I. P. Chernov

Identification and mapping of nuclear matrix‐attachment regions in a one megabase locus of human chromosome 19q13.12: Long‐range correlation of S/MARs and gene positions

The first draft human genome sequence now available allowed the identification of an enormous number of gene coding areas of the genomic DNA. However, a great number of regulatory elements such as enhancers, promoters, transcription terminators, or replication origins can not be identified unequivocally by their nucleotide sequences in complex eukaryotic genomes. One important subclass of these type of sequences is scaffold/matrix attachment regions (S/MARs) that were hypothesized to anchor chromatin loops or domains to the nuclear matrix and/or chromosome scaffold. We developed an experimental selection procedure to identify S/MARs within a completely sequenced one megabase (1 Mb) long gene‐rich D19S208‐COX7A1 locus of human chromosome 19. A library of S/MAR elements from the locus was prepared and shown to contain ∼20 independent S/MARs. Sixteen of them were isolated, sequenced, and assigned to certain positions within the locus. A majority of the S/MARs identified (11 out of 16) lie in intergenic regions, suggesting their structural role, i.e., delimitation of chromatin domains. These 11 S/MARs subdivide the locus into 10 domains ranging from 6 to 272 kb with an average domain size of 88 kb. The remaining five S/MARs were found within intronic sequences of APLP1, HSPOX1, MAG, and NPHS1 genes, and can be tentatively characterized as regulatory S/MARs. The correspondence of the chromatin domains defined by the S/MARs to functional characteristics of the genes therein is discussed. The approach described can be a prototype of a similar search of long sequenced genomic stretches and/or whole chromosomes for various regulatory elements. J. Cell. Biochem. 84: 590–600, 2002.

Identification, genome mapping, and CTCF binding of potential insulators within the FXYD5-COX7A1 locus of human chromosome 19q13.12.

Identification of insulators is one of the most difficult problems in functional mapping of genomes. For this reason, up to now only a few insulators have been described. In this article we suggest an approach that allows direct isolation of insulators by a simple positive-negative selection based on blocking enhancer effects by insulators. The approach allows selection of fragments capable of blocking enhancers from mixtures of genomic fragments prepared from up to 1-Mb genomic regions. Using this approach, a 1-Mb human genome locus was analyzed and eight potential insulators were selected. Five of the eight sequences were positioned in intergenic regions and two were within introns. The genes of the α-polypeptide H+/K+ exchanging ATPase (ATP4A) and amyloid β (A4) precursor-like protein 1 (APLP1) within the locus studied were found to be flanked by insulators on both sides. Both genes are characterized by distinct tissue-specific expression that differs from the tissue specificity of the surrounding genes. The data obtained are consistent with the conception that insulators subdivide genomic DNA into loop domains that comprise genes characterized by similar expression profiles. Using chromatin immunoprecipitation assay, we demonstrated also that at least six of the putative insulators revealed in this work could bind the CTCF transcription factor in vivo. We believe that the proposed approach could be a useful instrument for functional analysis of genomes.

Structure and Functions of Nuclear Matrix Associated Regions (S/MARs)

Modern concepts on the chromatin loop–domain organization and the role of the DNA regions specifically binding the nuclear matrix or nuclear scaffold (S/MARs) during its formation, maintenance, and regulation are discussed. Some S/MAR structural features, properties of binding the nuclear matrix, and probable mechanisms of their involvement in the gene regulation of activity are considered.

Therapeutic properties of a vector carrying the HSV thymidine kinase and GM-CSF genes and delivered as a complex with a cationic copolymer.

BackgroundGene-directed enzyme prodrug therapy (GDEPT) represents a technology to improve drug selectivity for cancer cells. It consists of delivery into tumor cells of a suicide gene responsible for in situ conversion of a prodrug into cytotoxic metabolites. Major limitations of GDEPT that hinder its clinical application include inefficient delivery into cancer cells and poor prodrug activation by suicide enzymes. We tried to overcome these constraints through a combination of suicide gene therapy with immunomodulating therapy. Viral vectors dominate in present-day GDEPT clinical trials due to efficient transfection and production of therapeutic genes. However, safety concerns associated with severe immune and inflammatory responses as well as high cost of the production of therapeutic viruses can limit therapeutic use of virus-based therapeutics. We tried to overcome this problem by using a simple nonviral delivery system.MethodsWe studied the antitumor efficacy of a PEI (polyethylenimine)-PEG (polyethylene glycol) copolymer carrying the HSVtk gene combined in one vector with granulocyte–macrophage colony-stimulating factor (GM-CSF) cDNA. The system HSVtk-GM-CSF/PEI-PEG was tested in vitro in various mouse and human cell lines, ex vivo and in vivo using mouse models.ResultsWe showed that the HSVtk-GM-CSF/PEI-PEG system effectively inhibited the growth of transplanted human and mouse tumors, suppressed metastasis and increased animal lifespan.ConclusionsWe demonstrated that appreciable tumor shrinkage and metastasis inhibition could be achieved with a simple and low toxic chemical carrier – a PEI-PEG copolymer. Our data indicate that combined suicide and cytokine gene therapy may provide a powerful approach for the treatment of solid tumors and their metastases.

Activity of the upstream component of tandem TERT/survivin promoters depends on features of the downstream component.

