[Plasmolipin and Its Role in Cell Processes].

The mechanisms involved in the origin and development of malignant and neurodegenerative diseases are an important area of modern biomedicine. A crucial task is to identify new molecular markers that are associated with rearrangements of intracellular signaling and can be used for prognosis and the development of effective treatment approaches. The proteolipid plasmolipin (PLLP) is a possible marker. PLLP is a main component of the myelin sheath and plays an important role in the development and normal function of the nervous system. PLLP is involved in intracellular transport, lipid raft formation, and Notch signaling. PLLP is presumably involved in various disorders, such as cancer, schizophrenia, Alzheimer's disease, and type 2 diabetes mellitus. PLLP and its homologs were identified as possible virus entry receptors. The review summarizes the data on the PLLP structure, normal functions, and role in diseases.

[Inhibition of Non-Receptor Tyrosine Kinase JAK2 Reduces Neuroblastoma Cell Growth and Enhances the Action of Doxorubicin].

Novel treatments for various types of malignant diseases are warranted. In this study, we evaluated JAK2 inhibitors (Janus kinase 2) for suppressing the growth of malignant neuroblastoma and glioblastoma cells as well as breast and non-small cell lung cancers. Neuroblastoma and glioblastoma cells are the most sensitive to the JAK2 inhibitor AG490. A study of the relative expression of receptors that can activate JAK2 suggests that cell line sensitivity to AG490 may be mediated by IL6-R, IL11-R and/or CSF1-R. AG490 enhances the effect of doxorubicin on neuroblastoma cells. Our findings suggest the possible relevance of JAK2 inhibitors for neuroblastoma therapy, especially in combination with doxorubicin.

[High Expression Level of SP1, CSF1R, and PAK1 Correlates with Sensitivity of Leukemia Cells to the Antibiotic Mithramycin].

Acute myeloid leukemia (AML) is a genetically heterogeneous group of oncological diseases of the hematopoietic system, which are extremely difficult to treat. The development of new targeted drugs (Hylteritinib, Venetoclax) significantly improved the survival of patients, but resistance, as well as cytotoxic anti-leukemia drugs, often occurs. The search for new molecular targets for the development of effective approaches for the treatment of AML is very urgent. In blast cells of patients with AML, mutations, chromosomal rearrangements, and increased expression of a number of non-mutant genes, including transcription factor genes, are detected. The transcription factor Sp 1 binds to GC-rich regions of regulatory regions of various genes and thus controls their expression. Sp1 targets include genes responsible for proliferation, cell cycle regulation, and differentiation. In many malignant diseases, a high level of Sp1 gene expression is associated with an unfavorable prognosis, therefore, Sp1 is considered as a promising therapeutic target for cancer. In this paper, we estimated the expression levels of Sp1 in various malignant tissues. Increased Sp1 expression was detected in samples obtained from patients with AML, acute lymphoblastic leukemia, Ewing sarcoma, ovarian and kidney cancer. It is also shown that Sp1 expression correlates with the expression of genes encoding cytokine receptors and growth factors (CSF1R and IL6R), intracellular kinases (CSK, SYK, PAK1, ILK, JAK2), and transcription factor LMO2. The correlation between expression levels of Sp1 and CSF1R, SYK, Jak2 and LMO2 is also characteristic of transplanted human leukemia cells. We measured expression levels of Sp1, CSF1R, ILK, PAK1 in the cells of three transplantable lines of human leukemia and found increased levels of expression of these genes in Kasumi-1 cells. In addition, we showed that Kasumi-1 cells are most sensitive to Mitramycin, a drug that displaces Sp1 from its targets with DNA. Our data indicate the need to identify AML cells that are most sensitive to inhibition of Sp1 activity in order to assess the possibility of suppressing its activity in vivo.

[Key molecular mechanisms associated with cell malignant transformation in acute myeloid leukemia].

Cancer, along with cardiovascular disorders, is one of the most important problems of healthcare. Pathologies of the hematopoietic system are the most prevalent in patients under 30 years of age, including acute myeloid leukemia (AML), which is widespread and difficult to treat. The review considers the mechanisms that play a significant role in AML cell malignant transformation and shows the contributions of certain genes to both remission and resistance of AML cells to various treatments.

