[Combination of ARE and HRE cis- Regulatory Elements Elevates the Activity of Tumor-Specific hTERT Promoter].

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.

[Human Antithrombin III Minigene with an Optimized Splicing Pattern].

Antithrombin III (AT3) belongs to the superfamily of serine protease inhibitors (serpins) and is a major anticoagulant in physiological conditions. Based on SERPINC1 gene, a minigene coding for human AT3, which is valuable for medicine and biotechnology, was constructed by minimizing the size of lengthy introns and preserving the splicing site-flanking sequences. An analysis of the minigene splicing pattern identified one correct AT3 transcript and two alternatively spliced transcripts, which formed either due to minigene exons 2 and 3 skipping or an aberrant exon insertion via splicing at cryptic splicing sites in intron 1 of the minigene. Site-directed mutagenesis of the cryptic splicing sites successfully optimized the splicing pattern of the AT3 minigene to completely prevent the generation of the alternative transcripts. The presence of the cryptic splicing sites in intron 1 of the minigene was confirmed with Human Splicing Finder v. 3.1 software, thus demonstrating that putative alternative splicing sites are possible to identify in minimized or hybrid introns of minigenes and to eliminate via mutagenesis before experimentally testing the minigene splicing patterns. The approach to the design of minigenes together with the bioinformatical analysis of the nucleotide sequences of minigene introns can be used to construct minigenes in order to generate transgenic animals producing economically valuable proteins in the milk.

Xenobiotic Response Elements (XREs) from Human CYP1A1 Gene Enhance the hTERT Promoter Activity.

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.

[Mutations in the Effector Domain of RhoV GTPase Impair Its Binding to Pak1 Protein Kinase].

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.

[Insertion of Multiple Artificial Introns of Universal Design into cDNA during Minigene Construction Assures Correct Transgene Splicing].

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.

Neonatal Lethality and Inflammatory Phenotype of the New Transgenic Mice with Overexpression of Human Interleukin-6 in Myeloid Cells.

To model human interleukin-6 (hIL-6) associated diseases, unique mice with transgenic overexpression of human IL-6 and reporter fluorescent protein EGFP in cells of macrophage-monocyte lineage were generated using loxP-Cre system. High level of hIL-6 production by macrophages and monocytes, as confirmed in vitro in primary culture of bone marrow-derived macrophages, in vivo resulted in early postnatal death in vivo, presumably, due to the effect of overexpression of hIL-6 on hematopoiesis.

[Optimal Artificial Mini-Introns for Transgenic Expression in the Cells of Mice and Hamsters].

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.

[Selection of microRNA for providing tumor specificity of transgene expression in cancer gene therapy].

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.

mTOR-dependent transcriptional repression of Pdcd4 tumor suppressor in lung cancer cells.

Programmed cell death 4 (Pdcd4) tumor suppressor is frequently lost in tumors of various origins including lung cancer, and its loss contributes to tumor progression. However molecular mechanisms underlying Pdcd4 suppression in lung cancer cells remain largely unexplored. Here we investigated molecular mechanisms of Pdcd4 suppression in lung cancer cells. Besides enhanced mTOR-dependent proteasomal degradation of Pdcd4 protein, we found that Pdcd4 transcription is negatively regulated by mTOR signaling, and localized cis-acting element in Pdcd4 promoter responsible for this effect. In conclusion, we described a novel molecular mechanism of Pdcd4 suppression in cancer cells consisting from mTOR signaling-dependent transcriptional repression of Pdcd4.

Characterization of Rabaptin-5 γ isoform.

Rab GTPases are key regulators of intracellular membrane traffic acting through their effector molecules. Rabaptin-5 is a Rab5 effector in early endosome fusion and connects Rab5- and Rab4-positive membrane compartments owing to its ability to interact with Rab4 GTPase. Recent studies showed that Rabaptin-5 transcript is subjected to extensive alternative splicing, thus resulting in expression of Rabaptin-5 isoforms mostly bearing short deletions in the polypeptide chain. As interactions of a Rab GTPase with different effectors lead to different responses, functional characterization of Rabaptin-5 isoforms becomes an attractive issue. Indeed, it was shown that Rab GTPase effector properties of Rabaptin-5 and its α and δ isoforms are different. This work focused on another Rabaptin-5 isoform, Rabaptin-5γ. Despite its ability to interact with Rab5, endogenously produced Rabaptin-5γ was absent from early endosomes. Rather, it was found to be tightly associated with trans-Golgi network and partially localized to a Rab4-positive membrane compartment. The revealed intracellular localization of Rabaptin-5γ indicates that it is not involved in Rab5-driven events; rather, it functions in other membrane transport steps. Our study signifies the role of alternative splicing in determination of functional activities of Rab effector molecules.

Pak6 protein kinase is a novel effector of an atypical Rho family GTPase Chp/RhoV.

