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A hybrid 6XRE-hTERT promoter consisting of the hTERT tumor-specific promoter and six copies of the XRE element from the CYP1A1 human gene promoter was created. Using a human lung cancer cells as a model, we showed that XRE elements in the hybrid promoter greatly increase the activity of the hTERT promoter and ensure the reporter gene transcriptional activation in response to the treatment of the cells with the AhR ligand benzo(a)pyrene. However, similar effects were also observed in normal human bronchial epithelial cells HBEpC, which indicates the loss of the tumor-specific activity by the 6XRE-hTERT hybrid promoter. XRE elements can be used for nonspecific transcription enhancement but are unsuitable for the creation of tumor-specific promoters with enhanced activity.

Tumor-specific promoters and cis-regulatory genetic elements are used for transcriptional control of therapeutic transgene expression in cancer gene therapy. HRE (hypoxia response element) and ARE (anti-oxidant response elements) cis-regulatory elements are targets for HIF1 and Nrf2 transcriptional factors, respectively, and mediate activation of gene transcription in a response to hypoxia and oxidative stress, characteristic of most solid tumors. Due to these features HREs and AREs are used in genetic constructs for cancer gene therapy to provide tumor-specific therapeutic transgene expression or replication of oncolytic adenovi-ruses. In this work on the basis of the tumor-specific promoter hTERT we have constructed hybrid promoters carrying combinations of HRE and ARE. We showed that upon imitation of hypoxia in human lung cancer cell lines the activity of the hybrid promoter HRE-ARE-hTERT is substantially higher compared to promoters carrying only ARE or HRE. Using in vitro suicide cancer gene therapy with the CD: UPRT/5-FC (cytosine deaminase; uracil phosphoribosyl transferase/5-fluorocytosine) enzyme-prodrug system as a model we showed an enhancement of the cytotoxic effect on human lung cancer cells upon imitation of hypoxia when cytosine deaminase: uracil phosphoribosyl transferase was expressed under the control of the HRE-ARE-hTERT promoter compared to HRE-hTERT and ARE-hTERT promoters. The novel hybrid promoter HRE-ARE-hTERT could be used for transcriptional targeting of therapeutic transgene expression or oncolytic adenovirus replication upon development of novel anti-cancer gene therapeutics.

Atypical RhoV GTPase (Chp/Wrch-2) is a member of the human Rho GTPase family, which belongs to the superfamily of Ras-related small GTPases. The biological functions of RhoV, regulation of its activity, and mechanisms of its action remain largely unexplored. Rho GTPases regulate a wide range of cellular processes by interacting with protein targets called effectors. Several putative RhoV effectors have been identified, including protein kinases of the Pak (p21-activated kinase) family: Pak1, Pak2, Pak4, and Pak6. RhoV GTPase activates Pak1 protein kinase and simultaneously induces its ubiquitin-dependent degradation. Pak1 regulates E-cadherin localization at adherens junctions downstream of RhoV during gastrulation in fish. The effector domain of RhoV mediates its binding to the CRIB (Cdc42/Rac1 interactive binding) motif in the N-terminal p21-binding domain (PBD) of Pak6 protein kinase. The role of the RhoV effector domain in mediating interaction with Pak1 has not been studied. This study has identified mutations in the effector domain of RhoV GTPase (Y60K, T63A, L65A, and D66A) that impair its interaction with Pak1 in the GST-PAK-PBD pull-down assay and coimmunoprecipitation. Our results suggest that the effector domain of RhoV mediates its binding to Pak1, complementing the current view of the molecular basics of RhoV binding to effectors of the Pak family. These data lay the basis for further studies on the role of Pak1 in RhoV-activated signaling pathways and cellular processes.

Nowadays increasing the accuracy and reliability of measuring natural gas flow-rate is an urgent problem. In article is offered to increase the reliability of measuring the gas volume and flow-rate in a wide range of flow-rates by constructing a measuring complex with a few measuring devices based on different principles of action. The most rational structure of the device or complex with reduction the influence of elements on the output error signal can be selected by structural methods. The results of investigation of the natural gas flow-rate measuring complex and evaluation the level of mutual influence of the design parameters on the metrological characteristics of the whole complex are given. Particular attention is paid to determining the definite location of serial measuring devices along the length of the instrumentation system, which provides a minimum loss of gas flow energy and high accuracy of measuring its volume and volume rate. The optimal dimensional geometric form of the measurement node in measuring complex with different physical types of primary transducers will provide high accuracy and reliability of measurement in a wide range with minimization of pressure loss and flowmeters price.