We spliced the promoters of the human telomerase and human survivin genes (PhTERT and PhSurv, respectively) widely used for gene therapy and known to have the broadest cancer type spectrum of activity. Two head-to-tail constructs were obtained: the PhTERT-PhSurv and PhSurv-PhTERT tandems. The splicing caused quantitative and qualitative changes in the promoter features. In both constructs, only the promoter proximal to the transcribed gene retained its ability to initiate transcription, whereas the distal promoter was silent, the phenomenon never reported before. However, the distal promoter modulated the activity of the proximal one by increasing its strength and causing an appearance of additional transcription start sites. We suggested that this suppression might be due to the presence of Sp1 transcription factor binding sites in both promoters and Sp1-bridges between these sites. Such Sp1-bridges might convert the tandem promoter linear DNA into a stem-loop structure. If localized inside the formed loop, the distal promoter could lose its ability to initiate transcription. To test this hypothesis, we constructed two modified double promoters, where the proximal PhSurv promoter was replaced either by a shortened variant of the survivin promoter (PhSurv269) or by the mouse survivin promoter. Both PhSurv substitutes were considerably shorter than PhSurv and had different numbers and/or positions of Sp1 sites. In modified tandems, transcription was initiated from both promoters. We also prepared two mutant forms of the PhSurv-PhTERT tandem with two or four Sp1 sites removed from the distal “long” PhSurv promoter. In the first case, the distal PhSurv promoter remained silent, whereas the removal of four Sp1 binding sites restored its activity. In the majority of studied cancer cell lines the efficiency of transcription from the hTERT-(shortened hSurv269) promoter tandem was markedly higher than from each constituent promoter. In normal lung fibroblast cells, the tandem promoter activity was considerably lower.

KLF5, a new player and new target in the permanently changing set of pancreatic cancer molecular drivers

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.

Cancer specificity of promoters of the genes controlling cell proliferation.

Violation of proliferation control is a common feature of cancer cells. We put forward the hypothesis that promoters of genes involved in the control of cell proliferation should possess intrinsic cancer specific activity. We cloned promoter regions of CDC6, POLD1, CKS1B, MCM2, and PLK1 genes into pGL3 reporter vector and studied their ability to drive heterologous gene expression in transfected cancer cells of different origin and in normal human fibroblasts. Each promoter was cloned in short (335–800 bp) and long (up to 2.3 kb) variants to cover probable location of core and whole promoter regulatory elements. Cloned promoters were significantly more active in cancer cells than in normal fibroblasts that may indicate their cancer specificity. Both versions of CDC6 promoters were shown to be most active while the activities of others were close to that of BIRC5 gene (survivin) gene promoter. Long and short variants of each cloned promoter demonstrated very similar cancer specificity with the exception of PLK1‐long promoter that was substantially more specific than its short variant and other promoters under study. The data indicate that most of the important cis‐regulatory transcription elements responsible for intrinsic cancer specificity are located in short variants of the promoters under study. CDC6 short promoter may serve as a promising candidate for transcription targeted cancer gene therapy. J. Cell. Biochem. 116: 299–309, 2015.

Master transcription regulators specifying cell-lineage fates in development as possible therapeutic targets in oncology

The transformation of normal precursors into cancer cells is an intricately regulated, multistep process. The master regulatory genes that play a crucial role in the process of organism development may also play a key role in carcinogenesis. From such a point of view, cancer is not simply a genetic disease that is due to a progressive accumulation of mutation—it is also a disorder of the developmental system of the tissue in which cancer emerges. Master regulators and their genes disturb stem cell differentiation upon mutation and thus may serve as targets for cancer therapy, in addition to the classic oncogenes and suppressors of tumor formation. This review is an attempt to give a modern concept of master genes and their functions in adult stem cells of the organism and in carcinogenesis, with pancreatic cancer as an example.

Maps of cis-Regulatory Nodes in Megabase Long Genome Segments are an Inevitable Intermediate Step Toward Whole Genome Functional Mapping.

The availability of complete human and other metazoan genome sequences has greatly facilitated positioning and analysis of various genomic functional elements, with initial emphasis on coding sequences. However, complete functional maps of sequenced eukaryotic genomes should include also positions of all non-coding regulatory elements. Unfortunately, experimental data on genomic positions of a multitude of regulatory sequences, such as enhancers, silencers, insulators, transcription terminators, and replication origins are very limited, especially at the whole genome level. Since most genomic regulatory elements (e.g. enhancers) are generally gene-, tissue-, or cell-specific, the prediction of these elements by computational methods is difficult and often ambiguous. Therefore, the development of high-throughput experimental approaches for identifying and mapping genomic functional elements is highly desirable. At the same time, the creation of whole-genome map of hundreds of thousands of regulatory elements in several hundreds of tissue/cell types is presently far beyond our capabilities. A possible alternative for the whole genome approach is to concentrate efforts on individual genomic segments and then to integrate the data obtained into a whole genome functional map. Moreover, the maps of polygenic fragments with functional cis-regulatory elements would provide valuable data on complex regulatory systems, including their variability and evolution. Here, we reviewed experimental approaches to the realization of these ideas, including our own developments of experimental techniques for selection of cis-acting functionally active DNA fragments from large (megabase-sized) segments of mammalian genomes.

Study of transactivation effect on transcription by Tat-TAR system of human immunodeficiency virus type I (HIV-I) in nonlymphoid cells HEK293 and Calu-1

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