[Determination of molecular glioblastoma subclasses on the basis of analysis of gene expression].

Two glioblastoma groups, which are distinguished from each other by expression level of 416 genes (P < 0.05), were determined using a mathematical model of linear Boolean programming on the basis of gene expression data, obtained by microarray analysis of the glioblastomas and available in Gene Expression Omnibus (GEO) data base. The expression level of 15 genes was more than two-fold higher in the first group of glioblastoma (80 samples) in comparison with the second group (144 samples) and 401 genes and--more than two-fold lower as compared to the second group. 10 of 15 genes, which expression level prevailed in the first group, encode the proteins involved in cell cycle regulation and cell proliferation. A significant percentage of 401 genes are the genes that encode proteins involved in the functioning of neural cells and participating in the processes such as synaptic transmission, neurogenesis, the formation of myelin sheath, axon formation. Kohonen map, built on the basis of the data of 15 genes with prevailed expression in the first group and 60 (of4 01) genes, whose expression level elevated in the second group, confirmed the existence of two glioblastoma groups with specific gene expression profiles. Distribution of the glioblastomas into two groups may reflect two pathways of astrocytic glioma development, one of which leads to the formation of tumors with higher levels of gene expression, which protein products are involved in cell cycle regulation and proliferation. On the other hand, the existence of two molecular variants may reflect different states of glioblastoma progression.

Immortalization and malignant transformation of eukaryotic cells.

The process of cellular transformation has been amply studied in vitro using immortalized cell lines. Immortalized cells never have the normal diploid karyotype, nevertheless, they cannot grow over one another in cell culture (contact inhibition), do not form colonies in soft agar (anchorage-dependent growth) and do not form tumors when injected into immunodeficient rodents. All these characteristics can be obtained with additional chromosome changes. Multiple genetic rearrangements, including whole chromosome and gene copy number gains and losses, chromosome translocations, gene mutations are necessary for establishing the malignant cell phenotype. Most of the experiments detecting transforming ability of genes overexpressed and/or mutated in tumors (oncogenes) were performed using mouse embryonic fibroblasts (MEFs), NIH3T3 mouse fibroblast cell line, human embryonic kidney 293 cell line (HEK293), and human mammary epithelial cell lines (mainly HMECs and MC-F10A). These cell lines have abnormal karyotypes and are prone to progress to malignantly transformed cells. This review is aimed at understanding the mechanisms of cell immortalization by different "immortalizing agents", oncogene-induced cell transformation of immortalized cells and moderate response of the advanced tumors to anticancer therapy in the light of tumor "oncogene and chromosome addiction", intra-/intertumor heterogeneity, and chromosome instability.

Expression of genes belonging to the IGF-system in glial tumors.

Increased expression of the insulin-like growth factor (IGF) family members, IGF1, IGF2, their receptors and binding proteins, or combinations thereof has been documented in various malignancies including gliomas. The results of multiple investigations suggest that the IGFs can play a paracrine and/or autocrine role in promoting tumor growth in situ during tumor progression but that these roles may vary depending on the tissue of origin. Enhanced IGF1 expression was not found in glioblastomas and it was supposed that IGF1 participation in the development of glial tumors may be substituted by protein products of highly expressed other genes, also participating in PI3K and MAPK pathways. Increased expression of IGF-binding protein genes in brain tumors makes the picture even more complicated. As other binding proteins, IGFBPs regulate the activity of their ligands by prolonging their half-life. The discrepancies arising from conflicting evidence on the results obtained by different laboratories in human gliomas are discussed. Our data highlight the importance of viewing the IGF-related proteins as a complex multifactorial system and show that changes in the expression levels of any one component of the system, in a given malignancy, should be interpreted with caution. As IGF targeting for anticancer therapy is rapidly becoming clinical reality, an understanding of this complexity is very timely.

Chitinase 3-like protein 2 (CHI3L2, YKL-39) activates phosphorylation of extracellular signal-regulated kinases ERK1/ERK2 in human embryonic kidney (HEK293) and human glioblastoma (U87 MG) cells.