The use of tumor-specific microRNA loss to inhibit transgene expression in normal cells is considered as a way to increase the specificity of gene-therapeutic antitumor drugs. This method assumes the introduction of recognition sites of suppressed in tumor cells microRNAs into transgene transcipt. In the presented work, the efficiency of the strategy for providing the tumor specificity of transgene expression depending on parameters of microRNA expression in normal and tumor cells was studied. It was established that microRNA suppression in tumor cells and the determination of absolute microRNA levels in tumor and normal cells are not sufficient for the adequate estimation of the possibility of specific microRNA usage in the scheme of cancer gene therapy, and particularly do not allow to exclude a significant decrease in the efficiency of the gene-therapeutic drug upon the introduction of microRNA recognition sites. These parameters are only suitable for the preliminary selection of microRNA. The effect of introduction of microRNA recognition sites on transgene expression level in target tumor cells should be validated experimentally. It is suggested that this should be done directly in the cancer gene therapy scheme with monitoring of the therapeutic transgene activity.

Atypical RhoV GTPase (Chp/Wrch-2) is a member of the human Rho GTPase family, which belongs to the superfamily of Ras-related small GTPases. The biological functions of RhoV, regulation of its activity, and mechanisms of its action remain largely unexplored. Rho GTPases regulate a wide range of cellular processes by interacting with protein targets called effectors. Several putative RhoV effectors have been identified, including protein kinases of the Pak (p21-activated kinase) family: Pak1, Pak2, Pak4, and Pak6. RhoV GTPase activates Pak1 protein kinase and simultaneously induces its ubiquitin-dependent degradation. Pak1 regulates E-cadherin localization at adherens junctions downstream of RhoV during gastrulation in fish. The effector domain of RhoV mediates its binding to the CRIB (Cdc42/Rac1 interactive binding) motif in the N-terminal p21-binding domain (PBD) of Pak6 protein kinase. The role of the RhoV effector domain in mediating interaction with Pak1 has not been studied. This study has identified mutations in the effector domain of RhoV GTPase (Y60K, T63A, L65A, and D66A) that impair its interaction with Pak1 in the GST-PAK-PBD pull-down assay and coimmunoprecipitation. Our results suggest that the effector domain of RhoV mediates its binding to Pak1, complementing the current view of the molecular basics of RhoV binding to effectors of the Pak family. These data lay the basis for further studies on the role of Pak1 in RhoV-activated signaling pathways and cellular processes.

The presence of introns is often required for efficient transgene expression. The use of full-length genes for transgenesis is associated with technical difficulties due to the large size of the genetic construct. To solve this problem, we recently suggested a universal design of small artificial introns that ensures efficient splicing. However, the insertion of more than one intron into cDNA might result in the aberrant splicing of the minigene with exon skipping. Here, we showed that the insertion of two artificial introns of universal design into cDNA resulted in a splicing pattern that corresponds to the excision of each intron with an exon between them remaining in the transcript. No transcript formation with exon skipping was detected. Therefore, the developed design of small artificial introns assures splicing solely between the donor and the acceptor splice sites of each single intron and results in the generation of a correct transcript from minigene pre-mRNA. These findings enable the construction of minigenes for transgenesis with more than one artificial intron, with no additional cis-elements required to prevent aberrant splicing.

Russian legislation lags behind the rapid developments witnessed in genetic engineering. Only a scientifically based and well-substantiated policy on the place of organisms that are created with the use of genetic engineering technologies and an assessment of the risks associated with them could guarantee that the breakthroughs achieved in modern genetic engineering technologies are effectively put to use in the real economy. A lack of demand for such breakthroughs in the practical field will lead to stagnation in scientific research and to a loss of expertise.

Introns can frequently enhance transgene expression, and sometimes they are absolutely substantial. Based on an analysis of murine genes, in which mRNA does not have alternative splicing, a universal design of the efficiently spliced artificial introns of small sizes has been proposed. These introns are shown to be efficiently spliced in CHO cells from hamster ovaries. The proposed strategy can be used to include introns in cDNA, which would elevate the production of recombinant proteins in cell culture, as well as in transgenic animals.

