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Cancer-associated fibroblasts (CAF) are attractive therapeutic targets in the tumor microenvironment. The possibility of using CAFs as a source of therapeutic molecules is a challenging approach in gene therapy. This requires transcriptional targeting of transgene expression by cis-regulatory elements (CRE). Little is known about which CREs can provide selective transgene expression in CAFs. We hypothesized that the promoters of FAP, CXCL12, IGFBP2, CTGF, JAG1, SNAI1, and SPARC genes, the expression of whose is increased in CAFs, could be used for transcriptional targeting. Analysis of the transcription of the corresponding genes revealed that unique transcription in model CAFs was characteristic for the CXCL12 and FAP genes. However, none of the promoters in luciferase reporter constructs show selective activity in these fibroblasts. The CTGF, IGFBP2, JAG1, and SPARC promoters can provide higher transgene expression in fibroblasts than in cancer cells, but the nonspecific viral promoters CMV, SV40, and the recently studied universal PCNA promoter have the same features. The patterns of changes in activity of various promoters relative to each other observed for human cell lines were similar to the patterns of activity for the same promoters both in vivo and in vitro in mouse models. Our results reveal restrictions and features for CAF transcriptional targeting.

The expression levels of the SOX9 gene in fetal, postnatal, and neoplastic pancreatic tissues were compared. In the fetal pancreatic samples, the mean relative level of the SOX9 gene expression was 8 times greater than the normal level. The tumor samples were divided into three groups depending on the SOX9 expression level. The first group showed a 6.5-fold increased expression level of SOX9 with respect to the normal one. The second and normal groups had approximately equal levels expression. The third group showed a 25-fold decreased expression level of SOX9. The discrepancy in the SOX9 expression, associated with the predominance of different functions of this master gene, depends on the poorly predictable individual factors and indicates that SOX9 should be excluded from the potential diagnostic biomarkers of pancreatic cancer.

Naturally occurring positively charged proteins can be promising carriers for nucleic acid transport in gene therapy. The most attractive alternative among them is histones. In this work, we describe expression and purification of recombinant human histones H2A and H2B and of chimeric histone H2A with HIV-1 TAT fragment (TAT-peptide). The proposed method of purification of histone proteins can significantly reduce the content of bacterial endotoxins in the target preparation, which makes it possible to use these proteins in in vivo experiments. The transfection ability of plasmid DNA complexes with core histones H2A and H2B and the chimeric histone was demonstrated. A highly specific and efficient transfection of human HT1080 cell line with the use of histones H2A and H2B was detected, whereas transfection by plasmid DNA complexes with chimeric H2A-TAT protein was observed for many cell lines.

The expression level of six transcription factor genes and the content of their protein products in five pancreatic cancer cell lines with parallel control of expression of three marker genes reflecting epithelial or mesenchymal state of cells was investigated. Cell lines MIA PaCa-2 and Capan-2 represented the best models of quasi-mesenchymal and epithelial, respectively, types of progression of the pancreatic ductal adenocarcinoma, according to the content of E-cadherin and vimentin and the expression of KLF5 and ZEB1 transcription factors.

Despite substantial progress in understanding the mechanisms of carcinogenesis and fighting oncology diseases, cancer mortality remains rather high. Therefore, there is a striving to reduce this mortality to the level determined by endogenous biological factors. The review analyzes the mutations that lead to cell malignant transformation and describes the contribution that self-renewal of adult tissues makes to tumorigenesis. Cancer progression is considered as a development of a complicated system where cells mutate, evolve, and are subject to selection. Cancer paradoxes are described in conclusion.

Pancreatic cancer is one of the most aggressive tumor types characterized by chemotherapy resistance and high metastatic activity. Recent studies revealed new genes, which are likely to be actively involved in the regulation of the processes occurring in the pancreas, as well as in the development of cancer in this organ. This review is devoted to the description of one of the recently revealed genes, KLF5, which seems to be a promising target for therapeutic intervention in the most widespread type of pancreatic cancer, ductal adenocarcinoma.