Human cartilage chitinase 3-like protein 2 (CHI3L2, YKL-39) is secreted by articular chondrocytes, also synoviocytes, lung, and heart. Increased levels of YKL-39 have been demonstrated in synovial fluids of patients with rheumatoid or osteoarthritis as well as in some other pathologies and in malignant tumors, particularly in glioblastomas. It belongs to glycosyl hydrolase family 18 and the most closely related to human cartilage glycoprotein 39 (HC gp-39 or chitinase 3-like protein 1, CHI3L1 or YKL-40), which as it was shown previously, promotes the growth of human synovial cells as well as skin and fetal lung fibroblasts. Dose-dependent growth stimulation was observed when the fibroblastic cell line was exposed to YKL-40 in a concentration range from 0.1 to 2 nM, which is similar to the effective dose of the well characterized mitogen, insulin-like growth factor 1. The use of selective inhibitors of the mitogen-activated protein kinase (MAP kinase) signaling pathway indicates that both, YKL-40 and IGF-I are involved in phosphorylation of extracellular signal-regulated kinases 1 and 2 (ERK1/ERK2). Thus YKL-40 initiates a signaling cascade which leads to increased cell proliferation, suggesting that this protein could play some role in the inhibition of apoptosis. We report here that YKL-39, which as YKL-40 has significantly increased expression in glioblastomas, also activates signal-regulated kinases ERK1/ERK2 in human embryonic kidney (HEK293) and human glioblastoma (U87 MG) cells.

[Expression of myelin basic protein and glial fibrillary acidic protein genes in human glial brain tumors].

Analysis of the expression of genes encoding myelin basic protein (MBP) and glial fibrillary acidic protein (GFAP) genes in human glial tumors was carried out for determination of the expression specificity of these genes according to tumor types and their malignancy. Low levels of MBP mRNA in astrocytoma specimens of malignancy grades II-IV and significantly higher levels in perifocal zone adjacent to them have been determined by Northern hybridization. Diffuse astrocytomas and anaplastic astrocytomas are characterized mostly by low level of MBP gene expression and high level of GFAP gene expression, but distinct subtypes of diffuse and anaplastic astrocytomas with high level of MBP gene and low level of GFAP gene expression can be also detected that may be the reflection of different oncogenic pathways. Very low levels or even absence of MBP mRNA were revealed in oligodendroglioma and all oligoastrocytomas. Thus, Northern hybridization data are correlated with Serial Analysis of Gene Expression (SAGE). Obtained results show that MBP is nonspecific marker for tumors of oligodendroglial origin, but determination of relative levels of MBP and GFAP mRNAs may be useful for glial tumors recognition. By such a way, these two genes together with previously found by us YKL-40 and TSC-22 can be included into the gene panel for the determination of so called "gene signatures" of brain tumors. However, severe requirements in relation to a clinical value of these "gene signatures" can not be formulated without their verification on plenty of clinical samples of tumors and valid control.

Comparison of microarray and sage techniques in gene expression analysis of human glioblastoma.

To enhance glioblastoma (GB) marker discovery we compared gene expression in GB with human normal brain (NB) by accessing SAGE Genie web site and compared obtained results with published data. Nine GB and five NB SAGE-libraries were analyzed using the Digital Gene Expression Displayer (DGED), the results of DGED were tested by Northern blot analysis and RT-PCR of arbitrary selected genes. Review of available data from the articles on gene expression profiling by microarray-based hybridization showed as few as 35 overlapped genes with increased expression in GB. Some of them were identified in four articles, but most genes in three or even in two investigations. There was found also some differences between SAGE results of GB analysis. Digital Gene Expression Displayer approach revealed 676 genes differentially expressed in GB vs. NB with cut-off ratio: twofold change and P < or = 0.05. Differential expression of selectedgenes obtained by DGED was confirmed by Northern analysis and RT-PCR. Altogether, only 105 of 955 genes presented in published investigations were among the genes obtained by DGED. Comparison of the results obtained by microarrays and SAGE is very complicated because authors present only the most prominent differentially expressed genes. However, even available data give quite poor overlapping of genes revealed by microarrays. Some differences between results obtained by SAGE in different investigations can be explained by high dependence on the statistical methods used. As for now, the best solution to search for molecular tumor markers is to compare all available results and to select only those genes, which significant expression in tumor combined with very low expression in normal tissues was reproduced in several articles. 105 differentially expressed genes, common to both methods, can be included in the list of candidates for the molecular typing of GBs. Some genes, encoded cell surface or extra-cellular proteins may be useful for targeting gliomas with antibody-based therapy.

