[Reduced Expression of the Tissue-Specific Oct-IL Isoform Exerts an Antitumor Effect on Namalwa Burkitt's Lymphoma Cells].

Increased expression levels of the Oct-1 transcription factor is considered to be one of the key markers of poor cancer prognosis. In addition to the ubiquitous Oct-1A isoform, which is found in all cells, there also exists a tissue-specific Oct-1L isoform, which is expressed in hematopoietic cells. Oct-1L increases cell resistance to different stresses and also regulates the expression of genes controlling differentiation of hematopoietic and immune system cells. The tissue-specific Oct-1L isoform levels are significantly increased in the B-cell lymphoblastoma Namalwa and Raji lines and the T-cell lymphoblastoma Jurkat line compared to normal B and T cells. Apparently, aberrant Oct-1L overexpression not only enhances stress resistance but also leads to the disruption of developmental pathways in the cells promoting their malignant transformation. We report here that targeted suppression of the tissue-specific Oct-1L isoform expression reduces the proliferation rate of Namalwa B-lymphoblastic Burkitt's lymphoma cells, significantly increases cell death rate under hypoxic conditions, and makes cells more sensitive to chemotherapeutic agents such as docetaxel and doxorubicin. These results indicate that targeted therapy aimed at the suppression of the Oct-1 isoforms with increased expression levels in tumor cells rather than the total Oct-1, thus avoiding the traumatic effects of total Oct-1 knockdown, may be promising. Selective suppression of Oct-1 isoforms is a promising strategy in the treatment of lymphoid tumors and may contribute to mitigating the disease course and increasing survival rates in cancer patients.

5-Azacytidine Suppresses the Expression of Tissue-Specific Oct-1 Isoform in Namalwa Burkitt's Lymphoma Cell Culture.

Overexpression of the transcription factor POU2F1 (Oct-1) increases the malignant potential of the tumor and determines the unfavorable prognosis for both solid and hematological cases of the disease in human carcinogenesis. The Oct-1 level determines the rate of development of the disease in acute myelodysplastic leukemia (AML), and a decrease in its expression significantly delays the development of leukemia in mice; however, a complete knockout of Oct-1 leads to the death of the animals. POU2F1 (Oct-1) is expressed as several isoforms transcribed from alternative promoters. They include both ubiquitous and tissue-specific isoforms. It was shown that in Burkitt's lymphoma Namalwa cells 5-azacytidine specifically suppresses the expression of the tissue-specific isoform Oct-1L mRNA (level of Oct-1L is abnormally increased in these cells), while not causing changes in the amount of the ubiquitous isoform Oct-1A mRNA. These results show that it is possible to selectively reduce the transcription level of the Oct-1L isoform aberrantly expressed in human tumor cells.

The Emergence of a New Isoform of POU2F1 in Primates through the Use of Egoistic Mobile Genetic Elements.

The emergence of new genes and functions is of paramount importance in the emergence of new animal species. For example, the insertion of the mobile element Tigger 2 into the sequence of the functional gene POU2F1 in primates led to the formation of a new chimeric primate-specific isoform POU2F1Z, the translation of which is activated under cellular stress. Its mRNA was found in all species of monkeys, starting with macaques. Analysis of the fragments of the Tigger2 copy corresponding to the human exon Z showed that the splicing sites of exon Z are homologous in humans and in most monkeys, with the exception of lemurs and galagos. The stop codon introduced into the mRNA by the Tigger2 sequence is present in all primates, starting with macaques. The internal ATG codon is also present in all primates, with the exception of lemurs and galagos. In the course of evolution, other MGEs, mainly of the SINE type, were inserted into the Tigger2 copy. In the course of evolution, both the location and the number of mobile SINE elements within the POU2F1 gene changed. Starting with macaques, the pattern of the arrangement of SINE elements within the Tigger2 copy in the studied region of the POU2F1 gene was fixed and then remained unchanged in other primates and humans, which may indicate its functional significance.

[POU2F1 (Oct-1) Differently Autoregulates the Alternative Promoters of Its Own Gene by Binding to Different Regulatory Sites].

