Development of molecular oncohematology in Ukraine

Disruption of the genetic component of cells are mandatory element of malignant transformation. For the majority of blood neoplasias genetic disorders have been discovered, and they can be used for diagnosis and appropriate therapy. The data obtained by authors about the role of domains of Bcr-Abl p...

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Дата:2013
Автори: Telegeev, G.D., Dybkov, M.V., Dubrovska, A.N., Miroshnichenko, D.A., Tyutyunnykova, A.P., Maliuta, S.S.
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Опубліковано: Інститут молекулярної біології і генетики НАН України 2013
Назва видання:Вiopolymers and Cell
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Цитувати:Development of molecular oncohematology in Ukraine / G.D. Telegeev, M.V. Dybkov, A.N. Dubrovska, D.A. Miroshnichenko, A.P. Tyutyunnykova, S.S. Maliuta // Вiopolymers and Cell. — 2013. — Т. 29, №. 4. — С. 277-282. — Бібліогр.: 31 назв. — англ.

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spelling irk-123456789-1526032019-06-13T01:25:25Z Development of molecular oncohematology in Ukraine Telegeev, G.D. Dybkov, M.V. Dubrovska, A.N. Miroshnichenko, D.A. Tyutyunnykova, A.P. Maliuta, S.S. Reviews Disruption of the genetic component of cells are mandatory element of malignant transformation. For the majority of blood neoplasias genetic disorders have been discovered, and they can be used for diagnosis and appropriate therapy. The data obtained by authors about the role of domains of Bcr-Abl protein (the main etiological factor in the pathogenesis of leukemia with Ph-chromosome) are presented in this review as well as approved diagnostic methods for myeloproliferative disorders and acute leukemias. Обов’язковим і характерним елементом злоякісної трансформації є порушення генетичного компонента клітини. Для більшості неоплазій системи крові генетичні порушення є відомими, що дозволяє використовувати їх для діагностики і відповідної терапії. Наведено авторські дані стосовно ролі доменів білка Bcr-Abl (головного етіологічного фактора в патогенезі лейкемій з філадельфійською хромосомою) та представлено апробовані методи діагностики мієлопроліферативних захворювань і гострих лейкемій. Обязательным и характерным элементом злокачественной трансформации являются нарушения генетического компонента клетки. Для большинства неоплазий системы крови известны генетические нарушения, что позволяет использовать их для диагностики и проведения соответствующей терапии. Приведены авторские данные о роли доменов белка Bcr-Abl (главного этиологического фактора в патогенезе лейкемий с филадельфийской хромосомой) и представлены апробированные методы диагностики миелопролиферативных заболеваний и острых лейкемий. 2013 Article Development of molecular oncohematology in Ukraine / G.D. Telegeev, M.V. Dybkov, A.N. Dubrovska, D.A. Miroshnichenko, A.P. Tyutyunnykova, S.S. Maliuta // Вiopolymers and Cell. — 2013. — Т. 29, №. 4. — С. 277-282. — Бібліогр.: 31 назв. — англ. 0233-7657 DOI: http://dx.doi.org/10.7124/bc.000822 http://dspace.nbuv.gov.ua/handle/123456789/152603 577.2.575 en Вiopolymers and Cell Інститут молекулярної біології і генетики НАН України
institution Digital Library of Periodicals of National Academy of Sciences of Ukraine
collection DSpace DC
language English
topic Reviews
Reviews
spellingShingle Reviews
Reviews
Telegeev, G.D.
Dybkov, M.V.
Dubrovska, A.N.
Miroshnichenko, D.A.
Tyutyunnykova, A.P.
Maliuta, S.S.
Development of molecular oncohematology in Ukraine
Вiopolymers and Cell
description Disruption of the genetic component of cells are mandatory element of malignant transformation. For the majority of blood neoplasias genetic disorders have been discovered, and they can be used for diagnosis and appropriate therapy. The data obtained by authors about the role of domains of Bcr-Abl protein (the main etiological factor in the pathogenesis of leukemia with Ph-chromosome) are presented in this review as well as approved diagnostic methods for myeloproliferative disorders and acute leukemias.
format Article
author Telegeev, G.D.
Dybkov, M.V.
Dubrovska, A.N.
Miroshnichenko, D.A.
Tyutyunnykova, A.P.
Maliuta, S.S.
author_facet Telegeev, G.D.
Dybkov, M.V.
Dubrovska, A.N.
Miroshnichenko, D.A.
Tyutyunnykova, A.P.