A growth hormone pseudogene in the salmon genome.

Representatives of the fish family Salmonidae were reported to possess two nonallelic growth hormone (GH)-encoding genes. In addition to those, we found a third GH-like sequence in a chum salmon genomic DNA library. A number of point mutations and large deletions abolished the possibility of expressing this sequence, showing that the chum salmon genomic DNA contains a GH pseudogene besides functional GH genes.

Nucleotide sequence analysis of the cloned salmon preproinsulin cDNA.

A cDNA library was constructed using polyadenylated RNA from salmon (Oncorhynchus keta) Brockmann bodies, plasmid vector pBR322, and in vitro recombinant DNA techniques. Insulin-related clones were identified with a cDNA probe generated from the same RNA and enriched for insulin sequences. Two recombinants were shown to contain the nucleotide sequence of the entire coding region and parts of the 5' and 3' untranslated regions. The salmon preproinsulin mRNA is about 760 nucleotides long, 315 of which code for the protein, while about 190 and 200 nucleotides belong to the 5' and 3' flanking regions, respectively. Comparison of the nucleotide sequences of salmon insulin mRNA with those from other species reveals that sequence conservation is limited to the regions coding for the B and A peptides and two segments of the signal peptide. The C-peptide region exhibits no significant sequence homology with the C-peptides of other vertebrates. The 5' and 3' untranslated regions of the salmon preproinsulin mRNA are homologous only with the anglerfish mRNA, whereas there is no evident homology with those of birds and mammals. In addition to establishing the sequence of the preproinsulin mRNA, cloned salmon insulin cDNA provides a specific probe for the analysis and isolation of genomic DNA fragments containing insulin genes.

Variability of polyadenylation sites in mRNAs from human fetal liver.

cDNA libraries of human fetal liver were constructed in pBR322 and lambda gt10 vectors. The libraries were screened for liver-specific clones by differential hybridization. This procedure revealed 25 and 32 liver-specific clones in plasmid and phage libraries, respectively. The majority of these clones were represented with serum albumin, fetal G gamma-globin and A gamma-globin cDNA inserts. Three types of 3'-non-coding region were found in 5 sequenced albumin cDNAs. In one type mRNA the distance between the AATAAA signal and polyadenylation site was 15 nucleotides, in 2 other types this distance was 10 and 6 nucleotides. The polyadenylation site in the G gamma-globin cDNA was located 2 nucleotides further from AATAAA signal, while in the A gamma-globin cDNA it was 2 nucleotides closer to the signal as compared with the results published previously.

Organization and expression of the chum salmon insulin-like growth factor II gene.

IGF-II plays an important role in growth and development of vertebrates. In the present study, the characterization of the first fish IGF-II gene, chum salmon IGF-II, is described. The sIGF-II gene consists of four exons, spanning a region of 9 kbp, that encode the 214 aa IGF-II precursor. While the amino acid sequences of fully processed IGF-II of salmon and mammalian species are very similar, the prepro-peptide sequence deviates extensively in the signal- and E-peptide domains. The transcription initiation site of the sIGF-II gene was localized within a 30 nt region employing RT-PCR. Using sIGF-II promoter-luciferase constructs it was demonstrated that the sIGF-II gene has a relatively strong promoter that contains tissue-specific regulatory elements.

The chum salmon IGF-II gene promoter is activated by hepatocyte nuclear factor 3beta.

IGF-II plays an important role in growth and development of vertebrates and is highly expressed in adult salmon liver. In the present study, we demonstrate that a liver-enriched transcription factor, hepatocyte nuclear factor 3beta (HNF-3beta), is an activator of the chum salmon IGF-II gene. Multiple binding sites for HNF-3beta were identified within the 5'-UTR using electrophoretic mobility shift assays and mutation of these sites prevents binding of HNF-3beta. In transient transfection assays it was shown that mutation of the HNF-3beta binding sites results in a substantial decrease of HNF-3beta-activated salmon IGF-II gene expression. This is the first identified transcription factor that is functionally involved in the regulation of fish IGF-II expression.