The POU2F1 gene, which plays an important role in regulating the mammalian genome and development, has both a ubiquitous (U) and a tissue-specific (L) promoter and is subject to intricate regulation. Regions of POU2F1 gene were found to contain multiple binding sites for its product POU2F1 (Oct-1), a transcription factor. Interspecies homology in these regions was found to exceed 90% among the human, mouse, rat, pig, and dog genomes, almost all of the Oct-1 binding sites being identical. Some of the sites cluster in the vicinity of each of the two alternative promoters, while others are in the 5' noncoding region 6 kb upstream of the transcription start site. The presence of Oct-1 at the sites was demonstrated by chromatin immunoprecipitation and the electrophoretic mobility shift assay (EMSA). A POU2F1 knockdown activated the U promoter and downregulated the L promoter in Namalwa cells, while Oct-1 overexpression exerted an opposite effect. Thus, Oct-1 acts via negative feedback to autoregulate the U promoter through low-affinity Oct-1 binding sites and positive feedback to autoregulate the L promoter through high-affinity canonical (oct) sites when increasing in concentration in a natural context.

Differentiation of IMR32 Neuroblastoma Is Accompanied by a Global Change in the Transcriptome.

Neuroblastoma is one of the most common cancers in infants and is often multidrug-resistant. One of the methods of treating neuroblastomas is to create conditions for their differentiation. In this work, we performed a full-transcriptome analysis of gene expression in an undifferentiated and differentiated in vitro human neuroblastoma cell line IMR-32 and identified the signaling pathways and biological processes that undergo the greatest changes during differentiation. The results obtained show that a complex heterogeneous population of nerve cells is formed at different stages of differentiation. In the cell population of differentiating neuroblastoma, the expression of genes in which cortical neuronal progenitor cells are enriched increases; at the same time, there are cells expressing markers of early postmitotic neurons. Cells differentiate in several different directions according to the type of synaptic mediator. At the same time, the differentiation of IMR-32 cells is accompanied by an increase in the transcription of genes that suppress the differentiation of nerve cells, Sox2 and PROM1, the expression of which is normally suppressed during in vivo differentiation.

GSK3 Kinase Inhibitor, CHIR, Suppress Transcription of Tissue Specific POU2F1 Isoform in Burkitt Namalwa Lymphoma Cells.

POU2F1 (Oct-1) is a transcription factor, the overexpression of which is found in many human malignant tumors; a significant increase in its level in cells determines the malignant potential of the tumor. POU2F1 is represented in cells by several isoforms that are transcribed from alternative promoters. In Burkitt's B-cell lymphoma Namalwa, the concentration of tissue-specific isoform Oct-1L is several times higher than in normal B cells. We tested the potential to inhibit the transcription of individual Oct-1 isoforms using the GSK3 kinase inhibitor CHIR, an aminopyrimidine derivative. We have shown that CHIR specifically affects the expression of the tissue-specific isoform Oct-1L, significantly reducing the level of mRNA and Oct-1L protein. However, CHIR does not change the amount of mRNA and protein of the ubiquitous isoform Oct-1A in Namalwa tumor cells. The results obtained show that it is possible to develop a system for selective inhibition of Oct-1 transcription factor isoforms in human cells to suppress drug resistance of tumor cells with a high POU2F1 content.

Thapsigargin, Inhibitor of Sarco-Endoplasmic Ca2+-ATPase, Effectively Suppresses the Expression of S100A4 Protein in Human Breast Cancer Cell Line.

Thapsigargin (SERCA ATPase inhibitor) inhibited the S100A4 metastatic marker expression in MDA-MB231 breast cancer cells. We found that S100A4 gene transcription is regulated by Ca2+ signaling pathways. We found that the synthesis of S100A4 mRNA and S100A4 protein in MDA-MB231 cells was effectively suppressed by thapsigargin at a concentration of 0.4-4 µM with retaining cell viability. We assume that the change in the gene transcription in response to disturbance of Ca2+ homeostasis is directly involved in the remodeling of Ca2+ signaling pathways.

[Multiple Interactions of the Oct-1 (POU2F1) Transcription Factor with PORE and MORE Sites].

The Oct-1 (POU2F1) transcription factor is one of the most important regulatory proteins in humans and other mammals. An increase in Oct-1 aids the resistance to oxidative and cytotoxic stresses and radiation exposure. A high level of Oct-1 is found in many human tumors and correlates with low survival. Oct-1 interacts with its binding sites as a monomer, a homodimer, or a multimer. The nucleotide sequence of the Oct-1 binding site determines the character of interaction and the conformation of Oct-1 on target DNA, thus influencing the binding of Oct-1 co-repressors and co-activators. Nucleotide substitutions were introduced in all positions of the PORE and MORE sequences and tested for effect on the Oct-1 capability of forming monomeric and dimeric DNA-protein complexes. The position and nature of nucleotide substitutions were found to affect the type of Oct-1 binding to DNA. Several substitutions suppressed the formation of dimers, while others stimulated the process. Certain nucleotide substitutions completely prevented the binding of both monomers and dimers. The Oct-1 concentration in the cell is another factor that affects the character of DNA-protein interactions. Based on the results, the nature and affinity of interaction with Oct-1 is possible to predict from the nucleotide sequence for PORE and MORE sites of the human genome.