This review was devoted to the use of the versatile component oftumoral stroma (fibroblast activation protein, FAP) as a target of the versatile tumor therapy. The tumor is a coevolution system, which includes the microenvironment or reactive stroma differing from the normal tissue by the phenotypic and genotypic features. Important elements of the tumor microenvironment are cancer-associated fibroblasts (CAFs), which contain typical marker FAP (serine proteinase with the enzymatic activity of dipeptidyl peptidase and endopeptidase). According to the literature, more than 90% of tumors contain FAP-positive activated fibroblasts. FAP is virtually absent in normal tissues, but it is present in the embryonic and tumor tissues, which makes it a selective and versatile model. In this work, basic approaches to affecting the CAF using FAP as a target were discussed. The use of FAP as a target provides an important advantage: its proteolytic activity can be used along with the protein-targeted agents. The main directions in the therapeutic use of FAP were discussed in this work.

Embryonic development and carcinogenesis are controlled by many transcription factors, and regulatory systems involved in embryogenesis of an organ also participate in tumor development in the same organ. The FOX family proteins belong to transcription factors, which play a key role in these processes. The representatives of the FOXA subfamily, being pioneer factors, act at the very early stages of embryonic development by interacting with condensed chromatin and open the way for the expression of formerly silent important transcription factors. The role of these factors in tumor development has not been fully elucidated, although recent studies indicate a significant influence of the FOXA subfamily proteins at the early stages of the tumor development. This review is limited by the description of the role of the FOXA factors in embryogenesis of the pancreas and their significance in the development of pancreatic ductal adenocarcinoma.

The expression level of some important master regulators of embryonic development of the pancreas in the tumor samples of this human organ was determined. We found that the transcription of SOX9, GATA4, PDX1, PTF1a, and HNF1b genes in the tumor samples was reduced as compared to the samples of normal pancreatic tissues, and the KLF5 gene expression in the tumor cells was elevated. We assume that all the studied genes, except KLF5, form a single regulatory module that supports the identity of tumor progenitor cells. A simultaneous suppression of expression of these master factors may be critical for the neoplastic transformation of pancreatic cells.

Nuclear proteins, like histone H2A, are promising non-viral carriers for gene delivery since they are biocompatible, biodegradable, bear intrinsic nuclear localization signal, and are easy to modify. The addition of surface-protein-binding ligand to histone H2A may increase its DNA delivery efficiency. Tumor microenvironment (TME) is a promising target for gene therapy since its surface protein repertoire is more stable than that of cancer cells. Cancer-associated fibroblasts (CAFs) are important components of TME, and one of their surface markers is beta-type platelet-derived growth factor receptor (PDGFR&beta, ). In this study, we fused histone H2A with PDGFR&beta, binding peptide, YG2, to create a novel non-viral fibroblast-targeting DNA carrier, H2A-YG2. The transfection efficiency of histone complexes with pDNA encoding a bicistronic reporter (enhanced green fluorescent protein, EGFP, and firefly luciferase) in PDGFR&beta, positive and PDGFR&beta, negative cells was estimated by luciferase assay and flow cytometry. The luciferase activity, percentage of transfected cells, and overall EGFP fluorescence were increased due to histone modification with YG2 only in PDGFR&beta, positive cells. We also estimated the internalization efficiency of DNA-carrier complexes using tetramethyl-rhodamine-labeled pDNA. The ligand fusion increased DNA internalization only in the PDGFR&beta, positive cells. In conclusion, we demonstrated that the H2A-YG2 carrier targeted gene delivery to PDGFR&beta, positive tumor stromal cells.

The mRNA content of the transcription factors KLF5 and ZEB1 was studied in pancreatic tumor tissues and in fetal and normal pancreas. Transcription of these factors was not high and similar in normal and fetal pancreatic tissues but greatly increased in the pancreatic ductal adenocarcinoma tissues. A significant positive correlation between the KLF5 and ZEB1 transcription levels in the pancreatic tumor tissues was observed.

