V. V. Pleshkan

Methylation of the prominin 1 TATA-less main promoters and tissue specificity of their transcript content

Prominin 1 (PROM1, CD133) is a unique transmembrane glycoprotein encoded by the PROM1 gene. It is a cell surface marker of various stem cells including hematopoietic, prostatic epithelial, pancreatic, leukemic, liver cancer, and colorectal cancer stem cells. Here, we studied tissue specificity of PROM1 transcription isoforms and the methylation level of its two main promoters (P1 and P2) in different human cell lines. Only transcripts lacking the 4th exon (the CD133.s1 form) were expressed in cell lines studied. Moreover, these transcripts, if sufficiently abundant, were initiated simultaneously and independently from both promoters P1 and P2. In cell lines with low levels of the total PROM1 transcript, the transcription was likely initiated from other promoters. Promoter P1 was hypermethylated in all cell lines under study, and therefore, methylation can hardly play an important role in its regulation. In contrast, the methylation of promoter P2 was tissue specific, and hypomethylation of this promoter is probably necessary but not sufficient for efficient transcription of the PROM1 gene. Therefore, we report an unusual instance of different mechanisms of transcription activity regulation for two closely located promoters of the same gene.

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.

Functional analysis of the HERV-K LTR residing in the KIAA1245/NBPF subfamily genes

Long terminal repeats (LTRs) of human endogenous retroviruses (HERVs) 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 Tera 1. 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 Tera1 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 LTR revealed 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 transcription regulation of the KIAA1245/NBPF subfamily genes.

The cause of cancer mutations: Improvable bad life or inevitable stochastic replication errors?

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.

Expression of the FAP gene in non-fibroblast human cell lines. Development of cancer-associated fibroblast models

The fibroblast activation protein (FAP) is selectively expressed in cancer-associated fibroblasts (CAFs) and facilitates tumor progression, which makes this protein an attractive therapeutic target. There are difficulties in obtaining CAFs for studying the function and suppression of FAP. In this work, the expression level of FAP was determined by PCR assay in 25 human cell lines and 8 surgical samples of tumor stroma. The expression of FAP was observed in all tumor stroma samples and in four cell lines: NGP-127, SJCRH30, SJSA-1, and A375. The level of FAP expression in NGP-127, SJCRH30, and SJSA-1 lines as well as in CAFs of patients was comparable, which makes these cell lines a possible model for studying FAP.

Are super-enhancers regulators of regulatory genes of development and cancer?

Enhancers make up a huge class of genome regulatory elements that play an important role in the formation and maintenance of specific patterns of gene transcriptional activity in all types of cells. In recent years, high-throughput methods for the genome-wide epigenetic analysis of chromatin have made it possible to identify structural and functional features of enhancers and their role in the spatial and functional organization of the genome and in the formation and maintenance of cell identity, as well as in the pathogenesis of certain diseases. Special attention has been focused on genome regions called super-enhancers, or stretch enhancers, which consist of clusters of elements with properties of classic enhancers. This review considers current data on specific properties of super-enhancers and their role in the formation of interconnected autoregulatory circuits with positive feedback that regulates the most important genes, the activity of which underlies the formation and maintenance of specialized cellular functions.

Melanoma: Surface markers as the first point of targeted delivery of therapeutic genes in multilevel gene therapy

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.

Fundamentally low reproducibility in molecular genetic cancer research

The review discusses the causes of multiple failures in cancer treatment, which might primarily result from the excessive variability of cancer genomes. They are capable of changing their spatial and temporal architecture during tumor development. The key reasons of irreproducibility of biomedical data and the presumable means for improvement of therapeutic results aiming at targeting the most stable tumor traits are suggested.

Fibroblast activation protein (FAP) as a possible target of an antitumor strategy

This review was devoted to the use of the universal component of tumoral stroma (fibroblast activation protein, FAP) as a target of the universal tumor therapy. A tumor is a coevolution system, which includes a microenvironment or reactive stroma differing from the normal tissue in its phenotypic and genotypic features. Cancer-associated fibroblasts (CAFs), which contain the typical marker FAP (serine proteinase with the enzymatic activity of dipeptidyl peptidase and endopeptidase), are important elements of the tumor microenvironment. 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 universal target. In this work, basic approaches to affecting CAFs using FAP as a target are 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 areas of development in the therapeutic use of FAP are discussed in this work.