaling pathway IL2-JAK-STAT2/5. We found that expression of genes, transactivated upon activation of these two signaling pathways, is reduced significantly at mRNA levels in CLL cells, compared to B cells of peripheral blood of healthy donors. Using the obtained microarray data, together with the data from a bioinformatics analysis, we demonstrated that the cellular signaling pathways TGFB-SMAD2/3 and IL-2-STAT2/5 (JAK-STAT5) are not functional at the basal level in B lymphocytes of CLL patients.
We found that one of the inactivation mechanisms of the TGFB-SMAD signaling pathway in leukemic cells is low expression (below the possible level of its detection by immunofluorescence analysis) of the SMAD2 protein, as well as the absence of nuclear heterodimers of SMAD3 and SMAD4 proteins. Inhibition of the IL-2-STAT signaling pathway (JAK-STAT5) at the basal level in CLL cells may be due to low phosphorylation level or complete absence of phosphorylation of STAT5 proteins (STAT5A and STAT5B) in leukemic cells. Unlike in B-cells of healthy donors, expression of the STAT5A protein was low in the patient CLL cells. As we have previously shown, the IL-2-STAT5 (JAK-STAT5) signaling pathway is inhibited in CLL cells. Noteworthy, the STAT5 protein (isoforms A and B) showed basal levels of phosphorylation in the control samples, i.e. B cells of healthy donors. The phosphorylated protein was observed almost exclusively in the nucleus. Moreover, STAT5 formed large nuclear inclusions, but a proportion of protein was observed in the cytoplasm as well. Of note, the signals of phosphorylated STAT5 and STAT5A were partially co-localized in the nucleus, indicating activation of the IL-2-STAT5 (JAK-STAT5) pathway in B cells of healthy donors. In contrast to the pattern observed in B-cells of healthy individuals, in CLL cells a very low signal of the phosphorylated STAT5 proteins was observed. The expression levels of the STAT5A protein were quite low as well. Noteworthy, when the STAT5A signal was rather high, phosphorylation was not detected. Moreover, the phosphorylated form was localized almost exclusively in the cytoplasm. The STAT5A protein shows cytoplasmic localization, indicating the absence of complexes in the nucleus that activate/repress transcription of the STAT5-dependent genes. Thus, we assumed that the STAT5A protein nuclear complexes, which usually transactivate STAT5-responsive genes, are not formed in B-lymphocytes of CLL patients. Hence, inhibition of the IL-2-STAT5 pathway in CLL cells is caused by a lack of the STAT5 proteins phosphorylation and/or the absence of the active STAT5A transcription complexes in the nucleus of CLL cells.
Thus, we have shown, for the first time, that two cellular signaling pathways - TGFB-SMAD2/3 and IL-2-STAT2/5 - are not active at the basal level in CLL cells. We proposed a scheme, explaining inhibition of these signaling cascades.