The use of tumor-specific microRNA loss to inhibit transgene expression in normal cells is considered as a way to increase the specificity of gene-therapeutic antitumor drugs. This method assumes the introduction of recognition sites of suppressed in tumor cells microRNAs into transgene transcipt. In the presented work, the efficiency of the strategy for providing the tumor specificity of transgene expression depending on parameters of microRNA expression in normal and tumor cells was studied. It was established that microRNA suppression in tumor cells and the determination of absolute microRNA levels in tumor and normal cells are not sufficient for the adequate estimation of the possibility of specific microRNA usage in the scheme of cancer gene therapy, and particularly do not allow to exclude a significant decrease in the efficiency of the gene-therapeutic drug upon the introduction of microRNA recognition sites. These parameters are only suitable for the preliminary selection of microRNA. The effect of introduction of microRNA recognition sites on transgene expression level in target tumor cells should be validated experimentally. It is suggested that this should be done directly in the cancer gene therapy scheme with monitoring of the therapeutic transgene activity.

Cancer-testis (CT) antigens are normally expressed mostly in human germ cells, there is also an aberrant expression in some tumor cells. This expression profile makes them potential tumor growth biomarkers and a promising target for tumor immunotherapy. Specificity of CT genes expression in oral malignant and potentially malignant diseases, e.g. oral leukoplakia, is not yet studied. Data on CT genes expression profile in leukoplakia would allow developing new diagnostic methods with potential value for immunotherapy and prophylaxis of leukoplakia malignization. In our study we compared CT genes expression in normal oral mucosa, oral leukoplakia and oral squamous cell carcinoma. We are the first to describe CT genes expression in oral leukoplakia without dysplasia. This findings make impossible differential diagnosis of oral leukoplakia and squamous cell carcinoma on the basis of CT genes expression. The prognostic value of CT genes expression is still unclear, therefore the longitudinal studies are necessary.

Inactivation of tumor suppressors and activation of protooncogenes are critical events in malignant cell transformation and tumor progression. Pdcd4 encodes a protein with tumor suppressor functions, which accounts for an increased interest to Pdcd4 as a potential diagnostic and prognostic marker, as well as a target for antineoplastic therapy. This review summarizes well-known properties and functions of Pdcd4 tumor suppressor and mechanisms of its regulation in tumor cells. It is also focused to the role of Pdcd4 in cellular transformation and tumor progression, as well as on its potential practical application in oncology.

VARP is a novel VPS9 domain-containing protein which acts as a guanine nucleotide exchange factor for small GTPases Rab21 and Rab5, regulators of early endocytosis. However, the molecular mechanisms underlying VARP activity regulation and intracellular localization remain unknown. Using protein interaction cloning in yeast we isolated multiadaptor proteins of 4.1 protein family and RanBP9 as putative VARP interaction partners. The interactions revealed might be important for proper intracellular localization of VARP and its functions in early endocytosis.

Changes in WIFI expression, an extracellular inhibitor of Wnt pathway, in non-small cell lung carcinomas were analyzed. Frequent (67% cases) suppression of WIFI transcript in non-small cell lung carcinomas were found. Our results, together with previously published data, suggest that inhibition of WIFI expression often occurs in squamous cell carcinomas and is less typical of adenocarcinomas. It was also found that a decrease in the WIFI transcript in tumors is parallel to concomitant suppression of the WIFI protein level. Our results provide further evidence that the WIFI suppression is a frequent event in the lung carcinogenesis, which might lead to disregulation of Wnt signaling pathway and contribute to tumor progression.

