Dependence of expression of regulatory master genes of embryonic development in pancreatic cancer cells on the intracellular concentration of the master regulator PDX1.

Exogenous expression of the gene encoding the pancreatic master regulator PDX1 in cell lines with different degrees of differentiation of pancreatic cancer cells is accompanied by changes in the expression of known master genes involved in cancer progression. In BxPC3PDX+ cells, as compared to BxPC3PDX-, we detected an increased expression of the following genes: NKX6.1 (2 times), NR5A2 (2.5 times), KLF5 (1.8 times), ZEB1 (3 times), and ONECUT1 (1.3 times), as well as a decreased expression of MUC1 and SLUG genes (3 and 2 times, respectively). In PANC1PDX+ cells, as compared to the control PANC1PDX- cells, we detected a decreased expression of ISL1 (2 times) and an increased expressed of KRT8 (2 times) and MUC1 (by 30%). In the high-grade cell lines (including the BxPC3 line studied), the total content of sites containing the marks of active enhancers was higher than that in the low-grade cell lines (PANC1).

Enhanced Vector Design for Cancer Gene Therapy with Hierarchical Enhancement of Therapeutic Transgene Expression.

A set of vectors for Cre recombinase-dependent expression of the hybrid suicidal FCU1 transgene was constructed, including a two-plasmid system wherein the FCU1 and Cre transgenes reside in separate vectors, and single-plasmid variants in which a single plasmid bears both transgenes. To improve the safety profile and specificity in cancer gene therapy applications, as well as to ensure stable propagation of plasmids in bacterial cells, the Cre/LoxP system components were optimized. A bicistronic vector with the Cre expression cassette placed between the LoxP sites unidirectionally with FCU1 cDNA resulted in higher therapeutic efficiency compared with the double-plasmid system in an enzyme-prodrug suicide cancer gene therapy scheme. Therefore, the feasibility of a single-plasmid approach in the development of cancer gene therapy with hierarchical enhancement of therapeutic transgene expression has been demonstrated.

Structural and Functional Characterization of Recombinant Isoforms of the Lentil Lipid Transfer Protein.

The recombinant isoforms Lc-LTP1 and Lc-LTP3 of the lentil lipid transfer protein were overexpressed in E. coli cells. It was confirmed that both proteins are stabilized by four disulfide bonds and characterized by a high proportion of the α-helical structure. It was found that Lc-LTP1 and Lc-LTP3 possess antimicrobial activity and can bind fatty acids. Both isoforms have the ability to bind specific IgE from sera of patients with food allergies, which recognize similar epitopes of the major peach allergen Pru p 3. Both isoforms were shown to have immunological properties similar to those of other plant allergenic LTPs, but Lc-LTP3 displayed a less pronounced immunoreactivity.

Cancer specificity of promoters of the genes involved in cell proliferation control.

Core promoters with adjacent regions of the human genes CDC6, POLD1, CKS1B, MCM2, and PLK1 were cloned into a pGL3 vector in front of the Photinus pyrails gene Luc in order to study the tumor specificity of the promoters. The cloned promoters were compared in their ability to direct luciferase expression in different human cancer cells and in normal fibroblasts. The cancer-specific promoter BIRC5 and non-specific CMV immediately early gene promoter were used for comparison. All cloned promoters were shown to be substantially more active in cancer cells than in fibroblasts, while the PLK1 promoter was the most cancer-specific and promising one. The specificity of the promoters to cancer cells descended in the series PLK1, CKS1B, POLD1, MCM2, and CDC6. The bidirectional activity of the cloned CKS1B promoter was demonstrated. It apparently directs the expression of the SHC1 gene, which is located in a "head-to-head" position to the CKS1B gene in the human genome. This feature should be taken into account in future use of the CKS1B promoter. The cloned promoters may be used in artificial genetic constructions for cancer gene therapy.

[Enhancer activity of DNA fragments from FXYD5-COX7A region of human chromosome 19].

