Characterization of Sp1, AP-1, CBF and KRC binding sites and minisatellite DNA as functional elements of the metastasis-associated mts1/S100A4 gene intronic enhancer.

The mts1/S100A4 gene encodes a small acidic calcium-binding protein that is expressed in a cell-specific manner in development, tumorigenesis and certain tissues of adult mice. A composite enhancer that is active in murine mammary adenocarcinoma cells was previously identified in the first intron of the mts1/S100A4 gene. Here we present a detailed analysis of the structure and function of this enhancer in the Mts1/S100A4-expressing CSML100 and non-expressing CSML0 mouse adenocarcinoma cell lines. In CSML100 cells the enhancer activity is composed of at least six cis-elements interacting with Sp1 and AP-1 family members and CBF/AML/PEBP2 and KRC transcription factors. In addition, a minisatellite-like DNA sequence significantly contributes to the enhancer activity via interaction with abundant proteins, which likely have been described previously under the name minisatellite-binding proteins. Extensive mutational analysis of the mts1/S100A4 enhancer revealed a cooperative function of KRC and the factors binding minisatellite DNA. This is the first example of an enhancer where two nuclear factors earlier implicated in different recombination processes cooperate to activate transcription. In Mts1/S100A4-negative CSML0 cells the strength of the enhancer was 7- to 12.5-fold lower compared to that in CSML100 cells, when referred to the activities of three viral promoters. In CSML0 cells the enhancer could be activated by exogenous AP-1 and CBF transcription factors.

Novel AP-1 binding site created by DNA-methylation.

DNA-methylation is known to repress transcription either by inactivation of positive regulatory cis-elements containing CpG dinucleotides or via the sequence-nonspecific and methylation-specific binding of inhibiting methyl-CpG dinucleotides or via the sequence-nonspecific and methylation-specific binding of inhibiting methyl-CpG binding protein 1 (MeCP1). In the present work we describe the novel way DNA-methylation can influence gene expression: a binding site for transcription factors AP-1 might be created by DNA-methylation. Such a DNA-methylation-dependent AP-1 binding site was found in the first intron of the metastasis-associated mts1 gene. The expression level of this gene correlates with the hypomethylation of the mts1 first intron sequence in mouse adenocarcinoma cells. The DNA - methylation-dependent AP-1 binding site was found to be functionally active in the nucleotide context of the mts1 gene. When methylated, this site reproducibly repressed transcription of CAT-containing DNA that had been transiently transfected into mouse adenocarcinoma CSML100 cells.

Studies on dissociation and reconstitution of nuclear 30-S ribonucleoprotein particles containing pre-mRNA.

Treatment of nuclear 30-S ribonucleoprotein (RNP) particles containing pre-mRNA (precursor of mRNA) with 2 M NaCl leads to dissociation of RNA and protein. The protein component is present either as an aggregate with a sedimentation coefficient close to 30 S (a free informofer) or as a slowly sedimenting material (monomers or oligomers of informatin). Most of the informofers and slowly sedimenting material are in the equilibrium state. Iodination or aging of the 30-S particles stabilizes informofers. Lowering of NaCl concentration in the mixture of RNA with informofers or informatin subunits leads to reconstitution of RNP particles. In both cases, the particles formed have a sedimentation coefficient of about 30 S and a buoyant density equal to 1.4-1.41 g/cm3 but their response to pancreatic RNAase (EC 3.1.27.5) and high salt treatment is very different. Both the particles reconstituted from RNA and informofers and the original particles are very sensitive to pancreatic RNAase and after high salt treatment free informofers are formed. In contrast, the RNA of the particles reconstituted from slowly sedimenting material is much more protected against pancreatic RNAase action. These particles are also rather stable to high salt treatment. Thus, only if a protein in the form of an informofer aggregate is used, faithful reconstitution takes place. The data obtained are discussed in terms of the structure of the nuclear ribonucleoprotein particles containing precursor of messenger RNA.

A minisatellite "core" element constitutes a novel, chromatin-specific activator of mts1 gene transcription.