Differentiation of Monocytic Cells Is Accompanied by a Change in the Expression of the Set of Oct-1 Isoforms.

Changes in the expression level of Oct-1A, Oct-1L, Oct-1X, and Oct-1Z isoforms and CD14 surface antigen during differentiation of HL-60 monocytic cells induced in vitro by dimethyl sulfoxide were studied, and the expression level of the four Oct-1 isoforms in vivo in human monocytes was determined.

Increased level of Oct-1 protein in tumor cells modulates cellular response to anticancer drugs.

The effect of overexpression of Oct-1 protein isoforms on the cell response to two anticancer drugs camptothecin and dexamethasone was studied. The effect of Oct-1 isoforms on regulated gene transcription was estimated by the difference in the level of mRNA in Burkitt's lymphoma cells (Namalwa line) untransfected and stably transfected with Oct-1 isoforms. The response to anticancer drugs of the Oct-1 target genes involved in the development of apoptosis depended, firstly, on the type of drug, secondly, on the concentration of Oct-1 in cells. and, thirdly, on the Oct-1 isoform with which these cells were transfected.

Involvement of transcription factor Oct-1 in the regulation of JAK-STAT signaling pathway in cells of Burkitt lymphoma.

We studied the role of transcription factor Oct-1 in the regulation of expression of genes of the JAK-STAT signaling pathway in the Namalwa Burkitt's lymphoma cell line. Overexpression of Oct-1 isoforms (Oct-1A, Oct-1L, and Oct-1X) causes a decrease in the activity of four genes involved in the JAK-STAT signaling pathway-IFNAR2, STAT1, STAT2, and STAT4. As a result of our research, it was found that genes STAT2 and STAT4 are direct targets for Oct-1 protein.

Transcription factor Oct-1 stimulates the release of Mts1/S100A4 protein by the cancer cells.

The effect of the transcription factor Oct-1 (POU2F1) on the expression of the tumor cell marker metastasin (Mts1/S100A4) was studied. Comparative analysis of various tumor lines showed no clear correlation between the expression level of Mts1/S100A4 and the content of Oct-1. However, at stable transfection of tumor cells with Oct-1A, Oct-1L, and Oct-1X isoforms we detected an elevated level of Oct-1, which stimulated Mts1/S100A4 secretion. These findings extend our understanding of the molecular mechanisms of the tumorigenic effect of Oct-1.

New alternative promoter in regulation of the Oct-1 human gene transcription.

For the first time, the presence of a new alternative promoter in the gene of the oct-1 transcription factor from which a previously unknown mRNA isoform Oct-1X, with 5'-terminus different from the previously described isoforms, was demonstrated. The nucleotide sequence of the Oct-1X cDNA was determined and the presence of a long open reading frame which starts with the first ATG codon of the second exon was demonstrated. A protein shortened on its N-terminus is assumed to be the product of a new mRNA isoform. Expression of the new isoform was studied in various human cell lines; the results indicate the ubiquitous nature of the Oct-1X expression. Therefore, in the work, the third alternative promoter of human oct-1 gene (earlier, a ubiquitous promoter U and an inducible tissue-specific promoter L were described) contributing to the fine regulation of mRNA isoform synthesis and formation of structural and functional diversity of Oct-1 protein isoforms in cells was revealed.

[The new isoform of Oct-1 transcription factor is transcribed from alternative promoter].

The Oct-1 transcription factor belongs to the family of highly conserved POU-domain proteins that participate in regulation of housekeeping and tissue-specific gene expression. Several isoforms of Oct-1 were described previously. We have found the new alternative promoter of Oct-1 gene and the corresponding mRNA Oct-1X. This mRNA encodes the alternative Oct-1 isoform that has truncated N-terminus comparing to isoforms Oct-1A and Oct-1L. We explored the abundance of this isoform in human tissues and cell lines and demonstrated that it is ubiquitously expressed but its expression strongly varies in different tissues. By co-transfection and double luciferase assay we have demonstrated that Oct-1X is the activator of housekeeping (histone H2B) and tissue-specific genes (B29) transcription.

[RNA editing in eukaryotic genome expression].

