Transcriptomic Analysis of DNA Repair Pathways in Human Non-Small Cell Lung Cancer Cells Surviving Multifraction X-Ray Irradiation.

Radioresistant sublines of non-small cell lung cancer cells differing in the p53 status were obtained: A549 (p53 wild type) and H1299 (p53 deficient). The exposure to ionizing radiation was carried out using a standard protocol developed on the basis of empirical clinical experience and consisting in exposure in a dose of 2 Gy once a day, 5 days a week up to total dose of 60 Gy. The cells survived after irradiation demonstrated reduced radiosensitivity, as well as changes in differential gene expression in comparison with parental cells. Some differences in the signaling pathways involved in DNA repair were revealed.

Potentialities of MicroRNA Diagnosis in Patients with Bladder Cancer.

Despite promising vista of the use of microRNA in molecular diagnosis of bladder cancer, there are few data on their expression profiles, which impedes assessment of diagnostic value of these marker molecules. In this study, suppression subtractive hybridization, on-chip hybridization, and high-throughput deep sequencing focused on profiling microRNA and assessing the diagnostic value of revealed marker molecules.

[Receptor tyrosine kinase KIT may regulate expression of genes involved in spontaneous regression of neuroblastoma].

Hallmark of neuroblastoma is an ability of this malignant tumor to undergo spontaneous regression or differentiation into benign tumor during any stage of the disease, but it is little known about mechanisms of these phenomena. We studied effect of receptor tyrosine kinase receptor KIT on expression of genes, which may be involved in tumor spontaneous regression. Downregulation of KIT expression by RNA interference in SH-SY5Y cells causes suppression of neurotrophin receptor NGFR expression that may promote the loss of sensibility of cells to nerve growth factors, also it causes upregulation of TrkA receptor expression which can stimulate cell differentiation or apoptosis in NGF dependent manner. Furthermore there is an upregulation of genes which stimulate malignant cell detection by immune system, such as genes of major histocompatibility complex HLA class I HLA-B and HLA-C, and interferon-γ receptors IFNGR1 and IFNGR2 genes. Thus KIT can mediate neuroblastoma cell sensibility to neurotrophins and immune system components--two factors directly contributing to spontaneous regression of neuroblastoma.

Silencing AML1-ETO gene expression leads to simultaneous activation of both pro-apoptotic and proliferation signaling.

The t(8;21)(q22;q22) rearrangement represents the most common chromosomal translocation in acute myeloid leukemia (AML). It results in a transcript encoding for the fusion protein AML1-ETO (AE) with transcription factor activity. AE is considered to be an attractive target for treating t(8;21) leukemia. However, AE expression alone is insufficient to cause transformation, and thus the potential of such therapy remains unclear. Several genes are deregulated in AML cells, including KIT that encodes a tyrosine kinase receptor. Here, we show that AML cells transduced with short hairpin RNA vector targeting AE mRNAs have a dramatic decrease in growth rate that is caused by induction of apoptosis and deregulation of the cell cycle. A reduction in KIT mRNA levels was also observed in AE-silenced cells, but silencing KIT expression reduced cell growth but did not induce apoptosis. Transcription profiling of cells that escape cell death revealed activation of a number of signaling pathways involved in cell survival and proliferation. In particular, we find that the extracellular signal-regulated kinase 2 (ERK2; also known as mitogen-activated protein kinase 1 (MAPK1)) protein could mediate activation of 23 out of 29 (79%) of these upregulated pathways and thus may be regarded as the key player in establishing the t(8;21)-positive leukemic cells resistant to AE suppression.

[Stimulation of proliferation by carnosine: cellular and transcriptome approaches].

Concentration of endogenous dipeptide carnosine in human muscle tissue reaches tens of millimoles. For more than 100 years of research, a lot of data concerning carnosine functions were accumulated, among which anti-aging effects are regarded most important. Heire, effect of carnosine in cell cultures was studied. It has been found that apart from the known action--an increase of the Hayflick limit and morphological rejuvenation--carnosine stimulates cell division in colony-forming assays and in the course of transition of cells to the quiescent state. The analysis of the transcriptome showed that carnosine-induced changes are mainly related to positive regulation of the cell cycle at all levels, from the onset of the DNA synthesis to chromosome condensation. One can suppose that the revealed stimulation of the cell cycle account for the carnosine-induced rejuvenation processes and a high concentration ofcarnosine in muscle tissue is required for the muscle recovery (regeneration) after excess loads.

