V. S. Prassolov

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.

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.

Organization and regulation of nucleocytoplasmic transport

Separation of DNA replication and transcription, which occur in the nucleus, from protein synthesis, which occurs in the cytoplasm, allows a more precise regulation of these processes. Selective exchange of macromolecules between the two compartments is mediated by proteins of the nuclear pore complex (NPC). Receptor proteins of the karyopherin family interact with NPC components and transfer their cargos between the nucleus and cytoplasm. Nucleocytoplasmic transport pathways are regulated at multiple levels by modulating the expression or function of individual cargoes, transport receptors, or the transport channel. The regulatory levels have increasingly broad effects on the transport pathways and affect a wide range of processes from gene expression to development and differentiation.Separation of DNA replication and transcription, which occur in the nucleus, from protein synthesis, which occurs in the cytoplasm, allows a more precise regulation of these processes. Selective exchange of macromolecules between the two compartments is mediated by proteins of the nuclear pore complex (NPC). Receptor proteins of the karyopherin family interact with NPC components and transfer their cargos between the nucleus and cytoplasm. Nucleocytoplasmic transport pathways are regulated at multiple levels by modulating the expression or function of individual cargoes, transport receptors, or the transport channel. The regulatory levels have increasingly broad effects on the transport pathways and affect a wide range of processes from gene expression to development and differentiation.

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.

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.

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.

Treatment with anti-cancer agents results in profound changes in lncRNA expression in colon cancer cells

Using real-time RT-PCR in combination with bioinformatics, we have shown for the first time that the treatment of HCT-116 and HT-29 colon cancer cells with two anti-cancer agents (doxycycline or 3,3′-diindolylmethane) results in profound changes in the intracellular content of several lncRNAs (by up to 100 times). Since many of these RNAs are secreted by tumors into the bloodstream, the obtained results provide a basis for developing more sensitive protocols for serological monitoring of tumor relapse and metastasis, as well as for search of new anti-cancer drugs.

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.

Comparative analysis of gene expression: Targeted antitumor therapy in neuroblastoma cell lines

In this study, we evaluated the level of c-kit, VEGFA, and MYC gene expression in seven neuroblastoma stable cell lines, i.e., SK-N-SH, SK-N-BE, SK-N-AS, SH-SY5Y, Kelly, IMR-32, and LAN-1. The level of expression of these genes can serve as a diagnostic factor for cancer progression and proteins encoded by these genes are promising targets for neuroblastoma treatment. SH-SY5Y and SK-N-AS cells have the highest MYC expression and the same VEGFA expression, although SH-SY5Y has tenfold higher c-kit expression than SK-N-AS cells. Both IMR-32 and LAN-1 cells have low levels of MYC expression, but differ in c-kit expression, while IMR-32 has significantly higher c-kit expression than any other neuroblastoma cell line. On the other hand, LAN-1 has the highest VEGFA expression. These data suggest that MYC, c-kit, and VEGFA genes can play different roles in the development and progression of neuroblastoma depending on other activated molecular mechanisms in malignant cells.