The investigation of molecular mechanisms contributing to cancer progression is the burning problem of current research. Considerable attention has been focused on the study of gene expression in cancer cells. Sphingomyelin synthase 1 gene (SGMS1) is one of the genes, the expression of which can be altered in cancer. SMS1 enzyme encoded by this gene catalyzes synthesis of sphingomyelin and diacylglycerol from phosphatidylcholine and ceramide. SMS1 may maintain the balance between cell death and survival by regulating the formation of the proaptotic mediator ceramide and anti-apoptotic mediator diacylglycerol. In addition, changes in the sphingomyelin level and sphingomyelin synthase activity have been observed in cancers of many tissues. However, the peculiarities of SGMS1 gene transcription have been insufficiently explored. In this work, the expression of transcripts of SGMS1 has been investigated by the method of real-time PCR in matched pairs of samples of human lung and esophagus cancer and adjacent tissues without pathology. A significant decrease in SMS1 transcript expression has been found in the samples of human lung cancer. At the same time, in the samples of human esophagus cancer and the adjacent tissues, the expression of SMS1 transcripts varies insignificantly, i.e., it is increased in seven and decreased in five of the fifteen samples. The obtained results indicate that SGMS1 gene is expressed differently in cancers of different genesis.

One of the problems of gene therapy of melanoma is effective expression of therapeutic gene in tumor cells and their metastases but not in normal cells. In this review, we will consider a two-step approach to a highly specific gene therapy. At the first step, therapeutic genes are delivered specifically to tumor cells using cell surface markers of melanoma cells as targets. At the second step, a specific expression of the therapeutic genes in tumor cells is ensured. Surface markers of melanoma cells were analyzed as potential targets for therapeutic treatment. Criteria for choosing the most promising targets are proposed. The use of specific melanoma promoters allows to further increase the specificity of treatment via transcriptional control of therapeutic gene expression in melanoma cells. Одной из проблем генной терапии злокачественной меланомы является целенаправленная экспрессия терапевтического гена в опухолевых клетках и их метастазах при минимизации ее в нормальных клетках. В обзоре рассмотрен двухэтапный подход к высокоспецифичной терапии. На первом этапе осуществляется адресная доставка терапевтических генов в опухолевые клетки с использованием в качестве мишени поверхностных маркеров меланомных клеток, на втором – обеспечивается специфичность экспрессии генов в опухолевых клетках. Проведен анализ поверхностных маркеров меланомных клеток с точки зрения применения их как мишеней для терапевтического воздействия. Предложены критерии выбора наиболее перспективных мишеней. Использование промоторов, активных только в меланомных клетках, позволяет еще больше увеличить специфичность воздействия за счет контроля транскрипции терапевтических генов в таких клетках. Однією з проблем генної терапії злоякісної меланоми є цілеспрямована експресія терапевтичного гена в пухлинних клітинах і їхніх метастазах за мінімізації її у нормальних клітинах. В огляді розглянуто двоетапний підхід до високоспецифічної терапії. На першому етапі здійснюється адресна доставка терапевтичних генів у пухлинні клітини з використанням як мішені поверхневих маркерів меланомних клітин, на другому – забезпечується специфічність експресії генів у пухлинних клітинах. Проведено аналіз поверхневих маркерів меланомних клітин з точки зору застосування їх як мішеней для терапевтичної дії. Запропоновано критерії вибору найперспективніших мішеней. Використання промоторів, активних лише в меланомних клітинах, дозволяє збільшити специфічність дії за рахунок контролю транскрипції терапевтичних генів у таких клітинах.

Melanoma is one of the most aggressive tumors. It develops from pigment-forming cells (melanocytes) and results in a high number of lethal outcomes. The use of genetic constructs with the ability to specifically kill melanoma cells, but not normal cells, might increase the lifespan of patients, as well as improve their quality of life. One of the methods to achieve a selective impact for therapeutic genes on cancer cells is to utilize a transcriptional control mechanism using promoters that are specifically activated only in cancerous cells. In this review, promoters of the genes that are preferentially expressed in melanoma cells are described. These promoters, and other highly melanoma-specific regulatory elements, reduce the unspecific expression of therapeutic genes in normal tissues. Moreover, cancer-specific promoters and their elements are advantageous for the development of universal anticancer drugs. Examples of the use of double promoters that have a high potential as instruments in cancer gene therapy are also given in this review.

