Dubrovska A. Investigation of molecular ang genetic mechanisms of tumor progression during Ph'-positive leukemias and diagnostics of them

Formation and expression of the fusion bcr/abl oncogene is the molecular hallmark of chronic myelogenous leukemia (CML) and Ph'-positive acute lymphoblastic leukemia (ALL). It is known two types of Bcr-Abl fusion protein: p190 Bcr-Abl which is mostly found in ALL and posses of greater transforming potential and p210 Bcr-Abl mostly found with CML. Dbl-homology (DH) domain of Bcr presented in p210 Bcr-Abl but not in p190 Bcr-Abl. This domain encodes a guanine nucleotide exchange factor activity specifically for Rho GTPase and modulates of the cell actin structure. The modulator effect of Dbl domain on actin structure may underlay the different transforming properties of two types of Bcr-Abl fusion proteins. Comparative analysis of actin distribution in polymorphic nuclear leukocytes of the different donors made possible to distinguish the following types of the cell staining: 1) diffuse distribution ( normal donors ); 2) paramembrane actin distribution in the cells of the patients C. (CML, blast crisis), F., Y. (ALL), cell line K562 and U937; 3) formation of the amorphous cytoplasm accumulation, "dot-like structures" in one case of the patient K. (CML, blast crisis)..We suppose that the revealing of dot-like distribution correlates with mutation in dbl-homology part of bcr/abl gene. Indeedly, the sequencing of the bcr/abl gene from patient K. confirmed the presence of mutation changes in dbl - homology region in positions 2127 cDNA of bcr/abl gene (replacement T - C, ) and 2449 (replacement C - A), that correspond to replacements 547 Phe-Leu and 654 Thr-Lys in protein molecule. Thus, the violation of the Bcr/Abl Dbl domain GEF function may be cause the magnification of the Bcr/Abl transforming potential and lead to progression of CML. The obtained data may cast light on the nature of CML blast crisis development and can be used for early detection of CML tumor progression as well as for elaboration more effective treatment protocols. Normal partner Bcr/Abl protein - Bcr may also be functionally concerned with modulation of the cytoskeleton. Bcr protein also is inhibitor of Bcr/Abl proteinkinase properties. It is interestingly whether expression level of the normal Bcr protein changes during the tumor progression. For providing an answer on this question we have obtained of antibodies against the Bcr part of the Bcr/Abl protein for theirs use for analysis of the Bcr and fused protein Bcr/Abl expression in patients with CML and ALL on the base of Western-blot test system. It is interestingly, in cases of patients B. and P. we observed the negative correlation between the levels of Bcr/Abl and Bcr proteins. Bcr/Abl is not detected in patient B. with CML, but present of Ph'-chromosome was proved by PCR method, that is evidence of the nessesary the complex approach to diagnostic of the Bcr/Abl-positive leukemias. Obtaining of the polyclonal anti-Bcr antibodies gives a possibility to analyze of the Bcr/Abl level and its normal cell partner Bcr in patients cell during the therapeutic course, evaluate of the therapy effectiveness, forecast of the next manner of the diseases.