Laboratory of Biosynthesis of Nucleic Acids

Head

Stanislav S. Avdieiev

Ph.D. (Mol. Biol.)
Phone: (380-44) 526-34-98;
fax: (380-44) 526-07-59;
E-mail: stas.avdieiev@imbg.org.ua; stasavdieiev@gmail.com

Research Area:

Investigation of the mechanisms of malignant tumors initiation and progression in order to create the general therapeutic approaches to cancer treatment

Clarification of the role of potential oncoproteins interactions and tumor suppressor proteins in RAS/MAPK and PI3K/AKT signaling cascades, involvement of these interactions in the malignant transformation of brain cells, proliferative and invasive properties by tumor cells and search for specific inhibitors of these signaling pathways

Ñurrent Research Activities and Recent Achievements:

Hundreds of genes expression changes have been revealed in tumors of glial and meningial origin by modern methods of expression genetics. Opposite changes of several genes expression suppose different mechanisms of these tumors development and can be used as molecular biomarkers. 129 genes with 5-fold changed expression were found in glioblastoma, the most aggressive human brain tumor. 44 of them were overexpressed genes, which participate in angiogenesis, immunity, ECM, cell signaling pathways, and related to the IGF-system. IGF1 is a key peptide in many tumors but its gene was not found as overexpressed in glioblastoma. It was shown that CHI3L1 gene with considerably increased expression could participate instead of IGF1 in the development of glial tumors.

The new human cell line stably producing CHI3L1 was constructed and found that these cells had an accelerated growth rate and could undergo anchorage-independent growth in soft agar that is one of the most consistent indicators of oncogenic transformation (Fig. 1).

Fig. 1. Cells with overexpressed CHI3L1 oncogene formed significantly more colonies in soft agar which is a sign of malignant transformation. A – 293 cells stable transfected by a pcDNA3.1_CHI3L1 plasmid which expresses CHI3L1 gene. B – 293 cells stable transfected by “empty” pcDNA3.1 plasmid vector.

293_CHI3L1 cells had activated PI3K and MAPK pathways; phosphorylated AKT was localized in cytoplasm, while ERK1/2 were localized in both cytoplasm and nuclei where they could activate different transcription factors with certain biological outcome. The formation of tumors in rats by 293 cells expressing CHI3L1, evidenced that CHI3L1 is an oncogene which is involved in tumorigenesis. It was the first animal model of human brain tumor which could be used for studying of various biological properties of brain tumors in the immunocompetent animals (Fig. 2).

Fig. 2. Malignant tumors in the rat brain after stereotactic intracerebral implantation of 293_CHI3L1 transformed cells. A – 293_CHI3L1 cells were implanted under ketamine anesthesia in kaudoptamen using Narishige stereotactic device, according to the coordinates of Swanson’s Brain Atlas. B – General view of tumor paraffin section, initiated by 293_CHI3L1 cells. Hematoxylin-eosin staining

It was found that CHI3L1 gene promotes chromosomal instability. Constitutive expression of CHI3L1 leads to qualitative and quantitative chromosomal abnormalities, contributes to the malignant phenotype by accelerating cell proliferation, and also increasing the genetic heterogeneity of cell populations (Fig. 3).

Fig. 3. Constitutive expression of the oncogene CHI3L1 promotes chromosomal instability in cells. Karyographs of the cell lines 293 and 293_CHI3L1 clone 2. X axis designates the chromosomes, axis Y – chromosomes copy number, axis Z – quantity of karyotyped cells (20 cells). Karyographs demonstrate variability and clonality of chromosomal changes within cell lines

CHI3L1 gene knockdown by CHI3L1 siRNA transfection gave noticeable CHI3L1 protein blockade (80-90 %) with significantly reduced. pERK1/2 and the colony-forming ability of 293_CHI3L1 cells in soft agar (Fig. 4). The obtained results demonstrate that activity of CHI3L1 mediated by pathways involved ERK1/2 and AKT has a growth-promoting role during tumorigenesis and indicate that efforts to inhibit its activity should be considered during cancer therapy.

Fig.4. Western blot analysis displayed CHI3L1 gene knockdown in 293_CHI3L1 cells

Along with superexpressed genes, it was found 85 genes relating to the potential tumor suppressor genes. The results show that CHI3L2 is an antagonist to CHI3L1 and if CHI3L1 is a real oncogene that may play an important role in tumorigenesis, CHI3L2 is anti-oncogene. A spatial model of CHI3L2 protein was constructed and it was revealed the main structural features that distinguish it from the homologous one but functionally opposite CHI3L1 protein (Fig. 5). Heparinbinding site of CHI3L1 has been identified using site-directed mutagenesis and it has been shown that it might be responsible for the oncogenic properties of CHI3L1.

