P. V. Spirin

Downregulation of activated leukemic oncogenes AML1-ETO and RUNX1(K83N) expression with RNA-interference

In the present study, we have applied the siRNA approach to reduce the expression of AML1-ETO and RUNX1(K83N) oncogenes, which are frequently found in leukemic cells. We have designed small hairpin RNAs (shRNA) for targeting AML1-ETO oncogene and a region close to the 5′-untranslated region of mRNA for the mutant RUNX1(K83N) oncogene and expressed the shRNAs in lentiviral vectors. We report a stable reduction in expression of oncogenes following the introduction of shRNAs into cells.

Modulation of activated oncogene c-kit expression with RNA-interference

Overexpression of oncogene c-kit is detected in 80% patients with acute myeloid leukemia (AML). A transgenic model cell line expressing oncogene c-kit was obtained by transduction with a recombinant retrovirus. We have designed small interfering RNAs (siRNAs) that efficiently suppress the expression of activated oncogene c-kit. Further, small hairpin RNAs (shRNAs) targeting c-kit mRNA were designed and expressed in lentiviral vectors. We report a stable reduction in c-kit expression following the introduction of shRNAs into model cells, as well as Kasumi-1 cells from a patient with AML.

Expression of the FLT3-ITD oncogene sensitizes murine progenitor B-cell line BAF3 to cytotoxic action of binase

Acute myeloid leukemia makes up about 30% of all leukemia cases in adults. Mutations in the genes of the receptor tyrosine kinases KIT and FLT3, along with chromosomal translocations, are frequently found in leukemic cells. In the current work, we show that the transgenic B-cells BAF3/FLT3-ITD are significantly more sensitive to cytotoxic action of the ribonuclease binase than original BAF3 cells. BAF3/FLT3-ITD cells differ from BAF3 in expression of the FLT3-ITD oncogene, which results in the alteration of normal signaling pathways. We observed a similar effect previously when studying binase cytotoxic action in cells Kasumi-1 and FDC-P1-N822K, in which the activated oncogene KIT-N822K was expressed. An elevated cytotoxicity of binase to the cells that express the FLT3-ITD oncogene indicates that, as in case of the FDC-P1 cells transduced by the KIT oncogene, the expression of an activated oncogene determines the cell’s sensitivity to the binase action.

Activated leukemic oncogenes responsible for neoplastic transformation of hematopoietic cells

Leukemias are a heterogeneous group of malignant blood diseases that are characterized by expansion of immature blast cells. The point molecular mechanisms of leukemogenesis are still unknown. Leukemia patients frequently have mutations of the genes responsible for normal proliferation and differentiation of hematopoietic stem cells. At present, scientific groups worldwide are engaged in biomedical studies of the structural and functional aspects of leukemic oncogenes and their role in human and animal leukemogenesis. The review describes the current concepts of the molecular properties of oncogenes whose activation may lead to CBF-AML, which results from mutations of the genes for the core binding factors AML1 (CBF6h) and CBFß.

Baculovirus vectors for efficient gene delivery and expression in mammalian cells

Baculovirus expression vectors are extensively used for the delivery of foreign genes and expression of recombinant proteins in insect and mammalian cells. Modified baculoviruses containing mammalian promoter elements (BacMam viruses) for an efficient transient and stable transduction of diverse mammalian cells ensure a high level of heterologous protein expression both in vitro and in vivo. Recombinant baculovirus vectors containing mammalian expression cassette with cytomegalovirus promoter, green or red fluorescent protein gene, SV40pA polyadenylation signal, and polylinker MCS were constructed for the delivery of genes encoding hepatitis C virus structural proteins into mammalian cells. In HEK293T and Huh7 cells, formation of glycoprotein complexes and HCV4ike particles was observed. A high efficiency of the baculovirus-medi-ated gene transfer and expression of the virus envelope proteins in mammalian cells was demonstrated using fluorescence, flow cytometry, and immunoblot techniques.

Receptor tyrosine kinase KIT may regulate expression of genes involved in spontaneous regression of neuroblastoma

Hallmark of neuroblastoma is an ability of this malignant tumor to undergo spontaneous regression or differentiation into benign tumor during any stage of the disease, but it is little known about mechanisms of these phenomena. We studied effect of receptor tyrosine kinase receptor KIT on expression of genes, which may be involved in tumor spontaneous regression. Downregulation of KIT expression by RNA interference in SH-SY5Y cells causes suppression of neurotrophin receptor NGFR expression that may promote the loss of sensibility of cells to nerve growth factors, also it causes upregulation of TrkA receptor expression which can stimulate cell differentiation or apoptosis in NGF dependent manner. Furthermore there is an upregulation of genes which stimulate malignant cell detection by immune system, such as genes of major histocompatibility complex HLA class I HLA-B and HLA-C, and interferon-γ receptors IFNGR1 and IFNGR2 genes. Thus KIT can mediate neuroblastoma cell sensibility to neurotrophins and immune system components--two factors directly contributing to spontaneous regression of neuroblastoma.