Expression of the mts1 gene is often associated with malignant transformation of tumor cells. Transcription of the gene is controlled by a number of positive and negative regulatory elements, all of them being localized in the first intron (+38 to +1215) of the mts1 gene. Through analysis of the distribution of DNase I hypersensitive sites in the first intron of the gene we revealed a structurally conserved region that consisted of a non-canonical NFkB binding site and a minisatellite "core" element. Deletion of the minisatellite core DNA in the context of the first intron had no effect on its regulatory capacity when assayed in transient transfections, while a fivefold decrease was observed in a pool of stably transfected cells. The minisatellite core sequence CTGGGCAGGCAG is involved in DNA-protein interactions in vivo, and is similar to a binding site for the previously identified minisatellite DNA sequence binding protein (Msbp-1). The core DNA interacted in vitro with a protein that had an apparent molecular mass of 40 kDa. These data indicate that the minisatellite DNA represents the novel, chromatin-specific element in the mts1 complex enhancer.

Activation of mts1 transcription by insertion of a retrovirus-like IAP element.

The mechanism of activation of mestatasis-associated mts1 gene transcription in the mouse myelomonocytic leukaemia WEHI-3 cell line is described. Northern blot analysis showed that WEHi-3 cells expressed two types of mts1-specific mRNA: standard (550 nt) and additional (about 800 nt). The steady-state expression level of the 800-nt RNA was isolated and sequence analysis showed that it contained a 357-bp fragment represented by long terminal repeat (LTR) sequences and a 5' untranslated region of the gag gene of the intracisternal A-particle (IAP) element. The chimeric IAP::mts1 800-nt mRNA is initiated from the 5' LTR promoter. The rearranged and normal loci of mts1 were cloned and partially sequenced. The results suggested that the insertion of the IAP sequences into the first intron of mts1 brings the gene under control of the strong IAP promoter. The additional chimeric 800-nt IAP::mts1 RNA transcript was the result of a splicing event linking IAP sequences with the coding part of mts1. We suggest that the chimeric IAP::mts1 RNA is capable of producing a functional Mts1 protein.

Structure of gene mts1, transcribed in metastatic mouse tumor cells.

Different oncogenes are implicated in the genesis of tumors. However, little is known so far about the genes which are activated at the latest stages of tumor progression. While studying two genetically related mouse lines, highly metastatic CSML-100 and nearly nonmetastatic CSML-0, we have cloned the cDNA of the gene, mts1, which is specifically expressed in different metastatic cells. The gene contains an open reading frame of 101 amino acids and shows homology with a family of Ca2(+)-binding proteins. Here, we present data on the structure of a 17-kb genomic clone of mts1 with surrounding sequences. The gene contains two introns and three exons. The mts1 upstream region has been cloned in a plasmid containing the cat gene. The results of transient expression of the mts1-cat plasmid in NIH3T3 cells indicate the presence of a transcription regulator of mts1.

The mts1 gene and control of tumor metastasis.

The main stream of biology today is the analysis of the molecular mechanisms of major biological phenomena through studies of the genes governing these processes and their protein products. An example is the problem of tumor metastasis which is extremely important both theoretically and practically. Here we describe the data obtained on the detection, cloning, structure and transcription control of the mts1 gene, that encodes metastasin 1, a protein which seems to play an important role in the control of metastasis in mouse tumors. In particular, the experiments on tumor cell transfection with constructions containing either a sense or antisense mts1 sequence under a strong promoter/enhancer element show the direct dependence of the metastatic phenotype on the expression of the mts1 gene at least in some systems. Gene mts1 encodes a protein belonging to the family of Ca(2+)-binding proteins and may be involved in the control of cell motility in different types of cells, such as macrophages and T-lymphocytes. The relationship between mts1 and other genes up- and down-regulated in metastatic cells is discussed.

The differentiation antigen Ly-6E.1 is expressed in mouse metastatic tumor cell lines.

We report the cloning of the mouse surface GPI-anchored Ly-6E.1 protein from a highly metastatic mouse adenocarcinoma cell line CSML-100 by differential display. The expression is specific for the metastatic cell line as the closely related, non-metastatic mouse adenocarcinoma cell line CSML-0 does not express Ly-6E.1. Northern blot analysis reveals expression in a number of mouse tumour cell lines, exclusively metastatic ones. To date, active Ly-6A/E has only been described in lymphoid cells. The correlation between Ly-6E.1 expression, and the ability to metastasize, is discussed.

Spectral studies on the calcium-binding properties of Mts1 protein and its interaction with target protein.

