V. S. Prasolov

Altered Expression of the SEMA3B gene in Epithelial Tumors

Tumor-specific expression downregulation may be indicative of a gene’s involvement in tumor suppression. For instance, SEMA3B mRNA levels are decreased in many cell lines of small-cell and non-small cell lung cancer, and SEMA3B was shown to suppress the growth of the NSCLC cell line NCI-H1299 and tumor formation in immunodeficient mice. In this work, SEMA3B expression levels were determined in epithelial tumors of different localizations. In cell lines of renal, breast, and ovarian cancer, SEMA3B mRNA levels were frequently (4/11, 36%) decreased as much as 10–250-fold according to semiquantitative RT-PCR assay. SEMA3B expression levels were also determined in primary tumor extracts of kidney, lung, breast, ovarian, and colorectal cancer. In clear cell renal cell carcinoma, SEMA3B expression was decreased 5–1000-fold in 25 of 51 extracts (49%) compared to 5/51 (10%) extracts with increased mRNA levels; the result was highly significant: P < 0.0001 by Fisher’s exact test. SEMA3B was frequently downregulated in ovarian (5/16, 31% vs. 2/16, 12%) and colorectal cancer (6/11, 54% vs. 2/11, 18%). These results suggest that SEMA3B is involved in the suppression of kidney, ovarian, and colon tumor growth.

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.

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.

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].

Lentiviral vector-based assay system for quantitative detection of intracellular translocations of recombinant proteins

An enzymatic assay system was developed to quantify the distribution of recombinant proteins over various cell structures. The system takes advantage of α-complementation of β-galactosidase. The large ω fragment of β-galactosidase is expressed in predefined cell structures with the aid of attached protein localization signals. The resulting reporter cell lines are infected with a second construct expressing a target protein fused with the shorter α fragment of β-galactosidase. The physical proximity of the two recombinant proteins carrying the β-galactosidase fragments results in the reconstitution of an active enzyme, and its activity is measured with a plate reader. The recombinant constructs are based on lentiviral vectors and can be rapidly and efficiently introduced into cells by infection with stocks of lentivirus particles. The efficiency of the system was demonstrated with the FOXO3A transcription factor, which shuttles between the cytoplasm and nucleus in the model colon carcinoma cell line RKO.

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

Envelope proteins E1 and E2 of the hepatitis C virus (HCV) play a major role in the life cycle of a virus. These proteins are the main components of the virion and 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 glycoprotein heterodimer, in protein folding, and in viral entry. The effects of N-linked glycosylation of HCV protein E1 on the assembly of structural proteins were studied using site-directed mutagenesis in a model system of Sf9 insect cells producing three viral structural proteins with the formation of virus-like particles due to the baculovirus expression system. The removal of individual N-glycosylation sites in HCV protein E1 did not affect the efficiency of its expression in insect Sf9 cells. The electrophoretic mobility of E1 increased with a decreasing number of N-glycosylation sites. The destruction of E1 glycosylation sites N1 or N5 influenced the assembly of the noncovalent E1E2 glycoprotein heterodimer, which is the prototype of the natural complex within the HCV virion. It was also shown that the lack of glycans at E1 sites N1 and N5 significantly reduced the efficiency of E1 expression in mammalian HEK293 T cells.

Functional characterization of two novel splicing mutations of glucokinase gene associated with maturity-onset diabetes of the young type 2 (MODY2)

Two novel mutations in the glucokinase gene (GCK) have been identified in patients with maturity-onset diabetes of the young type-2 (MODY2), i.e., a C-for-G substitution at position −1 of the acceptor splice site of intron 7 (c. 864-1G>C) and a synonymous c.666C>G substitution (GTC>GTG, p.V222V) at exon 6. An analysis of the splicing products obtained upon the transfection of human embryonic HEK293 cells with GCK minigene constructs carrying these mutations showed that both substitutions impaired normal splicing. As a result of c.864-1G>C, the usage of the normal acceptor site was blocked, which activated cryptic acceptor splice sites within intron 7 and generated several aberrant RNAs containing fragments of intron 7. The synonymous substitution c.666C>G created a novel donor splice site in exon 6, which results in the formation of an abnormal GCK mRNA with a 16-nucleotide deletion in exon 6. In vitro experiments on minigene splicing confirmed the inactivating effect of these mutations on glucokinase gene expression.

Dynamics of penetration of “Rigid” nanostructures of 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 thermal neutron capture by some nonradioactive elements (e.g., Gd157), which results in the sub-sequent emission of electrons and gamma rays. An effective instrument for delivering gadolinium into tumor tissue are “rigid” nanostructures (NSs) based on double-stranded DNA complexes with gadolinium (NS-Gd). The local concentration of Gd in these nanostructures may reach 40%. To optimize the process of neutron capture therapy, it is very important to investigate possible mechanisms of the penetration of NS-Gd particles into tumor cells. In this work, the dynamics of interaction between NS-Gd and cultivated Chinese hamster ovary cells (CHO) was studied by confocal and electron microscopy. NS-Gd were shown to be able to enter CHO cells. This process started after about 1 h of incubation. After 6 h, NS-Gd particles were detected in almost all cells. A further increase in the incubation time did not lead to significant changes in cell morphology, although the amount of NS-Gd inside cells continued to increase. The plasma membranes of the cells remained intact. Once entering the cells, NS-Gd particles remained there for a long time. The data show that NS-Gd has relatively low toxicity and suggest that the presence of NS-Gd in tumor cells does not prevent their division. The data are important for improving the efficiency of the method of neutron-capture therapy.