Maliuta, S.S.
author_sort Telegeev, G.D.
title Development of molecular oncohematology in Ukraine
title_short Development of molecular oncohematology in Ukraine
title_full Development of molecular oncohematology in Ukraine
title_fullStr Development of molecular oncohematology in Ukraine
title_full_unstemmed Development of molecular oncohematology in Ukraine
title_sort development of molecular oncohematology in ukraine
publisher Інститут молекулярної біології і генетики НАН України
publishDate 2013
topic_facet Reviews
url http://dspace.nbuv.gov.ua/handle/123456789/152603
citation_txt Development of molecular oncohematology in Ukraine / G.D. Telegeev, M.V. Dybkov, A.N. Dubrovska, D.A. Miroshnichenko, A.P. Tyutyunnykova, S.S. Maliuta // Вiopolymers and Cell. — 2013. — Т. 29, №. 4. — С. 277-282. — Бібліогр.: 31 назв. — англ.
series Вiopolymers and Cell
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fulltext UDC 577.2.575 Development of molecular oncohematology in Ukraine G. D. Telegeev, M. V. Dybkov, A. N. Dubrovska, D. A. Miroshnichenko, A. P. Tyutyunnykova, S. S. Maliuta Institute of Molecular Biology and Genetics, NAS of Ukraine 150, Akademika Zabolotnogo Str., Ukraine, 03680 g.d.telegeev@imbg.org.ua Disruption of the genetic component of cells are mandatory element of malignant transformation. For the majo- rity of blood neoplasias genetic disorders have been discovered, and they can be used for diagnosis and appro- priate therapy. The data obtained by authors about the role of domains of Bcr-Abl protein (the main etiological factor in the pathogenesis of leukemia with Ph-chromosome) are presented in this review as well as approved diag- nostic methods for myeloproliferative disorders and acute leukemias. Keywords: myeloproliferative neoplasms, Bcr-Abl, Jak2, molecular pathogenesis, diagnostics. Introduction. Malignant tumors are the second (after cardiovascular diseases) cause of deaths in developed countries. According to the Bulletin of National Cancer Registry of Ukraine [1], blood system neoplasms were 4.87 % of total cancer cases in 2011. Proportion of deaths from leukemias is 5.38 % of all cancer deaths cases. Each year about 7,500–8,000 cases of different blood neoplasms are recorded. Myeloproliferative diseases (neoplasms) (MPN) are a biologically heterogenous group of hematopoietic system diseases, the main featu- re of which is uncontrolled proliferation of stem hema- topoietic cells or myelopoiesis progenitor cells [2, 3]. The MPN part in total blood neoplasms is about 30–35 %. According to the WHO classification, there are cronic myeloid leukemia (CML), chronic neutrofilic leukemia, chronic eosinophilic leukemia, polycythemia vera, es- sential polycythemia, primary myelofibrosis, and un- classified myeloproliferative neoplasms [4, 5]. Sharing common morpho-clinical and hematologic features, these diseases, however, have different mole- cular basis. One of the most represented diseases in this group is CML. CML has a distinctive marker – Phila- delphia (Ph') chromosome, which is a result of recipro- cal translocation t(9;22) (q34; q11). This chromosome is present in CML and in some patients with acute lym- phoblastic leukemia (ALL). A fusion of two genes occurs at the molecular level: abl on chromosome 9 and bcr on chromosome 22. The breaks in abl occur mostly bet- ween 1st and 2nd exones and rarely between 2nd and 3rd. In contrast, bcr gene has three areas where the breaks happen frequently. Therefore, depending on the break- point in bcr gene there are three different forms of Bcr- Abl protein: p190, p210, p230. Each form was found in the certain type of leukemia: p210 Bcr-Abl – in 90–95 % of CML cases, p190 Bcr-Abl – mainly in ALL in adults (35 % of all ALL cases), while p230 is found in rela- tively benign neutrophilic form of myeloid leukemia. The impetus for the studying of molecular and ge- netic aspects of these diseases, which has been started at our institute in the 1990s, was a pure practical prob- lem – the development of a system for detecting chime- ric fusion of bcr-abl gene in the patients who were diag- nosed and treated in the hematology departments of Ky- iv hospitals. The cytogenetic methods used to confirm the presence of the Philadelphia chromosome required the bone marrow puncture, a long time for analysis, high- ly qualified staff, etc. Using the reverse transcriptase po- lymerase chain reaction with specific primers allowed 277 ISSN 0233–7657. Biopolymers and Cell. 2013. Vol. 29. N 4. P. 277–282 doi: 10.7124/bc.000822 � Institute of Molecular Biology and