[Experimental analysis of human specific protein coding open reading frame c11orf72].

Gene c11orf72 (also known as FLJ90834) included in human gene reference list was previously predicted on the basis oftranscriptome analysis. We show that c11orf72 predicted protein coding open reading frame is specific for human genome and that it is absent from DNAs of other investigated primate species (chimpanzee, macaque). For the first time, we systematically analyzed c11orf72 expression in five normal and two cancerous human tissues (testicles, heart, brain, lung, bladder, bladder tumor and testicular tumor) and found no transcriptional activity there. Promoter of c11orf72, located close to promoter of a housekeeping gene NDUFV1, has shown high methylation level, whereas NDUFV1 promoter was almost free from methylation. The protein product for cllorf72 was analyzed using heterologous expression in human cell lines NT2/D1 (Tera2) and HepG2, in N- and C-terminal fusion constructs with the fluorescent protein TurboGFP. C11orf72 protein showed no cytotoxic or promitotic activity and was distributed diffusely through the cell. Our data confirm the possibility of gain of new protein-coding genes during human evolution due to simple accumulation of point mutations. However, we found no evidence for the functional significance of gene c11orf72.

[Evolutionary recent insertions of mobile elements and their contribution to the structure of human genome].

Mobile elements are DNA fragments that are able to self-replicate within the genome of a host organism. Usually, mobile elements comprise about 40-50% of mammalian genome. In the present review, evolutionary recent insertions of mobile elements are considered which have occurred after divergence of human and chimpanzee ancestral forms, i.e. later than about 6 million years. Human-specific transposable elements are represented by relatively small number of copies that can be subdivided into four groups: HERV-K (HML-2), L1, Alu, and SVA. The number of human-specific copies of HERV-K (HML-2), L1, Alu, and SVA representatives amounts roughly to 150, 1200, 5500, and 860 copies per genome respectively. Furthermore, we succeeded in describing a new family of human-specific mobile elements that are present only in human genome and are absent in other primates. Insertions of human-specific mobile elements can be regarded as important candidates for the role of molecular-genetic agents of anthropogenesis--each new insertion of such a mobile element supplies the acceptor gene locus with the set of new functional sites for binding transcription factors that can make significant alterations to adjacent genes functioning. On the basis of known evidences confirming the influence of human-specific mobile elements on adjacent genes expression, total number of human genes regulated by them can be estimated like hundreds.

[Functional analysis of retroviral endogenous inserts in the human genome evolution].

Retroelements, mobile elements produced in DNA by reverse transcription, comprise about 40% of the human genome. A small part of these elements appeared in the genome quite recently after the divergence of humans and chimpanzees had occurred. Evolutionarily young retroelements are represented by the members of four groups, SVA, Alu, L1, and the endogenous HERV-K (HML-2) virus. These retroelements could play a functional role in the course of the molecular evolution of human DNA. We comprehensively studied the contribution of human-specific endogenous viruses (hsERV) to the structural modifications and regulation of the human genome. We found that hsERV presented in 134 copies occupied about 330 000 bp of human DNA. They added to genomic sequences the copies of 50 functional retroviral genes as well as 134 potential promoters and enhancers, 50% of which are located in the regions adjacent to known genes, and 22% in gene introns. At least 67% of these elements are human-specific promoters in vivo. hsERV viruses regulate the activity of known protein-encoding genes by means of RNA interference, function as enhancers, and provide new polyadenylation signals for mRNA.

Enhancer element potentially involved in human survivin gene promoter regulation in lung cancer cell lines.

We have revealed evolutionarily conserved regions in a 4500-bp DNA sequence 5'-adjacent to the survivin (BIRC5) gene. In the transcribed region of the BIRC5 gene we have detected and characterized in detail a 3'-fragment of the CpG island that stimulated in enhancer-like way the gene promoter activity in normal cells and in a number of cancer, in particular lung cancer, cell lines. When added to the initial 1498-bp survivin promoter region, a transcribed DNA fragment of a CpG island approximately twofold enhanced the promoter activity in cancer cells and in normal lung fibroblasts. The observed effect did not depend upon the orientation of the fragment and distances between the fragment and the transcription initiation site. In the case of a heterologous SV40 virus promoter, the effect was less pronounced. In addition to earlier reports, the results provide new information on the BIRC5 gene expression regulation and also demonstrate that this gene exon sequences can play a significant role in BIRC5 gene expression regulation. The data provide another possibility to increase survivin promoter activity without changing its cancer specificity for application in cancer (in particular, lung cancer) gene therapy.