Two calcium-binding sites of the Mts1 protein, a member of S-100 protein family, were distinguished with the Fluo-3 fluorescent technique. The geometric mean of the apparent dissociation constant (Kd) for these two sites is 2.6 microM; the Hill coefficient (nH) is 0.98. In the presence of a novel target protein p37, isolated from the mouse adenocarcinoma cell line CSML-100, Mts1 binds Ca2+ ions with higher affinity and with strong positive cooperativity (Kd = 0.2 microM, nH = 1.91). Interaction of Mts1 with p37 is confirmed by the fluorescent probe 2-p-toluidinylnaphthalene-6-sulfonate (TNS). Reaction with TNS shows that p37 interacts with the hydrophobic site of Mts1 which is exposed due to the binding of Ca2+ ions.

Separation of the pore-forming and cytotoxic activities from natural killer cell cytotoxic factor.

The influence of Ca2+ on the cytotoxic activity of natural killer cell cytotoxic factor (NKCF) was analyzed. When the natural killer susceptible cell line K562 was exposed to NKCF in the presence of 5 mM Ca2+, two peaks of cell damage were found. The first peak was observed after 30-40 min of incubation as a result of pore formation on the surface of target cells. The second was a peak of cytolytic activity which appeared after 24 h of incubation. Upon dilution of the NKCF preparation, only the first peak was observed. Therefore, NKCF produced by large granular lymphocytes in response to K562 consists of different proteins and represents pore-forming and cytolytic activities.

Time-dependent changes of LAK cell phenotypes correlate with the secretion of different cytotoxic proteins.

Human lymphokine-activated killer (LAK) cells were generated from peripheral blood lymphocytes (PBL) of normal volunteers by interleukin-2 (IL-2) stimulation for 1-8 days. During the first 3 days the surface marker CD16 characteristic for natural killer (NK) cells was expressed and later the CD3 marker characteristic for cytotoxic T cells became predominant. The conditioned media of LAK cells collected after interaction of LAK cells with K562 target cells was chromatographically separated into two cytotoxic fractions: F1 and F2. It was demonstrated that fraction F1 contained cytotoxic proteins having molecular weights of 30 and 40 kDa, and fraction F2 contained cytotoxic proteins having molecular weights of 22, 38 and 75 kDa. The presence of the proteins in each of these two fractions correlated with the phenotype changes of LAK cells: the F2 cytotoxic proteins were characteristic for NK-like cells, and the F1 proteins for cytotoxic T-lymphocyte (CTL)-like phenotypes.

Inhibitory effect of calf fetuin on the cytotoxic activity of LAK cell-derived factors and tumor necrosis factor.

A protein-inhibiting LAK cell-mediated cytotoxicity was isolated from a LAK cell-conditioned medium. The N-terminal amino acid sequence of this protein appeared to be identical to fetal calf fetuin. Pure isolated fetuin, as well as commercially available preparations of this protein, were shown to inhibit cytotoxic activity of both cytotoxic proteins released by LAK cells and TNF.

[A preparation of short DNA chains synthesized in vivo after introduction into DNA from live cells of trioxsalen crosslinks is not enriched by replication initiation regions]. / Preparat korotkikh tsepei DNK, sintezirovannykh in vivo posle vvedeniia v DNK zhivykh kletok trioksalenovykh shivok, ne obogashchen uchastkami nachala replikatsii.

The experiments undertaken in order to verify the trioxsalen-crosslinking method suggested by Russev and Vassilev for isolation of eukaryotic replication origins are described. It was found that the preparation of viral DNA isolated by the above mentioned method from CV-1 cells lytically infected with SV40 was not enriched in sequences including SV40 replication origin. The hybridization pattern of DNA preparation isolated by the trioxsalen-crosslinking procedure from chicken erythroblastosis cells with the cloned fragments of globin gene domain was found to be identical to those of the total DNA probe. The DNA fraction enriched in replication origins was isolated from the same cells with the aid of nascent DNA strand extrusion method by Zannis-Hadjopoulos et al. The hybridization pattern of this DNA fraction with the cloned fragments of chicken alpha-globin gene domain was different from those of total DNA. Taking together, the results of our experiments demonstrate that trioxsalen-crosslinking procedure does not lead to the isolation of replication origins from the objects studied in the present investigation.