3'-Hydroxymethyl 2'-deoxynucleoside 5'-triphosphates are inhibitors highly specific for reverse transcriptase.

dNTP(3'-OCH3), a 3'-O-methyl derivative of dNTP, is a chain terminator substrate for DNA synthesis catalyzed by AMV reverse transcriptase. The enzyme seems to be the only DNA polymerase susceptible to the inhibitor while all the other DNA polymerases tested are fully resistant to the nucleotide analog. The resistant polymerases are: E. coli DNA polymerase I, Klenow's fragment of DNA polymerase I, phage T4 DNA polymerase, calf thymus DNA polymerase alpha, rat liver DNA polymerase beta and calf thymus terminal deoxyribonucleotidyl transferase.

A comparison of the initiating abilities of ribo- and deoxyriboprimers in DNA polymerization catalyzed by AMV reverse transcriptase.

The difference in optimal conditions for DNA polymerization catalyzed by AMV reverse transcriptase on poly(A) and poly(dA) templates with d(pT)10 and (pU)10 primers has been found. A comparison of the initiating abilities of d(pT)10 and (pU)10 primers under optimal conditions for various template.primer complexes has been made. The best template.primer complex was poly(A).d(pT)10 and the worst was poly(A).(pU)10. The lengthening of d(pT)n primers by a mononucleotide unit (n = 2-10) increases their affinity by a factor of about 2 and 3 in the case of poly(dA) and poly(A) templates, respectively. The affinities of d(pT) to the enzyme does not change with the primer length.

5'-derivatives of oligonucleotides as primers of DNA polymerization catalyzed by AMV reverse transcriptase and Klenow fragment of DNA polymerase 1.

The Km and Vmax values for d(pT)8 and its derivatives containing various 5'-end groups were estimated in the reaction of polymerization catalyzed with AMV-RT and FK. The change in affinity of modified primers was more pronounced in the case of AMV-RT than in the case of FK. Introducing in d(pT)8 of intercalators such as phenazinium, ethidium and daunomycin residues results in 2.7-, 8.7- and 11-fold increases in the primer affinity to AMV-RT, respectively. However, in the case of hemin and cholesterol derivatives the Km values were 3 and 5 times higher than those for d(pT)8. Compared to d(pT)8, the affinity of FK to all the above analogs was 2.3-3.6 times higher with the exception of cholesterol derivative to which it was 2.4-fold lower. The effect of the 5'-end residues on the Vmax values of d(pT)8 was small and ranged from 44% to 120% of that for d(pT)8. Therefore such reactive derivatives of oligonucleotides can be used as effective primers of AMV-RT and FK. Possible reasons for various effects of the 5'-end residues of the primer on its interaction with FK or AMV-RT in the presence of poly(A) are discussed.

The chum salmon insulin-like growth factor II promoter requires Sp1 for its activation by C/EBPbeta.

The chum salmon insulin-like growth factor II (IGF-II) gene is highly expressed in liver tissue. In this study we demonstrate that two transcription factors, Sp1 and C/EBPbeta, are involved in the enhanced expression of the salmon IGF-II gene. The presence of the fish homolog for C/EBPbeta in salmon liver RNA was confirmed by Northern blotting. The sIGF-II promoter was activated up to 20-fold by co-transfection with C/EBPbeta. The functional importance of four out of the five putative C/EBPbeta binding sites was demonstrated with mutational analysis in transient transfection assays. The transcription factor Sp1 binds to two sites within the salmon IGF-II promoter. Interestingly, mutation of the Sp1 binding sites decreases not only the basal IGF-II promoter activity but also the C/EBPbeta-induced transactivation. These results demonstrate that liver-enriched C/EBPbeta and ubiquitously expressed Sp1 each activate the sIGF-II promoter and that Sp1 is required for full transactivation of the sIGF-II gene by C/EBPbeta. This suggests that C/EBPbeta and Sp1 act in synergy.

Isolation of a second nonallelic insulin-like growth factor I gene from the salmon genome.