Novel family of human transposable elements formed due to fusion of the first exon of gene MAST2 with retrotransposon SVA.

We identified a novel human-specific family of transposable elements that consists of fused copies of the CpG-island containing the first exon of gene MAST2 and retrotransposon SVA. We propose a mechanism for the formation of this family termed CpG-SVA, comprising 5'-transduction by an SVA insert. After the divergence of human and chimpanzee ancestor lineages, retrotransposon SVA has inserted into the first intron of gene MAST2 in the sense orientation. Due to splicing of an aberrant RNA driven by MAST2 promoter, but terminally processed using SVA polyadenylation signal, the first exon of MAST2 has fused to a spliced 3'-terminal fragment of SVA retrotransposon. The above ancestor CpG-SVA element due to retrotranspositions of its own copies has formed a novel family represented in the human genome by 76 members. Recruitment of a MAST2 CpG island was most likely beneficial to the hybrid retrotransposons because it could significantly increase retrotransposition frequency. Also, we show that human L1 reverse transcriptase adds an extra cytosine residue to the 3' terminus of the nascent first strand of cDNA.

[Molecular receptors of taste agents].

All representatives of higher eukaryotes can probably differentially perceive nutrients and poisonous substances. Molecular mechanisms of transduction of taste information have been best studied for mammals and for the fruit fly Drosophila. Here, we consider receptor mechanisms and conjugated primary signal processes of stimulation of taste receptor cells by stimuli of various taste modalities.

Functional significance of a putative sp1 transcription factor binding site in the survivin gene promoter.

We sequenced 1500-bp genomic DNA regions upstream from the survivin gene (BIRC5). DNA was isolated from human placenta and tumors of patients with diagnosed squamous cancer of the lung that showed high-level BIRC5 gene expression. We have revealed four new promoter allelic variants differing in single nucleotide substitutions, one variant with two nucleotide substitutions, and a variant with a TAAA tetranucleotide insertion. All promoter variants displayed low activity in cells with functionally active p53 protein and high activity in cell lines characterized by low level or absence of p53 protein function. The activity of the promoters with single nucleotide substitutions was comparable to that of the wild-type promoter, whereas two nucleotide substitutions markedly reduced the activity. We also demonstrated the functional significance of a putative Sp1 transcription factor-binding site at (-63...-54) upstream from the transcription initiation site. Mutation within this sequence led to a sharp decrease of promoter activity. The functional architecture of the survivin promoter is discussed based on results known from the literature and those obtained here.

Mouse retinal progenitor cell (RPC) cocultivation with retinal pigment epithelial cell culture affects features of RPC differentiation.

We provide evidence that coculturing of retinal progenitor cells (RPC) with retinal pigment epithelial cells significantly biases the standard in vitro RPC differentiation patterns. In particular, in cocultivation experiments RPCs lost the ability to differentiate spontaneously and displayed approximately 2.1-2.4-fold increase in immunoreactivity to the neural stem cell marker nestin and approximately 1.6-1.7-fold increase in rod photoreceptor cell rhodopsin marker immunoreactivity. The data suggest the influence of the intercellular interaction networks on RPC differentiation.

[New mechanism of retrogene formation in mammalian genomes: in vivo recombination during RNA reverse transcription].

L1 LINE retrotransposons play an important role in the shaping and permanent evolution of mammalian genomes. In particular, occupying about 20% of genomic DNA, they transduce their 3' flanking sequences to new genomic loci and create pseudogenes by reverse transcribing different kinds of cellular RNAs. Recently we discovered in mammalian genomes several families of chimeric pseudogenes, consisting of fused copies of various cellular transcripts. The characteristic peculiarities of such chimeric inserts suggest the involvement of L1 enzymatic machinery in their formation. The detailed sequence analysis revealed that 5' terminal parts of the chimeras are copies of nuclear RNAs, while 3' terminal sequences were formed on the templates of transcripts having cytoplasmic orientation. These data enabled us to propose the mechanism for the chimeras formation, comprising the switch of templates during RNA reverse transcription by L1 reverse transcriptase. The further identification of not only "double", but also "triple" chimeric retrogenes evidences the possibility of the double template switches as well. Some of the found chimeras are transcriptionally active, thus allowing to consider the discovered phenomenon as the new mechanism of the gene formation by "shuffling" of pre-existing transcribed sequences. Being active in mammals now, the mechanism appeared at least 75 million years ago and is evolutionary conserved.