Genetics, NAS of Ukraine, 2013 us to obtain the results within one day after taking pati- ents’ peripheral blood samples [6–8]. For the first time in Ukraine the guidelines «The monitoring of chronic myeloid leukemia with molecular methods» [9] have been created and approved by the Ministry of Health. This allowed us to introduce molecular genetic appro- aches to clinical practice. However, as it often happens, the practice highligh- ted a wide range of unsolved scientific problems. Of particular interest was the fact that different forms of Bcr-Abl protein (especially p190 and p210) are speci- fic for leukemias, which differ by the nature of clinical manifestations. The difference between these proteins is that protein Bcr incorporates DH (Dbl-homologous) and PH (pleckstrin-homologous) domains in the area distinguishing the forms of chimeric proteins p190 and p210 Bcr-Abl. The experimental data could not descri- be the functional activity of these domains as a part of Bcr, and in p210 Bcr-Abl. PH domain has not been stu- died at all. It was therefore decided to investigate the areas that distinguish these forms of Bcr-Abl protein in patients with Ph'-positive leukemia at different stages of the disease. The transformation of cells expressing fu- sion protein Bcr-Abl, is associated with impaired regu- lation of tyrosine kinase Abl, namely its elevated levels in tumor cells, but there are the unidentified factors that lead to an exacerbation of the chronic form of the dise- ase, its transition to the first stage of acceleration, and then to blast crisis. It has been shown that the Abl over- expression in cells does not lead to the tumor develop- ment, but rather results in inhibition of cell growth and apoptosis [10]. Therefore it was assumed that these specific featu- res of Ph'-positive leukemias are caused by Bcr protein domains. Analysis of dbl area of cDNA obtained from patients with CML, ALL. Based on the assumption that in some cases in the region of protein p210 Bcr-Abl, which is ab- sent in protein p190 Bcr-Abl, might occur the changes that lead to the functional similarity of these chimeric proteins, we have analyzed the structure of the site in patients with Ph'-positive leukemia at different stages of disease. As a result, the deletions of varying length were detected in the region, corresponding to Dbl do- main of Bcr protein, in several patients with acute occur- rence of the disease [11, 12]. In all cases, the deletions did not lead to disruption of reading frame. Similar changes were observed in the control group of healthy donors. Thus, we have shown that in some cases, in patients with CML and ALL with p210 Bcr-Abl at the acute stage of the disease there is a loss of sections of different length in DH region. So far, a number of sub- microscopic deletions of varying length on chromoso- me 9 th (abl gene), and on chromosome 22 (bcr gene) ha- ve been shown. In most cases, these changes reflected the progression of the disease and were an unfavorable prognostic factor. The changes in the area of chimeric bcr gene that differ from the «classical» have been described by seve- ral authors – e6a2, e8a2, e13a2, e15a2 [13–16]. In most cases these changes were detected during disease acce- leration. These transcripts have arisen as a result of non- canonical translocations that occurred in the areas other than the areas of M-, m-and�-Bcr of chimeric gene bcr- abl. The changes described by us were different. The main difference was that these changes were not a re- sult of an abnormal translocation type. The classic type of translocation was confirmed by RT-PCR at the stage of selecting samples for analysis. Disease progression could not be a result of the ef- fect of only one factor. However, it is likely that the lack of a domain or molecular changes in the region of exons 3–14 of chimeric gene bcr-abl (DH and PH do- mains) changes their functional activity, which leads to the formation of proteins acting differently. DH domains encode guanine exchange factors (GEFs) and work as a link between the surface cell receptors to cytokines, growth factors, G-protein receptors, adhesion, on one hand, and activation of Rho-like GTPases, on the other. Proteins p210 Bcr-Abl and p190 Bcr-Abl interact dif- ferently with GTPases RhoA, Rac1 and Cdc42 [17], which results in the implementation of their capacities, including influence on actin cytoskeleton, the NADPH- oxidase during ROS formation, cell signaling, etc. Study on actin cytoskeleton in leukocytes of patients with Ph'-positive leukemia. The actin-binding