The present review surveys the available data on the involvement of adenine deamination in RNA molecules in the formation of structurally and functionally diverse RNA and protein subforms in eukaryotic cells. Deamination of adenine by adenosine deaminases that act on RNA (ADARs) leads to the conversion of adenine into inosine (A-I editing) recognized by the splicing and translation systems as guanine. This may modify splicing sites in pre-mRNA and codons in translated regions ofmRNA and also affect the RNA secondary structure. Apart from mRNA, editing also involves microRNAs whose regulatory functions in multicellular animals are associated with the inhibition of transcription of target genes or with the degradation of certain RNA transcripts. ADARs can inhibit the production of mature microRNAs or modify microRNAs so that their specificity to target genes is altered. Adenosine deaminases editing adenines in transport RNAs (ADATs) convert adenine into inosine in tRNAs of all eukaryotes; as a result, the diversity of tRNA forms in the cell increases.

[Role of transcription factors binding sites in no-mediated histone H2B promoter repression].

Nitric oxide (NO) is an important signaling molecule with diverse actions in a wide variety of tissues. NO is a well-known inhibitor of cell growth, DNA replication and expression of cell-cycle implicated genes. In this study we analyzed the effect of NO on histone H2B expression in human HEK 293 cells. Using cell transfection with a plasmid expressing reporter gene under the control of histone H2B promoter, we showed that NO markedly attenuated the expression of the reporter gene indicating that NO inhibits the expression of the histone H2B gene at the level of transcription. Deletion and mutational analysis of the H2B gene promoter showed that the PPAR binding site and the region of "minimal" promoter (-65/+42 bp from transcription start) was an important for NO-dependent repression of histone H2B transcription. The peroxisome proliferator-activated receptor (PPAR) is a ligand-activated transcription factor that plays an important role in the regulation of lipid metabolism, cellular proliferation and inflammatory responses. It seems likely that NO-mediated H2B gene repression depends on modifications of endogenous PPAR ligands.

Cysteine 50 of the POU H domain determines the range of targets recognized by POU proteins.

The best target of POU proteins (Oct-1, Oct-2) is an octamer sequence ATGCAAAT. POU proteins also recognize, with weaker affinity, the TAAT-like targets of another group of regulatory factors, the homeoproteins. Up to now, it has not been known why Cys50 of the POUHdomain is absolutely conserved in contrast to that in homeoproteins. To assess the importance of Cys50 in determining the binding specificity of POU proteins, all possible amino acids were substituted for Cys at position 50, and the resulting mutants were tested with probes containing octamer (ATGCAAATNN) or homeospecific binding sites. Only the wild-type POU was shown to adequately discriminate between the octamer and homeospecific sites, and the protein affinity was only slightly affected by the nucleotide sequence flanking the octamer at the 3'-end. Any amino acid substitution at position 50 resulted in the mutant protein binding efficiently both to the octamer and the TAAT-like sequences. Moreover, in this case the 3'-flanking sequences influenced the binding to a much greater extent.

Conservative Val47 residue of POU homeodomain: role in DNA recognition.

Conservative Val47 residue, located in the third recognition helix of the Oct-2 POU domain, was alternately substituted with other 19 amino acids. Affinity and specificity of interaction with oct-site ATGCAAANGA and homeo-specific site ATAANGA were determined for all mutants. The wild type protein (with Val47) has maximal affinity and specificity in POU domain interaction with octamer sequence. However, V47I mutant showed stronger interaction with homeo-specific site. The highest specificity of interaction with homeo-site was recorded for V47S mutant. We conclude that only Val47 provides sequence-specific high-affinity binding of POU proteins with octamer targets other than the homeo-specific site. It is shown also that damages caused by point mutations may be at least partially compensated by participation in the oct-site recognition of both POUh and POUs domains.

Cell differentiation in vitro and the expression of Oct-2 protein and oct-2 RNA.

Expression of the oct-2 gene was studied in mouse tissues and during in vitro differentiation of embryocarcinoma PCC4, mouse neuroblastoma Neuro2A and NB41A3 cells in the presence of retinoic acid (RA) or 1% DMSO. oct-2 mRNA is characterized by a complex pattern and exists in both poly(A)+ and poly(A)- forms. oct-2 mRNA was found in many cell lines, whereas Oct-2 protein was detected only in some of these cells. oct-2 expression also changed during cell differentiation. The cell differentiation is likely to be controlled not only at the gene transcription level, but also at the level of regulation of nuclear transcription factor activity. Such a regulatory mechanism would provide for a finer regulation of cell differentiation.