[Ribonuclease binase induces death in T-cell acute lymphoblastic leukemia cells by apoptosis].

Bacterial ribonuclease binase is a potential anticancer agent. In the present study, we have determined the toxic effect of binase towards cell lines of T-cell acute lymphoblastic leukemia Jurkat and CEMss. We have shown that binase induces apoptosis in these cells. At the same time, binase does not cause toxic effects in leukocytes of healthy donors, which suggests that binase activity towards leukemic cells is selective. We have found that the treatment of cancer cells with binase leads to a reduction in reactive oxygen species and transcription factor NFκB levels, and demonstrated that these effects are a common feature of the action of RNases on cancer cells.

[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 study of therapy targeted genes expression in neuroblastoma cell lines].

In this study we evaluated c-kit, VEGFA, and MYC gene expression level in seven neuroblastoma stable cell lines: SK-N-SH, SK-N-BE, SK-N-AS, SH-SY5Y, Kelly, IMR-32, and LAN-1. Expression levels of these genes can serve as diagnostic factors of cancer progression, and proteins encoded by these genes are promising targets for neuroblastoma treatment. SH-SY5Y and SK-N-AS cells have highest MYC expression and the same VEGFA expression, although SH-SY5Y has 10 times higher c-kit expression than SK-N-AS cells. Both IMR-32 and LAN-1 cells have low MYC expression level, but differ in c-kit expression, IMR-32 has significantly higher c-kit expression, than any other neuroblastoma cell line. LAN-1 on the other hand has the highest VEGFA expression. These data suggest that MYC, c-kit, and VEGFA genes can play different roles in development and progression of neuroblastoma depending on other activated molecular mechanisms in malignant cells.

[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.

[Functional characterization of two novel splicing mutations in glucokinase gene in monogenic diabetes MODY2].

Two novel mutations in glucokinase (GCK) gene-G to C substitution at -1 position of intron 7 acceptor splice site (c. 864-1G>C) and synonymous substitution c. 666C>G (GTC>GTG, p.V222V) in exon 6--were identified in patients with monogenic diabetes MODY2 (Maturity Onset Diabetes of Young). GCK minigenes with these mutations were constructed. Analysis of splicing products upon transfection of minigenes into human embryonic cell line HEK293 has shown that each of these nucleotide substitutions impair normal splicing. Mutation c.864-1G>C blocks the usage of normal acceptor site which activates cryptic acceptor splice sites within intron 7 and generates aberrant RNAs containing the portions ofintron 7. Synonymous substitution c.666C>G creates novel donor splice site in exon 6 that leads to formation of defective GCK mRNA with deletion of 16 nucleotides of exon 6. Analysis of in vitro splicing of minigenes confirms the inactivating action of novel mutations on glucokinase expression.

[Universal modular system for in vitro screening of potential inhibitors of HIV-1 replication].

Here we describe a system based on recombinant lentiviral vectors for the safe screening of potential anti-HIV drugs. The system allows to evaluate the sensitivity of HIVl-1 reverse transcriptase and integrase (wild-type as well as mutant forms of these enzymes detected in drug-resistant virus isolates) towards different drugs and substances, but also to screen inhibitors of other stages of HIV-1 life cycle.

[Dynamics of penetration of "solid" nanoconstructions based on double-stranded DNA complexed with gadolinium into CHO cells].

