Telegeev G. Myeloproliferative diseases: molecular pathogenesis and diagnostics

The work is devoted to the study of the molecular pathogenesis of the chronic myelogenous leukemia (CML) so as to the development of diagnostics and monitoring methods for chronic myeloplasms. Nucleotide sequences of the DH domain of the hybrid bcr-abl gene of patients with CML and ALL is analysed at the different stage of the illness using the reverse transcription polymerase chain reaction (RT-PCR). The loss of various parts of different length of the DH domain of the p210 bcr-abl mRNA was found to occur for some patients suffering from ALL and CML so as point mutations at the acceleration illness stage. The new approach to the detection and differential diagnostics of the Ph - positive leukemias is proposed to reveal the classic translocation as well as the uncanonical translocation in the bcr region of the hybrid bcr-abl gene. In addition to this test systems for detection and monitoring of bcr-abl reorganisation, mutation V617F at the gene jak 2 was developped GEF activity of Bcr DH domain towards RhoA, Rac1 and Cdc42 hasn't been detected in both in vitro and in vivo experiments. A. Experimental data suggest that PH domain binds to phosphatidylinositol monophosphates PI(3)P, PI(4)P and PI(5)P with high affinity. There is uneven lipid content in intracellular cell membranes. Therefore, PH domain binding to phosphainositols might effect p210 Bcr-Abl protein localization. This was confirmed by comparison of p190 and p210 Bcr-Abl cell localization in immunofluorescent microscopy assay. Protein-protein interactions of PH domain were studied by pull-down assay. K562 cells were grown in [35S]-methionine medium therefore all K562 proteins were radioactively labeled and could be distinguished from bacterial protein contamination after Ni-NTA purification. After the comparison of the two gels spots corresponding to K562 proteins were excised and trypsinized. Peptide spectra were collected by MALDI TOF mass-spectrometry and corresponding proteins were identified by ProFound web resource. We identified 23 proteins that potentially bind to Bcr PH domain. Each of the detected proteins should be than verified by protein-protein binding assay in vitro or in vivo. For the further experiments we've chosen SMC1 (structure maintenance of chromosomes), tubulin, zizimin1 and PLC proteins and confirmed binding of them to PH domain by pull-down and the immunoprecipitation assays. Taken together, the study reveals important functions of Bcr DH and PH domains that could affect Bcr-Abl signal pathways. Keywords: chronic myelogenous leukemia, CML, acute lymphoblastic leukemia, ALL, bcr-abl gene, DH domain, jak 2 , PH domain, diagnostics