domains, present in the chimeric Bcr-Abl protein, interact with actin cytoskeleton and cellular localization [18]. To check the possible influence of these domains we have studied blood cells of patients with CML, leukemic cell cultures and leukocytes obtained from healthy donors. Comparative analysis of the actin cytoskeleton structu- res in polymorphonuclear leukocytes using FITC-la- beled falloidin allowed us to distinguish three types of 278 TELEGEEV G. D. ET AL. cell staining – cortical, diffuse and dot-like structures formation [19]. It has been shown that K562, U937 cells as well as cells of patients with Ph'-positive leuke- mia, have mostly cortical cytoskeleton distribution. In cells from healthy donors cortical cytoskeleton was al- most non-expressed. In some cases, in addition to cortical F-actin large dot-like structures were detected in the cells obtained from patients with CML (blast crisis stage). According to the literature data [18], changes in the organization of actin cytoskeleton may be associated with mutations in the DH region of bcr-abl gene and its dysfunction that affects Rho GTPases. It is well-known that DH domains are GEFs for Rho GTPases, causing actin cytoskeleton organization: the formation of phyllo- poda, lamellopoda, stress fibers etc. To test this Dbl re- gion of chimeric Bcr-Abl, the protein of patients with large punctates was used. The analysis revealed the pre- sence of point mutations in this region [20]: transver- sions at position 2127 (T/C) and at position 2449 (C/A). They lead to the substitution of phenylalanine for leu- cine at position 547 and threonine for lysine at position 654. The multiple alignment analysis of DH domains from eukaryotic proteins showed that the replacement of Rhe547/Leu is neutral and does not affect the cataly- tic DH domain function because it does not change the class of amino acid residues and belongs to �-helix H2A region. The latter is located on the inner side of �- helical DH domain group that is not involved in the in- teractions with GDP, GTPases and neighboring domains. Instead, Thr654 mutation leads to substitution of a neut- ral amino acid residue for basic residue and belongs to the most conservative area of DH domain – CR3. Muta- tion Thr654/Lys was found within the CR3 sequence that forms a hydrophobic pocket for GTPase binding. This fact suggests that the appearance of charged ami- no acids in this region may have a decisive influence on the biological activity of Dbl-homologous domain. Studying the GEF-activity of Bcr DH domain in vitro and in vivo. Currently more than 70 known proteins have DH domain in their structure. Most of them are GEF-fac- tors for different GTPases [21]. Therefore we have tested the GEF activity in vitro by radioactive GDP release and the incorporation of �-S35-GT recombinant proteins which contained DH and DHPH domains, and GTPa- ses RhoA, Rac1, Cdc42. The experiments with trans- formation of 293T cells with DH and DHPH recombinant constructs and GTPases RhoA, RhoB, Rac1, Rac3, Cdc42, Wrch1 have been also conducted. In both systems, no GEF activity on GTPases has been found [22]. Multiple alignment of amino acid sequences of DH family members showed that DH domain of Bcr has conserved amino acids that are responsible for forming the tertiary structure. It has also three conservative areas (CR1, CR2 and CR3) which may be involved in interac- tions with GTPases. Thus, the analysis of the primary sequence suggests that the DH domain of Bcr has all the structural elements that are present in the family of these domains, and therefore has the potential to func- tion as a GEF-factor, perhaps, it might have specificity for other members of Rho family or Ras superfamily GTPases. In particular, these potential partners can be the following GTPases: Rap1, Rab5, Arf1. They have been se- lected because of their ability to interact with PLC� (pre- sence of RA domain), the ratio of PH domain to memb- rane phospholipids (Rab5) and Golgi apparatus (Arf1). Study on p190 and p210 Bcr-Abl proteins by fluores- cent microscopy. As it was mentioned above, the diffe- rence between p190 and p210 Bcr-Abl is that p190 lacks the DH and PH part. It is known that there is a di- vision within lipids by cellular compartments, so the presence of phosphatydylinositol-monophosphate-bin- ding PH domain and DH domain in protein can most li- kely affect the localization of proteins and cytoskeletal organization. Cos-1 cells were transfected with the constructs ex- pressing proteins p190 or p210 Bcr-Abl. The difference in