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.

[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.

The Different Impact of ERK Inhibition on Neuroblastoma, Astrocytoma, and Rhabdomyosarcoma Cell Differentiation.

Aberrant ERK activity can lead to uncontrolled cell proliferation, immortalization, and impaired cell differentiation. Impairment of normal cell differentiation is one of the critical stages in malignant cell transformation. In this study, we investigated a relationship between ERK tyrosine kinase activity and the main differentiation features (changes in cell morphology and expression of genes encoding differentiation markers and growth factor receptors) in SH-SY5Y neuroblastoma, U-251 astrocytoma, and TE-671 rhabdomyosarcoma cells. ERK activity was assessed using a reporter system that enabled live measurements of ERK activity in single cells. We demonstrated that suppression of ERK activity by selective ERK inhibitors, in contrast to a commonly used differentiation inducer, retinoic acid, leads to significant changes in TE-671 cell morphology and expression of the myogenic differentiation marker genes PROM1, MYOG, and PAX7. There was a relationship between ERK activity and morphological changes at an individual cell level. In this case, SH-SY5Y cell differentiation induced by retinoic acid was ERK-independent. We showed that ERK inhibition increases the sensitivity of TE-671 cells to the EGF, IGF-1, and NGF growth factors, presumably by reducing basal ERK activity, and to the BDNF growth factor, by increasing expression of the TrkB receptor.

[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.

Establishment of a FDC-P1 Murine Cell Line with Human KIT N822K Gene Overexpression.

The mechanism of resistance of leukemia cells to chemotherapeutic drugs remains poorly understood. New model systems for studying the processes of malignant transformation of hematopoietic cells are needed. Based on cytokine-dependent murine acute myeloid leukemia (AML) FDC-P1 cells, we generated a new cell line with ectopic expression of the KIT gene encoding mutant human receptor tyrosine kinase (N822K). We investigated the role played by overexpression of the mutant KIT in the survival of leukemia cells and their sensitivity to therapeutic drugs. We also generated a co-culture system consisting of FDC-P1 murine leukemia cells and a HS-5 human stromal cell line. Our data can be used for a further comprehensive analysis of the role of KIT N822K mutation in the cellular response to anti-leukemic drugs, growth factors, and cytokines. These data are of interest in the development of new effective therapeutic approaches to the treatment of acute leukemia.

[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.

The Role of TAL1 in Hematopoiesis and Leukemogenesis.

TAL1 (SCL/TAL1, T-cell acute leukemia protein 1) is a transcription factor that is involved in the process of hematopoiesis and leukemogenesis. It participates in blood cell formation, forms mesoderm in early embryogenesis, and regulates hematopoiesis in adult organisms. TAL1 is essential in maintaining the multipotency of hematopoietic stem cells (HSC) and keeping them in quiescence (stage G0). TAL1 forms complexes with various transcription factors, regulating hematopoiesis (E2A/HEB, GATA1-3, LMO1-2, Ldb1, ETO2, RUNX1, ERG, FLI1). In these complexes, TAL1 regulates normal myeloid differentiation, controls the proliferation of erythroid progenitors, and determines the choice of the direction of HSC differentiation. The transcription factors TAL1, E2A, GATA1 (or GATA2), LMO2, and Ldb1 are the major components of the SCL complex. In addition to normal hematopoiesis, this complex may also be involved in the process of blood cell malignant transformation. Upregulation of C-KIT expression is one of the main roles played by the SCL complex. Today, TAL1 and its partners are considered promising therapeutic targets in the treatment of T-cell acute lymphoblastic leukemia.

Lentiviral gene delivery to plasmolipin-expressing cells using Mus caroli endogenous retrovirus envelope protein.

Gene therapy is a promising method for treating malignant diseases. One of the main problems is target delivery of therapeutic genes. Here we show that lentiviral vector particles pseudotyped with Mus caroli endogenous retrovirus (McERV) envelope protein can be used for selective transduction of PLLP-expressing cells. As a therapeutic gene in McERV-pseudotyped vector particles we used miniSOG encoding the cytotoxic FMN-binding protein, which can generate reactive oxygen species under illumination. Significant cytotoxic effect (up to 80% of dead cells in population) was observed in PLLP-expressing cells transduced with McERV-pseudotyped vector particles and subjected to illumination. We demonstrated that the McERV-pseudotyped HIV-1 based lentiviral vector particles are an effective tool for selective photoinduced destruction of PLLP-expressing cells.

[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.

[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.

[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.

Silencing AML1-ETO gene expression leads to simultaneous activation of both pro-apoptotic and proliferation signaling.

The t(8;21)(q22;q22) rearrangement represents the most common chromosomal translocation in acute myeloid leukemia (AML). It results in a transcript encoding for the fusion protein AML1-ETO (AE) with transcription factor activity. AE is considered to be an attractive target for treating t(8;21) leukemia. However, AE expression alone is insufficient to cause transformation, and thus the potential of such therapy remains unclear. Several genes are deregulated in AML cells, including KIT that encodes a tyrosine kinase receptor. Here, we show that AML cells transduced with short hairpin RNA vector targeting AE mRNAs have a dramatic decrease in growth rate that is caused by induction of apoptosis and deregulation of the cell cycle. A reduction in KIT mRNA levels was also observed in AE-silenced cells, but silencing KIT expression reduced cell growth but did not induce apoptosis. Transcription profiling of cells that escape cell death revealed activation of a number of signaling pathways involved in cell survival and proliferation. In particular, we find that the extracellular signal-regulated kinase 2 (ERK2; also known as mitogen-activated protein kinase 1 (MAPK1)) protein could mediate activation of 23 out of 29 (79%) of these upregulated pathways and thus may be regarded as the key player in establishing the t(8;21)-positive leukemic cells resistant to AE suppression.

[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.

Transfer and Expression of Small Interfering RNAs in Mammalian Cells Using Lentiviral Vectors.

RNA interference is a convenient tool for modulating gene expression. The widespread application of RNA interference is made difficult because of the imperfections of the methods used for efficient target cell delivery of whatever genes are under study. One of the most convenient and efficient gene transfer and expression systems is based on the use of lentiviral vectors, which direct the synthesis of small hairpin RNAs (shRNAs), the precursors of siRNAs. The application of these systems enables one to achieve sustainable and long-term shRNA expression in cells. This review considers the adaptation of the processing of artificial shRNA to the mechanisms used by cellular microRNAs and simultaneous expression of several shRNAs as potential approaches for producing lentiviral vectors that direct shRNA synthesis. Approaches to using RNA interference for the treatment of cancer, as well as hereditary and viral diseases, are under active development today. The improvement made to the methods for constructing lentiviral vectors and the investigation into the mechanisms of processing of small interfering RNA allow one to now consider lentiviral vectors that direct shRNA synthesis as one of the most promising tools for delivering small interfering RNAs.

[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.