The enhancer activity of four previously identified within the one megabase region of human chromosome 19 DNA fragments was investigated. All four fragments had similar tissue-specific profile--maximum of enhancer activity was observed in HEK293 and minimum in HeLa cells. Enhancers obtained had pronounced specificity toward cytomegalovirus promoter compared with SV40 promoter. Functional dissection of one of the fragments (enhancer 14) demonstrated that only its inner 127 b.p. part possessed enhancer activity. The negative regulators, i.e. silencers or insulators are probably located in flanking regions of enhancer 14 and limit its effect on promoter. At the same time, enhancer activity of enhancer 14 depends on its orientation relative to promoter that isn't typical to majority of enhancer elements. Inner 127 b.p. fragment contains 11 transcription factor binding sites; 8 of them are factors that take part in immune system regulation. Enhancer 14 is located 500 b.p. upstream of transcription start site of TYROBP (DAP12) gene that codes for of T-killer cells activator protein and possibly functions as tissue-specific enhancer for this gene.

[Functional analysis of the HERV-K LTR residing in the KIAA1245/NBPF subfamily].

Long terminal repeats (LTRs) of human endogenous retrovihuses (HERs) might affect transcription regulation of neighboring genes. In our previous study, we showed that the solitary LTR residing in the KIAA1245/NBPF gene subfamily displayed high enhancer activity in a transformed embryonal carcinoma cell line Tera1. In this study, we performed a functional dissection of the LTR and studied its deletion series. Using transient transfection assay, we confirmed the ability of the LTR to drive the expression of the luciferase reporter gene in Teral cells. At the same time, in two other transformed cell lines tested, NGP and NT2/D1, the full-size LTR and its fragments showed no or low enhancer activity, thus demonstrating cell type specificity of the LTR enhancer activity. The functional dissection of the LTRrevealed a specific region within the U3 part appeared to be responsible for the enhancer properties. We showed that the identified enhancer was able to work in a highly cell type specific manner. The data obtained are in line with the hypothesis suggesting that KIAA1245/NBPF LTR may affect the regulation of the KIAA1245/NBPF subfamily genes transcription.

Studies on functional role of DNA methylation within the FXYD5-COX7A1 region of human chromosome 19.

We used the Rapid Identification of Genomic Splits technique to get a detailed methylation landscape of a 1-megabase-long human genome region (FXYD5-COX7A1, chromosome 19) in normal and tumor lung tissues and in the A549 lung cancer cell line. All three samples were characterized by an essentially uneven density of unmethylated sites along the fragment. Strikingly enough, the distribution of hypomethylated regions did not correlate with gene locations within the fragment. We also demonstrated that the methylation pattern of this long genomic DNA fragment was rather stable and practically unchanged in human lung cancer tissue as compared with its normal counterpart. On the other hand, the methylation landscape obtained for the A549 cell line (human lung carcinoma) in the USF2-MAG locus showed clear differences from that of the tissues mentioned above. A comparative analysis of transcriptional activity of the genes in this region demonstrated the general absence of direct correlation between methylation and expression, although some data suggest a possible role of methylation in the regulation of MAG expression through cis-regulatory elements. In total, our data provide new evidence for the necessity of revising currently prevailing views on the functional significance of methyl groups in genomic DNA.

[Study of transactivation effect on transcription by Tat-TAR-system of human immunodeficiency virus type 1 (HIV-1) in non-lymphoid cells HEK293 and Calu-1].