Currently, neutron capture therapy is a promising cancer treatment. This method is based on the reaction of the thermal neutron capture by some non-radioactive elements (e.g., Gds57), which results in subsequent emission of electrons and gamma rays. An effective instrument for delivery of gadolinium into the tumor tissue are the particles of the "rigid" nanostructures (NS) based on double-stranded DNA complexes with gadolinium (NS-Gd). The local concentration of Gd in such nanostructures may reach 40%. To optimize the process of neutron capture therapy it is very important to investigate possible penetration mechanisms of NS-Gd particles into the tumor cells. In this work, the dynamics of interaction NS-Gd with cultivated chinese hamster ovary cells (CHO) was studied by confocal and electron microscopy. It is shown that NS-Gd are able to enter CHO cells. This process begins in about 1 hour after the start ofincubation. After 6 h NS-Gd particles were detected in almost all cells. A further increase of the incubation time does not lead to significant changes in cell morphology, although the number NS-Gd inside the cells increases. The plasma membrane of the cells remains intact. The NS-Gd particles, which entered the cells, remain inside the cells for a long time. The data obtained show that NS-Gd are relatively low-toxic and suggest that the presence of NS-Gd in the tumor cells does not prevent their division. The data obtained are important for improving the efficiency of the neutron capture therapy method.

[Role of N-linked glycans in HCV glycoprotein E1 in the folding of structural proteins and formation viral particles].

Envelope proteins of HCV play a major role in virus lifecycle. These proteins are main components of the virion. They are involved in virus assembly. Envelope proteins are modified by N-linked glycosylation which is supposed to play a role in their stability, in the assembly of the functional HCV glycoprotein heterodimer, protein folding and viral entry. The role of N-linked glycosylation sites in HCV E1 protein in structural proteins assembly was analyzed by site-directed mutagenesis in a model system--insect cells producing three viral structural proteins with formation of virus-like particles. Removing of single N-linked glycosylation sites in HCV E1 protein does not affect the efficiency of its expression in insect Sf9 cells. E1 electrophoretic mobility is increasing in parallel with decreasing the number of glycosylation sites. The destroying of glycosylation sites N1 or N5 in E1 influences the assembly of noncovalent glycoprotein heterodimer E1E2--the prototype of natural complex incorporated in virion. The lack of glycans in N1 and N5 sites of E1 was shown to affect the efficiency of its expression in mammalian HEK293 T cells.

[Expression of FLT3-ITD oncogene confers mice progenitor B-cells BAF3 sensitivity to the ribonuclease binase cytotoxic action].

Acute myeloid leukemia is the most common acute leukemia affecting adults, and its incidence increases with age. Along with chromosomal translocations in leukemic cells mutations in the genes of receptor tyrosine kinases KIT and FLT3 were found with a high frequency. Here we show that transgenic progenitor of B-cells BAF3/FLT3-ITD are much more sensitive to the ribonuclease binase cytotoxic effects than the original BAF3 cells. The principal difference between BAF3/FLT3-ITD and the original BAF3 cells is the expression of FLT3-ITD oncogene, which leads to a change in the normal cell signaling pathways. Earlier, we described a similar effect for the cytotoxic action of binase on Kasumi-1 and FDC-P1-N822K cells, which express the activated KIT-N822K oncogene. Increased binase cytotoxicity toward the cells, expressing FLT3-ITD oncogene, suggests that, as in the case of FDC-P1 cells, transduced by KIT oncogene, the expression of an activated oncogene determines the sensitivity of cells to binase.

The Role of Autophagy in the Development of Pathological Conditions of the Body.

Autophagy is the process of lysosomal elimination of the cell organelles, cytoplasmic sites, and pathogenic microorganisms that enter the cell. This process is associated with both cell death regulation and an increase in cell survival chances. Autophagy is involved in the development of various diseases (Crohn disease, cancer, atherosclerosis, etc.). For these reasons, it is of significant interest to establish the molecular targets involved in autophagy regulation and the factors that mediate its participation in pathogenesis. The review describes the potential molecular mechanisms involved in the regulation of autophagy, its contribution to the vital cell activity in a healthy organism, and pathologies.

[Replication-competent γ-retrovirus Mo-MuLV expressing green fluorescent protein gene as an efficient tool for screening of inhibitors of retroviruses that use heparan sulfate as the primary cell receptor].

The effect of sulfated polysaccharides on the efficiency of infection of mouse embryonic fibroblast cell lines SC-1 and NIH-3T3 by replication-competent recombinant Moloney murine leukemia virus (Mo-MuLV) carrying the eGFP gene was investigated. It was shown that used polysaccharides have no cytostatic and cytotoxic effects on SC-1 and NIH 3T3 cells inthe concentrations from 0.01 to 100 µg/ml and have virucidal activity against Mo-MuLV. Polysaccharides in the indicated concentrations inhibit cell infection by Mo-MuLV, that prevents further expansion of viral infection. It was detected that sulfated polysaccharides are effective inhibitors of other retroviruses, including lentiviruses, that use heparan sulfate as cell receptors for non-specific binding.