Melanoma is one of the most malignant tumors that metastasizes aggressively by lymphatic and hematogenous routes. Because of the resistance of melanoma cells to many types of chemotherapy, the disease causes a high mortality rate. High hopes are pinned on gene therapeutic approaches to melanoma treatment. At present, one of the main problems of the efficient use of postgenomic-generation therapeutic means is the lack of optimal techniques for delivering foreign genetic material to the patient’s target cells. Specific surface markers of melanoma cells can be considered as promising therapeutic targets. The review describes currently known melanoma-specific receptors and melanoma stem cells and considers the data on melanoma antigens presented on the cell surface by major histocompatibility complex proteins. The ability of surface proteins to be internalized might be successfully used to develop methods of targeted delivery of therapeutic gene constructs. In conclusion, a concept of multilevel gene therapy and the possible role of surface determinants as targets of gene system delivery to the tumor are discussed.

Objectives: Lysosomal proteases cathepsins B and D (CB and CD) play a significant part in cancer progression. For many oncological diseases protein expression levels of CB and CD have been investigated and correlations with tumour characteristics revealed. Meanwhile, there is very little information concerning mRNA expression level.Methods: In the present work, data about mRNA levels of CB and CD in human lung cancer was obtained using reverse transcription followed by real-time polymerase chain reaction.Results: For the first time CD and CB mRNA in human lung cancer tumours was quantified. It was shown that CB and CD mRNA levels do not correlate with any tumour characteristics. However, in most analysed tumours, expression of CD mRNA was downregulated compared with adjacent normal tissue (p

SUMMARY Here we directly compared gene expression profiles in human esophageal squamous cell carcinomas and in human fetal esophagus development. We used the suppression subtractive hybridization technique to subtract cDNAs prepared from tumor and normal human esophageal samples. cDNA sequencing and reverse transcription polymerase chain reaction (RT-PCR) analysis of RNAs from human tumor and the normal esophagus revealed 10 differentially transcribed genes: CSTA, CRNN, CEACAM1, MAL, EMP1, ECRG2, and SPRR downregulated, and PLAUR, SFRP4, and secreted protein that is acidic and rich in cysteine upregulated in tumor tissue as compared with surrounding normal tissue. In turn, genes up- and downregulated in tumor tissue were down- and upregulated, respectively, during development from the fetal to adult esophagus. Thus, we demonstrated that, as reported for other tumors, gene transcriptional activation and/or suppression events in esophageal tumor progression were opposite to those observed during development from the fetal to adult esophagus. This tumor ‘embryonization’ supports the idea that stem or progenitor cells are implicated in esophageal cancer emergence.

Numerous human cancers are accompanied with the increase of ferritin content in blood serum. Ferritin is composed of light and heavy chains, encoded by FTL and FTH genes, respectively. The analysis of EST database showed that the expression of FTL and FTH genes in lung tumors is decreased compared to normal tissues, and is not altered in renal cancer cells. The alteration of mRNA corresponding to FTL and FTH genes was estimated by real-time PCR in primary lung and kidney tumors. A significant and frequent inhibition of FTL and FTH gene expression (on average by 11 and 9 times, in 83% (33/40) and 73% (11/15) of cases, respectively) was detected in primary squamous lung carcinoma. The expression of these genes was not altered so significantly (by 6 and 3 times, in 58% and 27% of samples) in clear cell renal cell carcinoma. Our work reports for the first time the down-regulation of FTL gene expression at the first stage of lung cancer (10/10), and proposes this gene as a potential oncomarker for early diagnosis. The FTL mRNA content may be quantified by non-competitive hybridization on expression DNA microarrays. The possible causes of a serum ferritin increase in lung squamous cell carcinoma and clear cell renal cell carcinoma are discussed.

We, for the first time, directly compared gene expression profiles in human non-small cell lung carcinomas (NSCLCs) and in human fetal lung development. Previously reported correlations of gene expression profiles between lung cancer and lung development, deduced from matching data on mouse development and human cancer, have brought important information, but suffered from different timing of mouse and human gene expression during fetal development and fundamental differences in tumorigenesis in mice and humans. We used the suppression subtractive hybridization technique to subtract cDNAs prepared from human fetal lung samples at weeks 10-12 and 22-24 and obtained a cDNA library enriched in the transcripts more abundant at the later stage. cDNAs sequencing and RT-PCR analysis of RNAs from human fetal and adult lungs revealed 12 differentially transcribed genes: ADH1B, AQP1, FOLR1, SLC34A2, CAV1, INMT, TXNIP, TPM4, ICAM-1, HLA-DRA, EFNA1 and HLA-E. Most of these genes were found up-regulated in mice and rats at later stages than in human lung development. In surgical samples of NSCLC, these genes were down-regulated as compared to surrounding normal tissues and normal lungs, thus demonstrating opposite expression profiles for the genes up-regulated during fetal lung development.