We have characterized a second nonallelic insulin-like growth factor-I (IGF-I) gene in the chum salmon (Oncorhynchus keta) genome. This gene, IGF-I.2, differs from the previously described chum salmon IGF-I gene, IGF-I.1, in the E peptide-coding portion of exon 3; specifically, the IGF-I.2 gene lacks one codon present in the IGF-I gene and contains two potential splice donor sites at the 3' end of exon 3 rather than the single, more distal site present in the IGF-I.1 gene. The expression of these two IGF-I genes could give rise to as many as six IGF-I mRNA species, each of which would encode a unique E-peptide moiety of the IGF-I prohormone. Thus, the presence of multiple, distinct IGF genes adds an additional level of complexity to IGF-I gene expression and IGF-I biosynthesis in salmon.

Structure of the chum salmon insulin-like growth factor I gene.

Insulin-like growth factor I (IGF-I) plays a major role in development and metabolism. Currently, the cDNA-derived primary structure of IGF-I is known for some mammals and for chicken, frog, and salmon. Additionally, the organization of the human, rat, and chicken IGF-I genes has been established. The investigation of IGF-I gene structure in fish would extend the evolutionary picture for this hormone and facilitate our understanding of the features of the IGF-I gene that are common to all vertebrate species. The cloned chum salmon IGF-I gene appears to be much more compact than the mammalian and avian genes, being less than 20 kb in length. As in other species, however, the mature IGF-I peptide appears to consist of 70 amino acids and is encoded by exons 2 and 3. Intriguingly, exon 1-encoded 5'-untranslated region sequences are highly conserved, while the coding sequences at the 3' end of the same exon are less conserved. The amino terminus of the signal peptide is four amino acids shorter than in the mammalian and avian peptides. The end of the B domain, the C, A, and D domains, and the first part of the E peptide are encoded by exon 3, but the exon 3-encoded E peptide sequence is 27 amino acids longer than in other species. These extra 27 amino acids, encoded by both coho and chum salmon cDNAs, may be deleted by alternative splicing, as suggested from the sequence of a coho salmon IGF-I cDNA.(ABSTRACT TRUNCATED AT 250 WORDS)

[Reverse transcription of the RNA of eukaryotes and viruses. Conditions for obtaining a long product]. / Obratnaia transkriptsiia RNK éukariot i virusov. Usloviia polucheniia dlinnogo produkta.

The receiving of full-size DNA-copies of RNAs is necessary for molecular hybridisation experiments as well as for synthesis of recombinant bacterial plasmids with eucariotic DNA sequences. Some authors received such cDNAs for different RNAs with a help of variations in reaction conditions. In this article it is shown that such empirically chosen conditions mainly had an influence on a secondary structure of RNA-templates and that only in such a case the synthesis of the DNA-product with sizes corresponding to the RNA-template is possible.

[Splicing. I. Splicing of tRNA, rRNA and mRNA in organelles]. / Splaising. I. Splaising tRNK, pRNK i mRNK v organellakh.

Mosaic structure of genes is shown for three out of four known types of RNA: transfer, ribosomal and messenger. At least three different mechanisms are involved in maturation of transcripts from these genes. The peculiarity of tRNA splicing is connected with the possibility of existence of tRNA molecules free of ribonucleoprotein complexes. The mode of Tetrahymena pre-tRNA self-splicing may also take place during maturation of other RNAs (rRNA of protozoa, ribosomal and messenger rRNA of lower fungi, plant messenger rRNA), which share the similar structure. The third type of mechanism is involved in splicing messenger RNA in eukaryotic cell nuclei.

[Splicing. 2. Splicing of mRNA in the cell nucleus]. / Splaising. 2. Splaising mRNK v iadre.

The mosaic structure of genes is shown for three out of four known types of RNAs, namely transfer, ribosomal and messenger. At least three different splicing mechanisms are involved in maturation of transcripts of these genes, two of them had been described in the previous review. Well-developed effective in vitro splicing systems permit to provide a scheme for the nuclear pre-mRNA maturation processes which take place presumably in specific ribonucleoprotein complex particles. Alternative splicing character of these pre-mRNAs, yields in increased variety of the encoded proteins.