their localization in cells has been shown in [23]. Both proteins were found in the cytoplasm, but p190 Bcr-Abl has an equable distribution in the cytoplasm of cell while protein p210 is characterized by mainly perinuclear dis- tribution. So, Bcr part which is present in p210 and ab- sent in p190 Bcr-Abl, has certain influence on the distri- bution of proteins. It has been supposed that PH domain takes part in Bcr-Abl localization. Cos-1 cells morpholo- gy did not change during transfection. Commonly, cells are round-shaped and have uniform distribution of actin filaments, cell membranes form neither lamellopoda nor phylopoda. The influence of DH and PH domains was also tes- ted in NIH3T3 cells which have been transfected with GFP vectors carrying these domains. The absence of ob- vious morphological changes was confirmed. This may mean that Rho family GTPases, involved in the reor- ganization of actin cytoskeleton, in this case were not 279 DEVELOPMENT OF MOLECULAR ONCOHEMATOLOGY IN UKRAINE activated, which would be expected if the DH domain of p210 Bcr-Abl protein had GEF-activity. Determination of lipids interacting with PH domain of Bcr. PH domain has been described for more than 200 proteins, and it is involved in cell signaling, cell–cell in- teractions and cytoskeleton organization. It is known that in most proteins PH domain binds to phosphatydylino- sitols, so we decided to check if it is true for Bcr-Abl. In- deed, it was determined that Bcr-Abl binds to PI(3)P, PI(4)P and PI(5)P phosphatydylinositol-monophospha- tes [23, 24]. It has been shown that this interaction is highly affine [24]. The specificity of binding only phos- phatydylinositol-monophosphate is not typical for PH family. Usually, PH domains bind with high affinity three phosphatydylinositol-three- and bisphosphates that are the products of PI3-kinase. The cellular lipids provide the distribution of signaling molecules and organization of compartment – specific signaling complexes [25]. That is, binding of Bcr or p210 Bcr-Abl to PI(3)P and PI(4)P can be a result of early endosomal or pha- gosomal or Golgi complex membrane localization of these proteins. The role of the binding of PH domain to PIP(5), which is localized predominantly in the nuc- leus, may be significant. Investigation and analysis of the ability of Bcr PH do- main to bind cellular proteins. It is known that PH do- mains of some proteins in addition to binding to lipids may be involved in protein-protein interactions [26]. To test this fact, the «pull-down» approach was used for the precipitation of proteins from K562 cells lysate and their subsequent analysis by proteomics methods. As a re- sult, 26 proteins that interact with this domain were iden- tified [23]. The next step was an analysis of interactions with proteins SMC1 (structure maintenance of chromo- somes), �-tubulin, zizimin 1 and phospholipase C� (PLC �). To analyze the binding of PH domain, the DHPH con- struct, containing both PH and DH domains, was used. As a control, the DH only construct was used. Using a pull-down analysis, immunoblotting, and co-localiza- tion assay in HEK293T cells, the binding of PH domain to all proteins listed above was confirmed [23, 24]. The proteins identified belong to several functional groups, most proteins are involved in metabolic processes, cell proliferation, adhesion, signal transduction. Using bioinformatic methods, the network of pro- teins and signaling pathways that interact with the PH domain has been constructed. It has three nodal centers: ERK, NF�B and p38MAPK. Interaction with cortactin and protein FBP17, which are involved in the forma- tion of early endosoms, may suggest that Bcr and Bcr- Abl participate in this process. This assertion was sup- ported by established interaction with Bcr PH domain of Bcr with PIP(3) – typical endosomal and phagoso- mal membrane lipids [23] and with ESCRT complex (proteins TSG-101 and Vps28) [27]. PLC� also partici- pates in this process [23]. PLC� catalyzes the cleavage of phosphatydylinositol-4,5-bisphosphate to diacylgly- cerol and inositol-1,4,5-triphosphate (Ins(1,4,5)P3), which is a secondary messenger in many signaling path- ways. PLC� has a bifunctional role: it is involved in lipid metabolism and also acts as GEF-factor for Ras GTPases. So, PLC� can regulate Ras (through its GEF- domain) and bind Ras-GTP, which potentially creates a complex mechanism of feedback regulation of a multi- component system. Localization of p190 and p210 Bcr-Abl in the cell relative to the Golgi apparatus. As it has been mentio- ned