RNA interference and deep sequencing as tools for identifying new genes involved in leukemogenesis

49 The establishment of the molecular mechanisms of occurrence and progression of oncological diseases and the development of effective tools to combat them is an important field of modern biomedicine. Today, the complete set of genes involved in carcinogenesis is not known for the majority of types of malignant tumors [1]. Leukemia is a group of oncological diseases of the hemopoietic system. A considerable part of human leu kemias is represented by acute myeloid leukemias (AMLs) [2]. Malignization is a multistep process: the activation of proto oncogenes as a result of mutations triggers a cascade of genes and, consequently, disturbs the coordination of the molecular mechanisms respon sible for the differentiation of hematopoietic cells and normal hematopoiesis. All this significantly compli cates the diagnosis of leukemias and the selection of effective treatment schemes. The problem is addition ally complicated by the fact that many genes that con trol the growth and differentiation of hematopoietic cells and the degree of their involvement in the process of malignant transformation remain unknown [2, 3]. One of the most frequent mutations detected in AML patients is the chromosomal translocation t(8;21), which occurs in 8–20% of all AML patients and in approximately 40% of cases of M2 AML (according to the FAB classification) [4]. The t(8;21) translocation leads to the formation of the chimeric oncogene AML1 ETO, whose expression disturbs nor mal differentiation and cell status. Usually, this muta tion along is insufficient for the development of leuke mia. It is known that, in model cell systems, an increased expression of the AML1 ETO oncogene leads to an increased expression of the wild type c kit gene, encoding the receptor tyrosine kinase KIT. Importantly, an increased level of KIT is found in about 80% of all AML cases and is one of the most important and serious pathogenic factors [4]. We have shown that the inhibition of the expression of activated AML1 ETO oncogene in the Kasumi 1 cell line derived from the peripheral blood of an AML patient, reduces the expression of the c kit gene, the wild type form of which belongs to the genes whose expression is controlled by AML1 ETO. The results of studies performed in model cell lines do not always agree with the results obtained in the cells isolated from patients: model cells can signifi cantly differ in the initial expression profile of genes, particularly those that encode the proteins that are directly involved in cell differentiation. Gene expres sion constantly changes during the differentiation of hematopoietic cells, and the gene expression profile in the blast cells strongly depends on the nature of these cells. In this study, we used Kasumi 1 blast cells obtained from an AML patient with the t(8;21) translo cation, because these cells are devoid of the disadvan tages characteristic of the model cells derived in vitro. To suppress the expression of the AML1 ETO gene, we used recombinant lentiviral vectors that direct the synthesis of small hairpin RNAs (shRNAs), which suppress the activity of the AML1 ETO oncogene at the posttranscriptional level. DNA of lentiviral vectors (recombinant proviruses) is integrated into the genome and ensures a stable long term expression of shRNA in transduced cells [5].

A new bidirectional promoter from the human genome

Both human and other mammalian genomes contain a number of closely linked gene pairs transcribed in opposite directions. Bioinformatic analysis suggests that up to 10% of human genes are arranged in this way. This work reports cloning of a human genome fragment that separates two head-to-head oriented genes located at 2p13.1 and encoding hypothetical proteins with unknown functions: CCDC (Coiled Coil Domain Containing) 142 and TTC (TetraTricopeptide repeat Containing) 31. The intergenic region CCDC142-TTC31 overlaps with a CpG island and contains a number of potential binding sites for transcription factors. This fragment functions as a bidirectional promoter in the system of luciferase reporter gene expression upon transfection of human embryonic kidney (HEK293) cells. Vectors containing oppositely oriented genes of two fluorescent proteins: green (EGFP) and red (DsRed2), separated by a fragment of the CCDC142-TTC31 intergenic region, were constructed. Transfection of HEK293 cells with these vectors resulted in simultaneous expression of both fluorescent proteins. The promoter activity was also determined for truncated versions of the intergenic region. The minimal promoter fragment contained Inr, BRE, and DPE elements characteristic for TATA-less promoters. Thus, a novel bidirectional promoter was cloned from the human genome; it can be used for simultaneous constitutive expression of two different genes in human cells.

Replication-competent gamma-retrovirus Mo-MuLV expressing green fluorescent protein as efficient tool for screening of inhibitors of retroviruses that use heparan sulfate as primary cell receptor

The effect of sulfated polysaccharides on the efficiency of the infection of mouse transplantable fibroblast SC-1 and NIH-3T3 cell lines by replication-competent recombinant Moloney murine leukemia virus (Mo-MuLV), which carries the eGFP gene, was investigated. It was found that sulfated polysaccharides have no cytostatic and cytotoxic effects on SC-1 and NIH-3T3 cells in the concentration range of 0.01–100 μg/mL and have virucidal activity against Mo-MuLV. Polysaccharides in the indicated concentrations inhibit Mo-MuLV infection that prevents the further development of viral infection. It was shown that sulfated polysaccharides are also effective inhibitors of other retroviruses, including lentiviruses, which use sulfated polysaccharides as primary cell receptors.

Abnormal expression of genes that regulate retinoid metabolism and signaling in non-small-cell lung cancer

Retinoids are signaling molecules that control a wide variety of cellular processes and possess antitumor activity. This work presents a comprehensive description of changes in the expression of 23 genes that regulate retinoid metabolism and signaling in non-small-cell lung cancer tumors compared to adjacent normal tissues obtained using RT-PCR. Even at early stages of malignant transformation, a significant decrease in ADH1B, ADH3, RDHL, and RALDH1 mRNA levels was observed in 82, 79, 73, and 64% of tumor specimens, respectively, and a considerable increase in AKR1B10 mRNA content was observed in 80% of tumors. Dramatic changes in the levels of these mRNAs can impair the synthesis of all-trans retinoic acid, a key natural regulatory retinoid. Apart from that, it was found that mRNA levels of nuclear retinoid receptor genes RXRγ, RARα, RXRα, and gene RDH11 were significantly decreased in 80, 67, 57, and 66% of tumor specimens, respectively. Thus, neoplastic transformation of lung tissue cells is accompanied with deregulated expression of key genes of retinoid metabolism and function.