Abstract
The abnormal activation of tyrosine kinases are a common finding in chronic myeloproliferative disorders. Perturbation of RTK signalling by genetic alterations results into an abnormal proliferation advantage and finally into a malignant transformation. c-Ros is an orphan RTK displaying transforming activity whose role has been established in the development of neuronal neoplasia. The aim of this study was to evaluate the involvement of c-Ros in the pathogenesis of chronic myelomonocytic leukemia (CMML) and to establish the effects of c-Ros activation. c-Ros expression was evaluated by RQ-PCR in 133 samples collected from 96 CMML patients at diagnosis (96 BM and 37 PB) and 60 healthy donors (30 PB and 30 BM). The protein amount and localization was analyzed by westen blot and immunofluorescence assay. In order to establish the effects of c-Ros activation we generated a chimeric receptor containing the extracellular domain derived from epidermal growth factor receptor (EGFR) and the transmembrane and cytoplasmic domains from c-ros (ER). The chimeric receptor was then transfected in NIH3T3 and HEK293T cells. Transfected and control cells were then stimulated with 100 nM EGF ligand and proliferation and apoptosis evaluated by incorporation of 3H timidine and MTT assay and by FACS for the detection of annexin V, respectively. We found that Ros is undetectable in healthy subjects but it is overexpressed in CMML (p<0,0001) in both BM and PB cells with a median value of 2−ΔΔCt in BM of 380 (range 10–63303) and 212 in PB (range 6–30012). WB confirmed the presence of c-Ros protein in CMML cells but not in normal controls. Immunofluorescence staining localized the protein within the cytoplasm. We found that ROS is highly expressed in CD34+ cells and monocytes from CMML patients but not in their normal counterparts. Sequence analysis revealed the absence of mutations of c-Ros promoter. SNPs analysis exclude the presence of duplications or deletions of the gene. Moreover we found that the EGF induced activation of c-Ros affects proliferation by increasing of 3.5 folds the proliferation rate as compared to cells transfected with the empty vector and stimulated with EGF under the same conditions. Furthermore cell adhesion was 4 folds decreased as compared to control. By contrast apoptosis is not affected by c-ROS activity (p=0,2). This study demonstrates that c-Ros is abnormally expressed in patients with CMML. The abnormal activation of c-Ros is responsible for loss of adhesion and increased proliferation. In conclusion, we identified a new tyrosine kinase which may be responsible for the proliferation defect typical of CMML cells and could represent a target for molecular therapies.
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