above we have shown that the PH domain has the ability to bind PI(4)P, which is the main component of lipid membranes of Golgi complex. It was therefore decided to investigate the co-localization of proteins p190 and p210 Bcr-Abl with Golgi apparatus. To do this, Cos-1 cells were transfected with the constructs ex- pressing proteins p190 and p210 Bcr-Abl. For visualiza- tion of the Golgi apparatus the antibodies to the matrix protein GM130 were used. It has been noticed that the protein p210 was co- localized with the Golgi apparatus, which typical shape was observed; in addition, p210 was also localized in cytoplasm. However, the highest concentration of pro- tein was observed in the Golgi complex. p190 Bcr-Abl did not have such obvious location around the nucleus, it was evenly distributed throughout the cytoplasm of cells [23]. The specificity of the interaction was tested by adding wortmannin which led to delocalization of p210 Bcr-Abl in the area where it has been detected before treatment. The results of co-transfection with costructs bearing shRNA specific to PI4K and PTEN al- so showed the effect of reduced binding of p210 to Gol- gi complex. Thus, it was shown that the presence of PH domain affects the subcellular localization of this protein, cau- ses the interaction with various proteins and organelles of the cell. First, it is a participation in the endosomes 280 TELEGEEV G. D. ET AL. formation. The proteins that interact with the PH-do- main of Bcr (cortactin, FBP 17, PLC�, �-tubulin) take part in this process. Thus, the interaction of p210 Bcr- Abl protein with TSG10, a component of the complex ESCRT1, might be explained [27]. Despite the lack of C2 domain of Bcr protein, which is responsible for Bcr binding to the protein TSG101, the interaction is still observed in the cells expressing p210 Bcr-Abl. This fact is well explained by the data established by binding PH domain to PI(3)P, a dominant early endosomal li- pid. The absence of this domain in protein p190 Bcr- Abl alters endosomal sorting and direction of growth factors receptors transport and differentiation. Differen- tiation of hematopoietic cells depends not only on the growth factors receptors, but also on their internaliza- tion and degradation in time. Thus, aggravation of en- dosomal transport and accumulation of differentiation factors, which cannot be degraded, on the cell surface may affect the differentiation of hematopoietic cells. The presence of PH domain determines the high affini- ty of binding Bcr to PI(3)P on early endosomes and pha- gosomes. Lack of GAP domain at COOH end of Bcr part leads to deregulation of Rac GTPase, resulting in the constitutive activity of Rac in hematopoietic cells, increased formation of reactive oxygen species and emerging of «oxidative burst» [28]. Improving the detection of Philadelphia chromosome in patients with CML and ALL and monitoring patients during treatment with Abl kinase inhibitors. One of the main problems in the treatment of CML is emerging of new mutations that make the drugs, including Abl kina- se inhibitors, unable to work. More than 50 mutations within the kinase domain of Bcr-Abl protein, most of which lead to lost or reduced efficiency of Imatinib, one of the main drugs for CML treatment, have been descri- bed. Therefore, detection of these mutations is impor- tant for selecting the treatment strategy. Since most of these mutations arise in region 200– 400AK of Abl, and mutations M244V, G250E, Y253F/H, E255K/V, T315I, M351T, F359V are more than 85 % of the mutational changes, the primers for nested PCR of this site were designed. The most common mutation is T315I, which leads to the replacement of threonine by isoleucine and causes a loss of effectiveness of the imatinib [29]. Thus, in this study, it has been shown for the first ti- me that Bcr part of the Bcr-Abl fusion protein plays a significant role in the mechanisms of tumor develop- ment during CML progression. It determines the speci- ficity of different oncoproteins (p190, p210, p230), due to their different localization in the cell and participa- tion in different signaling cascades. This fact, in addi- tion to the traditional use of Abl-kinase inhibitors, in- creases the possibility of targeted therapy of Ph'-positi- ve leukemia. Given the high genetic instability in pati- ents with myeloproliferative disorders, the test systems for a) detection and differential diagnosis of Ph'-positi- ve leukemias by RT-PCR, and b) monitoring changes in ATP-binding region of Abl protein were developed, al- lowing