[Multiple template switches on LINE-directed reverse transcription: the most probable formation mechanism for the double and triple chimeric retroelements in mammals].

It was shown that the shuffling mechanism for transcribed genome components, which involves a template switch on the RNA reverse transcription using the L1 retroelement enzymatic machinery, is common in mammals. The occurrence frequency of the resulting chimeric retroelements in the genomes of rodents is twice as high as in the DNA of primates. Moreover, we proved that not only single but also double switches may occur in vivo, which result in the fusion of copies of three different transcripts. Many of the identified chimeras are transcribed in mammals.

Retroelements and formation of chimeric retrogenes.

It is very likely that formation of new genes is the main pathway of molecular evolution in living organisms. Many such genes are products of preexisting reshuffling of genetic material. In these processes a very important role is played by mutations associated with the activity of transposable elements, mostly retroelements (REs) for higher eukaryotes. The life cycle of REs involves a stage of so-called reverse transcription of their RNA intermediates, i.e. synthesis of complementary DNA on an RNA template. Transcriptionally active sequences of RE origin are referred to as retrogenes. REs create chimeric genes by a variety of mechanisms: new RE insertions, recombinations between RE sequences, formation of functional gene active pseudogenes and template switches during reverse transcription of messenger RNA. The abovementioned events are also able to give rise to new RE families. These mechanisms are reviewed here along with the description of major RE groups.

[Human genome-specific HERV-K intron LTR genes have a random orientation relative to the direction of transcription, and, possibly, participated in antisense gene expression regulation]. / Spetsifichnye dlia genoma cheloveka LTR HERV-K v intronakh genov imeiut nesluchainuiu orientatsiiu otnositel'no napravleniia transkriptsii i, vozmozhno, prinimaiut uchastie v antisens-reguliatsii ékspressii genov.

A consensus nucleotide sequence of long terminal repeats (LTRs) of endogenous human-specific retroviruses of the K family (HERV-K) was constructed and used for the genome-wide search for homologies in international databases. There were revealed 142 LTRs, 12 of which were localized in introns of unique human genes. It was found for the first time that ten intron LTRs are absent in the orthologic loci of the chimpanzee genome and the orientation of nine of them is opposite to the transcription direction of the corresponding human genes. A hypothesis was propounded that the found LTRs affect the gene expression by initiation of the antisense RNA synthesis.

Interaction of 65- and 62-kD proteins from the apical membranes of the Aedes aegypti larvae midgut epithelium with Cry4B and Cry11A endotoxins of Bacillus thuringiensis.

A protein with the molecular weight of 65 kD is the only component of Aedes aegypti larvae BBM capable to specifically bind mosquitocidal toxins Cry4B and Cry11A of Bacillus thuringiensis. This protein lacks the leucine aminopeptidase activity which is characteristic for the toxin-binding proteins from the membranes of caterpillars. Cry-toxins inactive against A. aegypti larvae either fail to bind to the 65-kD protein and to a putative product of its proteolysis with the molecular weight of 62 kD (Cry1Ab), or bind but do not compete for this binding with mosquitocidal proteins (Cry9A). The proteolytic splitting out of the first five alpha-helices in the Cry4B toxin molecule does not affect its binding to the 65- and 62-kD proteins, but an additional removal of 20-30 amino acids from the C-terminal of the molecule sharply spoils this binding. Monosaccharide residues are not involved in the binding of the 65- and 62-kD proteins with Cry4B, Cry11A, and Cry9A.

Membrane proteins of Aedes aegypti larvae bind toxins Cry4B and Cry11A of Bacillus thuringiensis ssp. israelensis.

Proteins of molecular weight 65 and 62 kD and having affinity for toxins Cry4B and Cry11A produced by Bacillus thuringiensis ssp. israelensis have been isolated from brush border membranes of Aedes aegypti larvae using affinity chromatography. Using a ligand blotting technique, we show that the binding of these proteins to the biotinylated toxins is reversible and that the two toxins compete for binding to the two proteins. These proteins are likely to be Cry4B and Cry11A toxin receptors in gut epithelial cells of Aedes aegypti larvae.