Alterations in the c-Met/HGF system are frequently observed in various types of tumors and can directly influence tumor progression. In particular, the c-Met/HGF system can influence progression of lung tumors. The altered c-Met and HGF expression occurs in non-small-cell lung carcinomas. In this work we analyzed changes in c-Met and HGF expression in non-small-cell lung carcinomas by comparing expression levels with those in adjacent non-malignant tissues using semi-quantitative PCR. The c-Met transcript was detected in 50% of squamous cell carcinoma samples with an elevated level observed in 2 out of 25 cases (8%). HGF expression was detected only in two squamous cell carcinomas. At the same time, the c-Met transcript was observed in all 5 studied adenocarcinoma samples with an increased level compared to adjacent non- malignant tissue in 3 cases. HGF transcript was found in 2 adenocarcinoma samples. Therefore, c-Met rather than HGF transcript is frequently observed in non-small-cell lung carcinomas, especially in adenocarcinomas. According to the results of other studies, the c-Met transcript can serve as an indicator of the aggressive behavior and progression of non-small-cell lung carcinomas.

MAK-V/Hunk is a recently isolated MARK/Par-1-related mammalian protein kinase with yet unknown function. To investigate transcriptional regulation of the mouse mak-v/Hunk gene, we isolated genomic fragment of the mouse mak-v/Hunk promoter region. The mak-v/Hunk promoter has no typical TATA box or CAAT box, is GC-rich and contains CpG-island. Amplification of cDNA ends suggested that transcription initiation site is 156 nt upstream translation initiation site. The 5'-flanking region of the mak-v/Hunk gene was ligated to luciferase reporter gene and possessed functional promoter activity. Luciferase assay with a series of truncated 5'-flanking regions demonstrated the region between nt -508 and -347 has a pronounced stimulating effect on transcription activity. In addition, our data suggest that mak-v/Hunk promoter region might be a target for CpG methylation.

We propose a system for detection of overproduction of protein kinase MAK-V/Hunk in tumours. MAK-V/ Hunk overproduction is observed in about 50% breast carcinomas. Positive staining is obseved in tumour cells only and has mainly cytoplasmic characteristics. Increased production of MAK-V/Hunk does not correlate with histological type of carcinoma, metastasizing, steroid receptor status (estrogen and progesterone), proliferative activity. Tumours positive for MAK-V/ Hunk were more frequently observed in c-erbB2-positive (3+) tumours than in c-erbB2-negative ones. Overproduction of MAK-V/Hunk in human breast cancer may be used fore more precise molecular typing of this tumour, in particular in case of c-erbB2-positive tumors, however, diagnostic-prognostic value of this new molecular marker needs further studies.

Identification of interaction partners opens a way to direct functional characterization of proteins. Several cDNAs coding for potential partners of protein kinase MAK-V/Hunk were isolated using two-hybrid cloning in yeast. Based on the partner properties, MAK-V/Hunk was assumed to play a role in tumorigenesis and tumor progression. With the previous results of two-hybrid cloning, MAK-V/Hunk was shown to participate in vesicular transport.

Production of gamma-interferon was increased considerably by priming with homologous gamma- or alpha-interferons as well as by T-activin or vitamins of group B. The factors under study were effective both in the population of mononuclears and in the total fraction of nucleated cells of the donor blood. The maximal yields of gamma-interferon, up to 10,000 IU/ml, could be obtained by priming with gamma-interferon in a dose of 5 IU/ml or alpha-interferon in a dose of 1000 IU/ml. T-activin and vitamin B12 also enhanced production 8-10-fold. The combined use of priming, T-activin, and vitamin B12 in various combinations gave no further increase in the yield of activity.

The antitoxic activity of leucocytic injection interferon I and immune interferon was shown in experimental erythrocyte hemolysis in the presence of staphylococcus alpha-toxin. The antitoxic effect was directly proportional to the interferon concentration in the medium and inversely proportional to the toxin concentration. Neutralization of the antiviral activity of leucocytic interferon did not lower its antitoxic effect. The highly purified and concentrated preparation of leucocytic injection interferon I and recombinant interferon had no antitoxic effect.

The highest yields of gamma-interferon activity were obtained by using a fraction of mononuclears recovered from freshly collected donor blood in ficoll-verografin density gradient without using hemolysis. Unification of mononuclears from individual donors into a common pool stimulated interferon production. Staphylococcal enterotoxins A and B, concanavalin A, and lentyl-lectin were found to be the most effective inducers. Immobilization of inducers on neutral carriers reduced their effectiveness. Upon induction with lectin the synthesis was complete within 24 hours, and with enterotoxin in 3 days. In the latter instance the synthesis dynamics was of a two-phase nature. Gamma-interferon produced the antiviral condition later (in 10 hours) than alpha-interferon.