Chp/RhoV is an atypical Rho GTPase whose functions are far from being fully understood. To date several effector proteins of Chp have been identified, including p21-activated kinases Pak1, Pak2, and Pak4. Using a yeast two-hybrid system and co-immunoprecipitation, here we show that another p21-activated kinase, Pak6, is a novel Chp-binding protein. Interaction between Chp and Pak6 depends on the activation state of the GTPase, suggesting that Pak6 is an effector protein for Chp. Point mutations in the effector domain of Chp or in the CRIB motif of Pak6 significantly impair the interaction between Chp and Pak6 upon co-immunoprecipitation, suggesting that the binding interface involves the effector domain of Chp and the CRIB motif in Pak6. We found that Chp does not affect the phosphorylation status of the S560 residue in the catalytic domain of Pak6 when Chp and Pak6 are co-expressed in HEK293 cells. Therefore, similarly to Cdc42, Chp is not likely to activate Pak6. In NCI-H1299 cells, Chp co-localizes with Pak6 on vesicular structures in activation state-dependent manner. Taking the data together, we report here the identification of p21-activated kinase Pak6 as a novel effector of the atypical Rho GTPase Chp. Our data suggest further directions in elucidating biological functions of these proteins.

Nanoantibodies for detection and blocking of bioactivity of human vascular endothelial growth factor A(165).

Nanoantibodies (single-domain antibodies, nanobodies) derived from noncanonical single-chain immunoglobulins provide an attractive tool for in vitro and in vivo diagnostics as well as for development of targeted drugs for clinical use. Nanoantibodies against several clinically important targets have been developed and are actively investigated. However, no development of nanoantibodies against vascular endothelial growth factor VEGF-A(165) has been reported. We describe here the generation of nanoantibodies derived from single-chain Bactrian camel immunoglobulins directed against VEGF-A(165). We demonstrate that these nanoantibodies are suitable for enzyme-linked immunoassay to quantify human VEGF-A(165) as well as for blocking its activity. Our results provide a basis for diagnostic kit development for quantification of VEGF-A(165), which emerges as a biomarker useful in various pathological conditions. In addition, the nanoantibodies might be used for development of therapeutic molecules targeting VEGF-A(165)-dependent pathological neoangiogenesis.

[Study of testicular cancer gene expression in samples of oral leukoplakia and squamous cell carcinoma of the mouth].

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.

Interaction between MAK-V protein kinase and synaptopodin.

MAK-V protein kinase (also known as HUNK) was discovered more than decade ago but its functions and molecular mechanisms of action still remain mostly unknown. In an attempt to associate MAK-V with particular chains of molecular events, we searched for proteins interacting with the C-terminal domain of MAK-V protein kinase. We identified synaptopodin as a protein interaction partner for MAK-V and confirmed this interaction in various ways. Because synaptopodin is important for dendritic spine formation and plays a role in synaptic plasticity, our results might have significant impact on future studies for understanding the role of MAK-V in cells of the nervous system.

[Pdcd4 tumor suppressor: properties, functions, and their application to oncology].

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.

Production of Recombinant Proteins in the Milk of Transgenic Animals: Current State and Prospects.

The use of transgenic animals as bioreactors for the synthesis of the recombinant proteins secreted into milk is a current trend in the development of biotechnologies. Advances in genetic engineering, in particular the emergence of targeted genome editing technologies, have provided new opportunities and significantly improved efficiency in the generation of animals that produce recombinant proteins in milk, including economically important animals. Here, we present a retrospective review of technologies for generating transgenic animals, with emphasis on the creation of animals that produce recombinant proteins in milk. The current state and prospects for the development of this area of biotechnology are discussed in relation to the emergence of novel genome editing technologies. Experimental and practical techniques are briefly discussed.

[Multiadaptor 4.1 and RanBP9 protein family members as putative interaction partners for VARP, a Rab21 GTPase guanine nucleotide exchange factor].

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.

[c-Met and HGF expression in non-small-cell lung carcinomas].

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.

[Suppression of WIFI transcript and protein in non-small cell lung carcinomas].

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.

A Novel Hybrid Promoter ARE-hTERT for Cancer Gene Therapy.

describe a novel hybrid tumor-specific promoter, ARE-hTERT, composed of the human TERT gene promoter (hTERT) and the antioxidant response element (ARE) from the human GCLM gene promoter. The hybrid promoter retains the tumor specificity of the basal hTERT promoter but is characterized by an enhanced transcriptional activity in cancer cells with abnormal activation of the Nrf2 transcription factor and upon induction of oxidative stress. In the in vitro enzyme-prodrug cancer gene therapy scheme, ARE-hTERT promoter-driven expression of CD : UPRT (yeast cytosine deaminase : uracil phosphoribosyltransferase) chimeric protein induced a more pronounced death of cancer cells either upon treatment with 5-fluorouracil (5FC) alone or when 5FC was combined with chemotherapeutic drugs as compared to the hTERT promoter. The developed hybrid promoter can be considered a better alternative to the hTERT promoter in cancer gene therapy schemes.