Survivin (BIRC5) belongs to the family of inhibitors of apoptosis proteins (IAPs). BIRC5 is expressed in embryonic tissues and in most malignant tumors but not in fully differentiated adult tissues. BIRC5, acting as an apoptosis inhibitor, presumably plays an important part in carcinogenesis, thus providing an attractive target for antitumor therapy. The mechanisms regulating the survivin level are insufficiently understood, but a significant role is ascribed to natural survivin inhibitors, SMAC and PML. The transcription levels of BIRC5, SMAC, and PML and the levels of their protein products were assessed by RT-PCR and immunoblotting in normal and tumor human tissues in non-small cell lung cancer and esophageal squamous cell carcinoma. BIRC5 transcription was observed only in tumor tissues, whereas SMAC and PML were expressed in tumor and normal tissues at similar levels. The protein levels corresponded to the mRNA levels. Thus, increased levels of survivin in tumor tissues are not a consequence of downregulation of its inhibitors SMAC and PML, whose levels do not differ between tumor and normal cells.

The KLRB1 gene encodes the CD161 receptor in natural killer cells (NK cells). The gene is also expressed in the NKT cells. Both types of cells are cytotoxic and capable of recognizing and eliminating various tumor, virus-infected, and allotransplant cells. The biological function of human CD161 is not clear. It is suggested that it can be involved in the regulation of both cytotoxic cell functions and cytokine production. As the majority of cancer patients show down-regulation of the immune system, we hypothesized that the level of KLRB1 expression might also be suppressed in cancer cells. Here, we show that KLRB1 gene expression was suppressed in tumor tissues in 68% of patients with non-small-cell lung cancer (p < 0.0001) and in 57% of patients with esophageal squamous-cell carcinoma (p = 0.0003). The high frequency of KLRB1 gene suppression in cancer patients suggests that this gene can represent a highly informative marker of lung and esophageal cancers.

Secreted polymeric immunoglobulins, IgA dimers and IgM pentamers, are unique in having not only the L and H chains but also the J chain, which is responsible for oligomerization. These antibodies belong to the local adaptive immune system, located in the mucosas of the respiratory and digestive systems and acting first to protect the body from infectious agents. Polymeric immunoglobulins are produced by highly specific B cells and are actively transported to the mucosa surface through epithelial cells. Hence, their production and, accordingly, the J-chain content are associated with the transporting function of the epithelium and depend on its state, which dramatically changes upon malignant transformation. The content of the J chain and the transcription level of its gene were studied in normal and tumor cells at various stages of squamous cell cancer and adenocarcinoma of the lungs by RT-PCR and immunoblotting.

PIWI pathway proteins are expressed during spermatogenesis where they play a key role in germ cell development. Epigenetic loss of PIWI proteins expression was previously demonstrated in testicular germ cell tumors (TGCTs), implying their involvement in TGCT development. In this work, apart from studying only normal testis and TGCT samples, we also analyzed an intermediate stage, i.e. preneoplastic testis tissues adjacent to TGCTs. Importantly, in this study, we minimized the contribution of patient-to-patient heterogeneity by using matched preneoplastic/TGCT samples. Surprisingly, expression of germ cell marker DDX4 suggests that spermatogenesis is retained in premalignant testis tissues adjacent to nonseminoma, but not those adjacent to seminoma. Moreover, this pattern is followed by expression of PIWI pathway genes, which impacts one of their functions: DNA methylation level over LINE-1 promoters is higher in preneoplastic testis tissues adjacent to nonseminomas than those adjacent to seminomas. This finding might imply distinct routes for development of the two types of TGCTs and could be used as a novel diagnostic marker, possibly, noninvasively. Finally, we studied the role of CpG island methylation in expression of PIWI genes in patient samples and using in vitro experiments in cell line models: a more complex interrelation between DNA methylation and expression of the corresponding genes was revealed.