clinicians to adjust therapy when mutations in this region occure during tyrosine kinase inhibitor treatment. Under the current classification of the World Health Organization (4.5) molecular diagnostics is an obligatory component of modern diagnostics of leuke- mias. In collaboration with the colleagues from the Im- munocytochemistry department of Kavetsky Institute of Experimental Pathology, Oncology and Radiobiolo- gy of NAS of Ukraine) we have developed a protocol for detection of the most characteristic MPN mutations, namely Jak2, mpl, tet2, asxl1 [30], and mutations in acute leukemia mll/af4 (t (4, 11) (q21; q23); mll/af9 (t (9; 11) (p22; q23); cbfb/myh11 (inv16) [31]. The effec- tiveness of this protocol was tested on a large number of patients. This greatly improved diagnostic and therapeu- tic protocols for leukemias of various origins in Ukraine. Ã. Ä. Òåëåãåºâ, Ì. Â. Äèáêîâ, À. Ì. Äóáðîâñüêà, Ä. Î. ̳ðîøíè÷åíêî, À. Ï. Òþòþííèêîâà, Ñ. Ñ. Ìàëþòà Ðîçâèòîê ìîëåêóëÿðíî¿ îíêîãåìàòîëî㳿 â Óêðà¿í³ Ðåçþìå Îáîâ’ÿçêîâèì ³ õàðàêòåðíèì åëåìåíòîì çëîÿê³ñíî¿ òðàíñôîðìà- ö³¿ º ïîðóøåííÿ ãåíåòè÷íîãî êîìïîíåíòà êë³òèíè. Äëÿ á³ëüøîñò³ íåîïëàç³é ñèñòåìè êðîâ³ ãåíåòè÷í³ ïîðóøåííÿ º â³äîìèìè, ùî äîç- âîëÿº âèêîðèñòîâóâàòè ¿õ äëÿ ä³àãíîñòèêè ³ â³äïîâ³äíî¿ òåðàﳿ. Íàâåäåíî àâòîðñüê³ äàí³ ñòîñîâíî ðîë³ äîìåí³â á³ëêà Bcr-Abl (ãî- ëîâíîãî åò³îëîã³÷íîãî ôàêòîðà â ïàòîãåíåç³ ëåéêåì³é ç ô³ëàäåëü- ô³éñüêîþ õðîìîñîìîþ) òà ïðåäñòàâëåíî àïðîáîâàí³ ìåòîäè ä³àã- íîñòèêè 쳺ëîïðîë³ôåðàòèâíèõ çàõâîðþâàíü ³ ãîñòðèõ ëåéêåì³é. Êëþ÷îâ³ ñëîâà: 쳺ëîïðîë³ôåðàòèâí³ íåîïëàçìè, Bcr-Abl, Jak2, ìîëåêóëÿðíèé ïàòîãåíåç, ä³àãíîñòèêà. Ã. Ä. Òåëåãååâ, Ì. Â. Äûáêîâ, À. Í. Äóáðîâñêàÿ, Ä. À. Ìèðîøíè÷åíêî, À. Ï. Òþòþííèêîâà, Ñ. Ñ. Ìàëþòà Ðàçâèòèå ìîëåêóëÿðíîé îíêîãåìàòîëîãèè â Óêðàèíå Ðåçþìå Îáÿçàòåëüíûì è õàðàêòåðíûì ýëåìåíòîì çëîêà÷åñòâåííîé òðàíñôîðìàöèè ÿâëÿþòñÿ íàðóøåíèÿ ãåíåòè÷åñêîãî êîìïîíåí- 281 DEVELOPMENT OF MOLECULAR ONCOHEMATOLOGY IN UKRAINE òà êëåòêè. Äëÿ áîëüøèíñòâà íåîïëàçèé ñèñòåìû êðîâè èçâåñòíû ãåíåòè÷åñêèå íàðóøåíèÿ, ÷òî ïîçâîëÿåò èñïîëüçîâàòü èõ äëÿ äè- àãíîñòèêè è ïðîâåäåíèÿ ñîîòâåòñòâóþùåé òåðàïèè. Ïðèâåäåíû àâòîðñêèå äàííûå î ðîëè äîìåíîâ áåëêà Bcr-Abl (ãëàâíîãî ýòèî- ëîãè÷åñêîãî ôàêòîðà â ïàòîãåíåçå ëåéêåìèé ñ ôèëàäåëüôèéñêîé õðîìîñîìîé) è ïðåäñòàâëåíû àïðîáèðîâàííûå ìåòîäû äèàãíî- ñòèêè ìèåëîïðîëèôåðàòèâíûõ çàáîëåâàíèé è îñòðûõ ëåéêåìèé. Êëþ÷åâûå ñëîâà: ìèåëîïðîëèôåðàòèâíûå íåîïëàçìû, Bcr-Abl, Jak2, ìîëåêóëÿðíûé ïàòîãåíåç, äèàãíîñòèêà. REFERENCES 1. Fedorenko Z. P., Goulak L. O., Gorokh Y. L., Ryzhov A.Yu., Soumki- na O. V., Koutsenko L. B. Cancer in Ukraine, 2011–2012. Inciden- ce, mortality, activities of oncological service // Bull. Nat. Can- cer Registry of Ukraine / Ed. I. B. Shchepotin.– Kyiv, 2013.–73 p. 2. Tefferi A., Vainchenker W. Myeloproliferative neoplasms: mole- cular pathophysiology, essential clinical understanding and treat- ment stra- tegies // J. Ñlin. Încol.–2011.–29, N 5.–P. 573–582. 3. Glusman D. F., Sklyarenko L. M., Nadgornaya V. A. Diagnostic oncohematology.–Kyiv: DIA, 2011.–254 p. 4. Tefferi A., Vardiman J. W. Classification and diagnosis of myelo- proliferative neoplasms. The 2008 World Health Organization criteria and point-of-care diagnostic algorithms // Leukemia.– 2008.–22, N 1.–P. 14–22. 5. Swerdlow S. H., Campo E., Harris et al. WHO Classification of Tu- mours of Haematopoietic and Lymphoid Tissues / Fourth Edition.– Lyon: IARC Press, 2008.–439 p. 6. Telegeev G. D., Dybkov M. V., Karpenko O. I., Cherepenko H. I. Molecular basis of Ph'- leukemia and finding the way to treat them // Biopolym. Cell.–1994.–10, N 5.–P. 78–92. 7. Telegeev G. D., Dybkov M. V., Bozhko M. V., Tretiak N. M., Ma- liuta S. S. Molecular-biology approaches to detection of Phila- delphia chromosome in patients with leukemia // Biopolym. Cell.–1996.–12, N. 6.–P. 63–68. 8. Telegeev G. D., Dybkov M. V., Bojko M. V., Òretyak N. M., Ma- liuta S. S. Molecular biological diagnostics of blood neoplastic diseases // Tsitol Genet.–1998.–32, N 1.–P. 71–78. 9. Telegeev G. D., Dybkov M. V., Bojko M. V., Demidenko D. V., Maliuta S. S., Òretyak N. M., Bondar M. V. Monitoring of chro- nic myeloid leukemia by molecular-biological methods [guide- lines].– Kyiv: The National Centre for Scientific Health Infor- mation Health of Ukraine publ., 1997.–16 p. 10. Sawyers C. L., McLaughlin J., Goga A., Havlik M., Witte O. The nuclear tyrosine kinase c-Abl negatively regulates cell growth // Cell.–1994.–77, N 1.–P. 121–131. 11. Telegeev G. D., Dybkov M. V., Dubrovska A. N., Maliuta S. S. De- letion of the fifth exon of bcr/abl gene by acute lymphoblastic leukosis with Ph' chromosome // Biopolym. Cell.