Fig. 5. Three-dimensional structure of CHI3L2 protein obtained by a modeling on the base of homology with CHI3L1 protein. A – CHI3L1 protein. B – CHI3L2 protein

The antiproliferative properties of two distinct classes of molecules, namely bradykinin (BK) antagonists and azolidinones, were shown in three different in vitro models of malignant transformation: 293 cells, stably transfected by CHI3L1 oncogene (293_CHI3L1), human glioblastoma cells U373 and mantle cell lymphoma (MCL) cells Granta, JeKo, Mino, and UPN1 (Fig. 6). For drugs delivery into the brain tumors are used the nanocojugates of Polycefin on the basis of polymaleic acid which can penetrate across the blood-brain barrier.

Fig. 6. Inhibition of 293_CHI3L1 and U373 cells growth by bradykinin antagonists

Artificial intellect approach was used for diagnostics of glial brain tumors by self-organized Kohonen’s map (SOM). Obtained data clearly show the clusterization of glioblastoma and normal brain samples (Fig. 7).

Fig. 7. Classification of brain tumors using artificial neural network. Distribution of glioblastoma and normal brain samples using Kohonen map

National Grants:

Projects of National Academy of Sciences of Ukraine:

  • 2013–2017 Project “From Molecular to Cellular Events in Human Pathologies” (scientific co-supervisor – Dmitrenko V. V.)
  • 2012–2013 Project “Identification of transcriptome changes and the search of genes for intellectual classification of human brain tumors” (scientific supervisor – Dmitrenko V. V. )
  • 2010–2014 Project “New molecular genetic markers for gene expression signatures of brain tumors and their interaction with signaling pathways” (scientific supervisor – Dmitrenko V. V.)
  • 2010–2014 Project “Creation of system for brain tumors growth inhibition based on nanoconjugates of antysense oligonucleotides and antibodies against to oncoproteins, with natural biopolymers” (scientific supervisor – Dmitrenko V. V.)

Grants of State Fund for Fundamental Researches:

  • 2013–2014 Project “Characterization of new biomarkers of human glial tumors” (scientific supervisor – Dmitrenko V. V.)
  • 2013–2014 Project “Identification of perspective molecular markers for monitoring of human neurodegenerative and oncological diseases” (scientific co-supervisor – Dmitrenko V. V.)
  • 2011–2014 Project “Molecular mechanisms of cell signaling in normal and pathological conditions: the focus on ion channels” (SKL Molecular and Cell Biology, scientific co-supervisor – Dmitrenko V. V.)

Grant of State Committee of Ukraine for Science, Innovation and Information:

  • 2014–2015 Project “Identification and characterization of new biomarkers of human glial tumors” (scientific supervisor – Dmitrenko V. V.)

International Grants:

  • 2011–2014 FP7-INCO-2011-6, ERA-WIDE Project “Strengthening cooperation in Molecular Biomedicine between EU and UKRAINE”, COMBIOM (scientific supervisor - Kavsan V. M.)
  • 2011–2013 STCU Project, 5446 “Nanoconjugates of natural biopolymers with antisense oligonucleotides and antibody for inhibition of glial tumors” (scientific supervisor – Kavsan V. M.)

Collaboration:

with Ukrainian organizations:

  • State Institution “Institute of Neurosurgery named after A. P. Romodanov of NAMS of Ukraine” (Kyiv)
  • R. E. Kavetsky Institute of Experimental Pathology, Oncology and Radiobiology, NASU (Kyiv)
  • Danylo Halytsky Lviv National Medical University (Lviv)

with foreign organizations:

  • Belarusian State University (Minsk, Belarus)
  • Serbsky State Scientific Center for Social and Forensic Psychiatry (Moscow, Russia)
  • Institute of Chemical Biology and Fundamental Medicine, SB RAS (Novosibirsk, Russia)
  • University of Colorado Denver (Denver, USA)
  • Cedars-Sinai Medical Center (Los Angeles, USA)
  • Institute Gustave-Roussy (Paris, France)
  • Service de Neurologie Mazarin, INSERM U 711 (Paris, France)

Selected publications:

  1. Stepanenko A.A., Andreieva S.V., Korets K.V., Mykytenko D.O., Baklaushev V.P., Chekhonin V.P., Dmitrenko V.V. mTOR inhibitor temsirolimus and MEK1/2 inhibitor U0126 promote chromosomal instability and cell type-dependent phenotype changes of glioblastoma cells. Gene. 2016, 579 (1):58-68.
  2. Stepanenko A.A. Andreieva S.V. Korets K.V. Mykytenko D.O. Baklaushev V.P. Huleyuk N.L. Kovalova O.A. Kotsarenko K.V. Chekhonin V.P. Vassetzky Y.S. Avdieiev S.S. Dmitrenko V.V. Temozolomide promotes genomic and phenotypic changes in glioblastoma cells. Cancer Cell Int., 36, doi: 10.1186/s12935-016-0311-8.
  3. Dergai M., Iershov A., Novokhatska O., Pankivskyi S., Rynditch A. Evolutionary changes on the way to clathrin-mediated endocytosis in animals. Genome Biol. Evol. 2016, 8(3):588-606.
  4. Finiuk N. S., Senkiv J. V., Riabtseva A. O., Mitina N. Y., Molochii N. I., Kitsera M. O., Avdieiev S. S., Zaichenko O. S., R. S. Stoika. Modulation of temozolomide action towards rat and human glioblastoma cells in vitro by its combination with doxorubicin and immobilization with nanoscale polymeric carrier. Ukr.Biochem. J.2016, 88 (special issue): 87-98.
  5. Stepanenko AA, Dmitrenko VV. HEK293 in cell biology and cancer research: phenotype, karyotype, tumorigenicity, and stress-induced genome-phenotype evolution. Gene. 2015;569(2):182-190.
  6. Stepanenko AA, Baklaushev VP, Vassetzky YS, Dmitrenko VV. Cisplatin treatment of C6 rat glioma in vivo did not influence copy number alterations and growth pattern of tumor-derived resistant cells. Biopolym. Cell. 2015; 31(3):209–217.
  7. Avdieiev S, Gera L, Hodges RS, Dmitrenko VV. Glioma-associated protein CHI3L2 suppresses cells viability and induces G1/S transition arrest. Biopolym. Cell. 2015;31(4):316-320.
  8. Stepanenko A, Andreieva S, Korets K, Mykytenko D, Huleyuk N, Vassetzky Y, Kavsan V. Step-wise and punctuated genome evolution drive phenotype changes of tumor cells. Mutation Res. (Mutation Research-Fundamental and Molecular Mechanisms of Mutagenesis). 2015;771:56-69.
  9. Avdieiev S, Gera L, Havrylyuk D, et al. Bradykinin antagonists and thiazoli-dinone derivatives as new potential anti-cancer compounds. Bioorg. & Med. Chem. 2014;22:3815-3823.
  10. Stepanenko AA, Kavsan VM. Karyotypically distinct U251, U373, and SNB19 glioma cell lines are of the same origin but have different drug treatment sensitivities. Gene. 2014;540:263-265.
  11. Kavsan VM, Kulagova TA, Kuznetsova TA, et al. Structure and function of oncogene-transfected immortal cells. Biopolym. Cell. 2014;30(1):25-28.
  12. Avdieiev SS, Gera L, Hodges R, et al. Growth suppression activity of bradykinin antagonists in glioma cells. Biopolym. Cell. 2014;30(1):77-79.
  13. Stepanenko AA, Vassetzky YS, Kavsan VM. Antagonistic functional duality of cancer genes. Gene. 2013; 529: 199-207. doi: 10.1016/j.gene.2013.07.047
  14. Stepanenko AA, Kavsan VM. Cancer genes and chromosome instability. Oncogene and Cancer – From Bench to Clinic, InTech Publisher, 2013; 151-182.
  15. Dmitrenko VV, Avdieiev SS, Areshkov PO, et al. From reverse transcription to human brain tumors Biopolym. Cell. 2013; 29(3):221-233 doi: 10.7124/bc.00081C
  16. Stepanenko AA, Kavsan VM. Evolutionary karyotypic theory of cancer versus conventional cancer gene mutation theory. Biopolym. Cell. 2012; 28(4):267–280. doi:10.7124/bc.000059
  17. Baklaushev VP, Kavsan VM, Balynska OV, Yusubalieva GM, Abakumov VA, Chekhonin VP. New experimental model of brain tumors in brains of adult immunocompetent rats. Brit. J. Med. & Med. Res. 2012; 2 (2): 206-215.
  18. Areshkov PO, Avdieiev SS, Balynska OV, LeRoith D, Kavsan VM. Two closely related human members of chitinaselike family, CHI3L1 and CHI3L2, activate ERK1/2 in 293 and U372 cells but have the different influence on cell proliferation. Int. J. Biol. Sci. 2012; 8: 39-48.
  19. Kavsan VM, Baklaushev VP, Balynska OV, et al. Gene encoding chitinase 3like 1 protein (CHI3L1) is a putative oncogene. Int. J. Biomed. Sci. 2011; 7: 230237.
  20. Kavsan VM, Iershov AV, Balynska OV. Immortalized cells and one oncogene in malignant transformation: old insights on new explanation. BMC Cell Biol. 2011; 12:23. doi: 10.1186/1471-2121-12-23
  21. Iershov A, Odynets K, Kornelyuk A, Kavsan V. Homology modeling of 3D structure of human chitinaselike CHI3L2 protein. Central Eur. J. Biol. 2010; 5(4):407-420. doi:10.2478/s11535-010-0039-8
  22. Moureau C, Moynier M, Kavsan VM, Montagnier L, Bahraoui E. Specificity of antiNef antibodies produced in mice immunized with DNA encoding the HIV1 nef gene product. Vaccine. 2000; 18: 333-341. doi: 10.1016/S0264-410X(99)00203-0
  23. Palamarchuk AY, Kavsan VM, Sussenbach JS, Holthuizen PE. The chum salmon IGFII gene promoter is activated by hepatocyte nuclear factor 3ß. FEBS Lett. 1999; 446:251-255. doi:10.1016/S0014-5793(99)00219-7