An important problem in the development of gene therapy approaches in oncology is the necessity of using promoters providing specific and high level of gene expression in tumor cells. To solve this problem, we used inducible system of gene expression regulation (Tat-TAR-system), which is utilized by human immunodeficiency virus (HIV). tat and tk-HSV genes, as well as a fragment of LTR HIV-1, were cloned in the retrovirus vector, tk-HSV gene was under control of the LTR HIV-1 fragment. Potential capacity of these constructions for transactivating tk-HSV gene transcription was studied. Basal expression level of this gene was defined in transient transfection of HEK293 cells. It was shown that specific transactivation of the tk-HSV gene was controlled by the LTR HIV-1 fragment in lung carcinoma cells Calu-1, permanently transfected by the tat gene construction. The effect of transactivation of tk-HSV transcription in Tat-TAR-system was demonstrated in Calu-1 cells in conditions of control of cancer-specific tat gene over BIRC5 promoter.

[Leiden, G20210A mutations in prothrombin gene and antiphospholipid antibodies in systemic lupus erythematosus and antiphospholipid syndrome]. / Mutatsii Leiden, G2021A v gene protrombina i antifosfolipidnye antitela pri sistemnoi krasnoi volchanke i antifosfolipidnom sindrome.

AIM: To estimate incidence rate of Leiden mutation in factor V gene, prothrombin gene mutation responsible for replacement of G for A in position 20210 (G20210A) of its 3'-end noncoding part in patients with systemic lupus erytheamtosus (SLE) and antiphospholipid syndrome (APS) and their relationships with antiphospholipid antibodies (aPL): lupus anticoagulant (LA) and anticardiolipin antibodies (aCL). MATERIALS AND METHODS: The trial included 33 patients (2 males and 31 females) aged 20-62 years (mean age 32.7 +/- 9.8 years). 29 patients suffered from SLE, 17 of them had also APS (criteria by G. R. V Hudhes). 3 patients had primary APS, 1 female had hemorrhagic vasculitis. IgG and IgM-aCL were detected by enzyme immunoassay. In revealing mutation, DNA was used isolated from peripheral blood by standard methods based on polymerase chain reaction (PCR). Primary screening of Leiden mutation was made according to Bertina et al. with Mnl I restrictase. The mutation was confirmed by an original technique with allele-specific primers. G20210A mutation in the prothrombin gene was determined with restrictase Taq I after introduction of artificial restriction site in PCR product. The patients were divided into 2 groups. Group 1 incorporated 12 patients with SLE without APS. Group 2--21 patients with APS (17 with SLE + APS, 3 with primary APS and 1 with hemorrhagic vasculitis). RESULTS: Patients of group 1 had neither thrombotic complications nor Leiden mutation. Two patients of group 2 had heterozygous Leiden mutation. Both females were aPL-positive and had previously recurrent thrombophlebitis. One of them had also recurrent disorders of cerebral circulation. None of the examinees had any G20210A mutations in the prothrombin gene. CONCLUSION: Detection of genetic defects in APS provide arguments in the dispute about the necessity, duration and choice of anticoagulant therapy. If patients with APL syndrome appeared to have besides aPL also genetic defects in coagulation, this will enable identification of patients at high risk of thrombosis which need permanent administration of anticoagulants among which only low-molecular ones or glycosaminoglycanes are indicated. Mutation in gene of factor V is absolute contraindication to phenilin and quamarines.

Control of bacteriophage phi X 174 gene expression by the transcription termination factor rho in vitro.

The effect of rho factor on bacteriophage phi X 174 gene expression was studied in a cell-free, DNA-dependent, RNA-directed protein synthesizing system (transcription-translation coupled system) using S30 extracts of wild-type and rho-mutant Escherichia coli cells. It has been found that in the presence of a functionally active and endogenous or added rho factor, only a limited number of the viral genes (A, B, and D) are expressed effectively, whereas the expression of genes, F, G, and H, which encode capsid proteins, is strongly inhibited. Mutational or thermal inactivation of rho factor results in a considerable activation of capsid protein synthesis and, at the same time, in some depression of the synthesis of gene A, B, and D products. From the results of this study it is suggested that phage phi X 174 has two classes of gene, late and early, which are separated on the chromosome by a rho-dependent transcription terminator. We propose that the expression of these classes is regulated by a termination-antitermination mechanism.