–2001.–17, N 4.–P. 298–301. 12. Dybkov M. V., Telegeev G. D., Dubrovskaya A. N., Maliuta S. S. Deletion in dbl domain of bcr/abl gene in leukemia patients with Ph' chromosome // Exp. Oncol.–2002.–24, N 2.–P. 153–154. 13. Popovici C., Cailleres S., David M., Lafage-Pochitaloff M., Sain- ty D., Mozziconacci M. J. E6a2 BCR-ABL fusion with BCR exon 5-deleted transcript in a Philadelphia positive CML res- ponsive to Imatinib // Leuk. Lymphoma.–2005.–46, N 9.–P. 1375–1377. 14. Colla S., Sammarelli G., Voltolini S., Crugnola M., Sebastio P., Giuliani N. E6a2 BCR-ABL transcript in chronic myeloid leuke- mia: is it associated with aggressive disease? // Haematologica.– 2004.–89, N 5.–P. 611–613. 15. How G. F., Lim L. C., Kulkarni S., Tan L. T., Tan P., Cross N. C. Two patients with novel BCR/ABL fusion transcripts (e8/a2 and e13/a2) resulting from translocation breakpoints within BCR exons // Br. J. Haematol.–1999.–105, N 2.–P. 434–436. 16. Moreno Mdel P., Cortinas M. N., Bonomi R., Cardeza A., Uriar- te Mdel R. A novel BCR-ABL fusion transcript (e15a2) in 2 pa- tients with atypical chronic myeloproliferative syndrome // Blood.–2001.–97, N 11.–P. 3668–3669. 17. Harnois T., Constantin B., Rioux A., Grenioux E., Kitzis A., Bour- meyster N. Differential interaction and activation of Rho family GTPases by p210 bcr-abl and p190 bcr-abl // Oncogene.– 2003.–22, N 41.–P. 6445–6454. 18. McWhirter J. R., Wang J. Y. Effect of Bcr sequences on the cel- lular function of the Bcr-Abl oncoprotein // Oncogene.–1997.– 15, N 14.–P. 1625–1634. 19. Dubrovskaya A. N., Telegeev G. D., Dybkov M. V., Volosha- nenko O. S., Shved V. V., Maliuta S. S. Mutation analysis and bac- terial expression of the chimerical oncoprotein Bcr/Abl Dbl-ho- mology domain // Biopolym. Cell.–2002.–18, N 2.–P. 96–101. 20. Telegeev G. D., Dubrovska A. N., Dybkov M. V., Maliuta S. S. In- fluence of Bcr/Abl fusion proteins on the course of Ph leukemias // Acta Biochim. Polon.–2004.–51, N 3.–P. 845–849. 21. Oleksy A., Opalinski L., Derewenda U., Derewenda Z. S., Otlew- ski J. The molecular basis of RhoA specificity in the guanine nucleotide exchange factor PDZ-RhoGEF // J. Biol. Chem.– 2006.–281, N 43.–P. 32891–32897. 22. Miroshnychenko D. O, Teleheiev H. D., Maliuta S. S. Analysis of GEF activity of Bcr protein DH domain // Ukr. Biokhim. Zh.– 2007.–79, N 5.–P. 116–121. 23. Miroshnychenko D., Dubrovska A, Maliuta S., Telegeev G., As- penstrom P. Novel role of pleckstrin homology domain of the Bcr-Abl protein: analysis of protein-protein and protein-lipid in- teractions // Exp. Cell Res.–2010.–316, N 4 P. 530–542. 24. Miroshnychenko D. O., Dubrovska A. M., Telegeev G. D., Maliu- ta S. S. Protein-lipid and protein-protein interactions of Bcr PH domain // Biopolym. Cell.–2007.–23, N 5.–P. 405–409. 25. Czech M. P. Dynamics of phosphoinositides in membrane retri- eval and insertion // Annu. Rev. Physiol.–2003.–65.–P. 791–815. 26. Lemmon M. A., Ferguson K. M. Molecular determinants in pleck- strin homology domains that allow specific recognition of phospho- inositides // Biochem. Soc. Trans.–2001.–29, Pt 4.– P. 377– 384. 27. Olabisi O. O., Mahon G. M., Kostenko E. V., Liu Z., Ozer H. L., Whitehead I. P. Bcr interacts with components of the endosomal sorting complex required for transport-I and is required for epidermal growth factor receptor turnover // Cancer Res.–2006.– 66, N 12.–P. 6250–6257. 28. Reddy M. M., Fernandes M. S., Salgia R., Levine R. L., Griffin J. D., Sattler M. NADPH oxidases regulate cell growth and mig- ration in myeloid cells transformed by oncogenic tyrosine kina- ses // Leukemia.–2011.–25, N 2.–P. 281–289. 29. Cortes J., Jabbour E., Kantarjian H. et al. Dynamics of BCR- ABL kinase domain mutations in chronic myeloid leukemia af- ter sequential treatment with multiple tyrosine kinase inhibitors // Blood.–2007.–110, N 12.–P. 4005–4011. 30. Dybkov M. V., Gartovska I. R., Telegeev G. D., Maliuta S. S. De- velopment of test system for detection of V617F mutation of Jak2 gene in patients with chronic myeloproliferative disorders // Science and Innovation.–2009.–5, N 6.–P. 59–63. 31. Glusman D. F., Sklyarenko L. M., Nadgornaya V. A., Zavelevich M. P., Poludnenko L. Yu., Ivanovskaya T. S., Ukrainskaya N. I., Òele- geev G. D., Dybkov M. V., Polischuk L. A. Development of comp- lex of immunocytochemical and molecular genetic technologies of acute leukemias diagnostics and their implementation into clini- cal practice // Science and Innovation.–2013.–9, N 1.–P. 44–54. Received 20.05.13 282 TELEGEEV G. D. ET AL.