In preparation of the therapeutic genetic constructs aimed to the gene-programmed enzymatic transformation of the non-toxic prodrug into toxin within cancer cells the right choice of regulatory elements (promoters and enhancers) is essential. This is widely accepted that the efficiency of the gene therapy constructions is dependent, in particular, on the strength of promoters driving the expression of the therapeutic genes. In this work we demonstrated, using the melanoma-specific promoters and enhancers of human melanoma inhibitory activity and mouse tyrosinase gene, that for the development of cytotoxic effect the promoter strength is not of primary importance. In the case of HSVtk, coding for the herpes simplex virus thymidine kinase, and FCU1, coding for cytosine deaminase/uracil phosphoribosyltransferase hybrid protein genes, their cytotoxic activity was determined by the quantity of the added prodrug.

Recently, more evidence supporting common nature of promoters and enhancers has been accumulated. In this work, we present data on chromatin modifications and non-polyadenylated transcription characteristic for enhancers as well as results of in vitro luciferase reporter assays suggesting that PIWIL2 alternative promoter in exon 7 also functions as an enhancer for gene PHYHIP located 60Kb upstream. This finding of an intragenic enhancer serving as a promoter for a shorter protein isoform implies broader impact on understanding enhancer-promoter networks in regulation of gene expression.

ISSN 16076729, Doklady Biochemistry and Biophysics, 2011, Vol. 437, pp. 105–108. © Pleiades Publishing, Ltd., 2011.Original Russian Text © K.N. Kashkin, E.A. Musatkina, A.V. Komelkov, D.A. Sakharov, E.V. Trushkin, E.A. Tonevitsky, T.V. Vinogradova, E.P. Kopantzev, M.V. Zinovyeva,O.V. Kovaleva, K.A. Arkhipova, I.B. Zborovskaya, A.G. Tonevitsky, E.D. Sverdlov, 2011, published in Doklady Akademii Nauk, 2011, Vol. 437, No. 6, pp. 837–841.

Background: Stromal cells are a functionally important component of human carcinomas. The aim of this study was to obtain and characterise primary cultures of stromal cells from human carcinomas and the corresponding surrounding normal tissue. Methods: Primary stromal cell cultures from tumours of lung, oesophagus and pancreas were obtained using a mild tissue dissociation method and a medium for culturing mesenchymal cells. Immunofluorescence staining and western blotting were used to analyse the expression of differentiation markers and selected known oncoproteins in the cell cultures obtained. Results: A panel of stromal primary cultures was prepared from different human tumours and from matched normal cancer-free tissues. The in vitro proliferative potential of tumour-associated fibroblasts was shown to be higher than that of matched normal stromal cells. A mutational analysis of the TP53 and KRAS2 genes in a number of stromal cultures did not reveal known mutations in most cells of the cultures studied. Western blot analysis showed that stromal cells of lung tumours were characterised by a statistically significantly lower expression level of the p16 protein as compared with that in normal lung stromal cells. An important finding of our study was that, according to immunofluorescence assay, a fraction of fibroblast-like vimentin-positive cells in some tumour and normal stromal cell cultures expressed an epithelial marker – cytokeratins. Conclusions: Proliferating stromal cells from the carcinomas studied proved to be genetically normal cells with altered expression profiles of some genes involved in carcinogenesis, as compared with normal stromal cells. Epithelial-mesenchymal transition may lead to the emergence of transdifferentiated fibroblast-like cells in tumour stroma and in the tumour-surrounding tissue.

Proprotein convertases (PCs) is a protein family which includes nine highly specific subtilisin-like serine endopeptidases in mammals. The system of PCs is involved in carcinogenesis and levels of PC mRNAs alter in cancer, which suggests expression status of PCs as a possible marker for cancer typing and prognosis. The goal of this work was to assess the information value of expression profiling of PC genes. Quantitative polymerase chain reaction was used for the first time to analyze mRNA levels of all PC genes as well as matrix metalloproteinase genes MMP2 and MMP14, which are substrates of PCs, in 30 matched pairs of samples of human lung cancer tumor and adjacent tissues without pathology. Significant changes in the expression of PCs have been revealed in tumor tissues: increased FURIN mRNA level (p