[Novel bidirectional promoter from human genome].

In human and other mammalian genomes a number of closely linked gene pairs transcribed in opposite directions are found. According to bioinformatic analysis up to 10% of human genes are arranged in this way. In present work the fragment of human genome was cloned that separates genes localized at 2p13.1 and oriented "head-to-head", coding for hypothetical proteins with unknown functions--CCDC (Coiled Coil Domain Containing) 142 and TTC (TetraTricopeptide repeat Containing) 31. Intergenic CCDC142-TTC31 region overlaps with CpG-island and contains a number of potential binding sites for transcription factors. This fragment functions as bidirectional promoter in the system ofluciferase reporter gene expression upon transfection of human embryonic kidney (HEK293) cells. The vectors containing genes of two fluorescent proteins--green (EGFP) and red (DsRed2) in opposite orientations separated by the fragment of CCDC142-TTC31 intergenic region were constructed. In HEK293 cells transfected with these vectors simultaneous expression of two fluorescent proteins is observed. Truncated versions of intergenic region were obtained and their promoter activity measured. Minimal promoter fragment contains elements Inr, BRE, DPE characteristic for TATA-less promoters. Thus, from the human genome the novel bidirectional promoter was cloned that can be used for simultaneous constitutive expression of two genes in human cells.

[Modulation of activated oncogene c-kit expression with RNA-interference].

Hyperexpression of oncogene c-kit is found in 80% patients with acute myeloid leukemia (AML). The transgenic model cell line expressing the oncogene c-kit was obtained by transduction with recombinant retrovirus. We have designed small interfering RNAs (siRNA) efficiently suppressing the expression of activated oncogene c-kit. Further small hairpin RNAs (shRNA) 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 the patient with AML.

[Characterization of new splicing mutation in steroid 21-hydroxylase gene].

Novel mutation in CYP21A2 gene causing the steroid 21-hydroxylase deficiency - C to G substitution in 7-position ofintron 2 acceptor splice site (c.290-7C>G) was identified. The effect of the mutation on splicing was checked in the system of CYP21A minigene expression in the cultured mammalian cells. The mutation impairs the usage of intron 2 acceptor splice site resulting in intron retention.

The Role of the MCTS1 and DENR Proteins in Regulating the Mechanisms Associated with Malignant Cell Transformation.

The mutations associated with malignant cell transformation are believed to disrupt the expression of a significant number of normal, non-mutant genes. The proteins encoded by these genes are involved in the regulation of many signaling pathways that are responsible for differentiation and proliferation, as well as sensitivity to apoptotic signals, growth factors, and cytokines. Abnormalities in the balance of signaling pathways can lead to the transformation of a normal cell, which results in tumor formation. Detection of the target genes and the proteins they encode and that are involved in the malignant transformation is one of the major evolutions in anti-cancer biomedicine. Currently, there is an accumulation of data that shed light on the role of the MCTS1 and DENR proteins in oncogenesis.

Plasmolipin and Its Role in Cell Processes.

The mechanisms involved in the origin and development of malignant and neurodegenerative diseases are an important area of modern biomedicine. A crucial task is to identify new molecular markers that are associated with rearrangements of intracellular signaling and can be used for prognosis and the development of effective treatment approaches. The proteolipid plasmolipin (PLLP) is a possible marker. PLLP is a main component of the myelin sheath and plays an important role in the development and normal function of the nervous system. PLLP is involved in intracellular transport, lipid raft formation, and Notch signaling. PLLP is presumably involved in various disorders, such as cancer, schizophrenia, Alzheimer's disease, and type 2 diabetes mellitus. PLLP and its homologs were identified as possible virus entry receptors. The review summarizes the data on the PLLP structure, normal functions, and role in diseases.