[Spatial organization of replicons in the eukaryotic nucleus: attachment of replication initiation regions to the nuclear skeleton]. / Prostranstvennaia organizatsiia replikonov v éukarioticheskom iadre: prikreplenie uchastkov nachala replikatsii k iadernomu skeletu.

The DNA fraction enriched in replication origins was isolated from chicken erythroblastosis cells with the aid of nascent DNA strand extrusion method. In renaturation experiments, the sequence specificity of this DNA fraction (oriDNA) was compared to the sequence specificity of the nuclear matrix DNA. The oriDNA was shown to comprise a specific subset of unique genes all of which being represented also in the nuclear matrix DNA. The same unique DNA sequences were found to be depleted from the oriDNA and from the nuclear matrix DNA. A conclusion has been drawn that replication origins are located at the basements of DNA loops. The putative positions of replication origins within the domain of chicken alpha globin genes were mapped by hybridization experiments and were found to coincide with the positions of stable (not depending on the transcriptional state of globin genes) sites of DNA attachment to the nuclear skeleton.

[Enhanced transcription of genes of the intracisternal A particles and B2 elements in mouse tumors]. / Povyshennyi uroven' transkriptsii genov intratsistral'nykh A-chastits i B2-élementov v opukholiakh myshei.

A comparison of the expression of two mobile genetic elements A1 and B2 was studied in normal and tumor tissues. The A1 element is a chromosomal homolog of IAP genes, and B2 is a short ubiquitous repetitive sequences of the mouse genome. These sequences were earlier cloned in our laboratory and in this study were used as probes in hybridization experiments with RNA isolated from different mouse tumor and normal tissues. Both elements were efficiently transcribed in tumor cells. The level of expression of A1 sequences in tumors was 100-200 times higher than in normal tissues. The amount of B2 small cytoplasmic RNA significantly varied in different normal tissues. The content of this RNA was much higher in tumors. Closed circular DNA molecules containing IAP sequences were found in Ehrlich carcinoma cells. These DNA molecules are considered as intermediate forms of the mobile elements. The role of these mobile elements in the regulation of RNA expression and tumor progression is discussed.

[The effect of catalytic subunit of cAMP-dependent protein kinase on the activity of cytotoxic factor from natural killer cells]. / Vliianie kataliticheskoi sub''edinitsy cAMP-zavisimoi proteinkinazy na aktivnost' tsitotoksicheskogo faktora natural'nykh killerov.

The catalytic subunit of cAMP-dependent protein kinase modifies proteins of NKCF. Several proteins of the treated NKCF can be labelled with 32P. Treatment of NKCF with protein kinase enhance the cytotoxicity of NKCF.

[Preparation of recombinant metastazin and characteristics of it]. / Poluchenie rekombinantnogo metastazina i ego kharakteristika.

The recombinant mts-1 protein has been obtained with the use of the pMAL-c expression vector. This protein was shown to bind calcium ions and interact with melittin, a polypeptide from bee venom.

[Circular variants of long dispersed repeats in the mouse genome. The role of reverse transcriptase in their formation]. / Kol'tsevye formy dlinnykh dispergirovannykh povtorov v genome myshi. Rol' revertazy v ikh obrazovanii.

Extrachromosomal closed circular IAP gene DNA was cloned from Ehrlich ascites carcinoma cells. Two clones were analysed in detain and the following conclusions were drawn: 1) there is closed circular IAP gene DNA in mouse cells; 2) the formation of circular DNA and the transposition of IAP gene by means of reverse transcriptase are documented.

[Analysis of homologous DNA sequences within the first intron of the mouse and human mts1 genes: kB-like site and microsatellite DNA]. / Analiz gomologichnykh posledovatel'nostei DNK v pervom introne gena mts1 myshi i cheloveka: kB-podobnyi sait i mikrosatellitnaia DNK.

Homologous regions were revealed and analyzed within the first intron of the mouse and human mts1 genes. Maximal homology was detected between microsatellite DNA sequences from the first intron of the mouse gene (+804, +863) and the human gene (+600, +645). DNase I hypersensitive sites were revealed within these regions in both mouse and human cell lines tested, thus showing functional significance of the homology detected. In both the mouse and the human genes, the 5' end of homologous regions is flanked by a DNA sequence similar to a NF-kB/Rel protein-binding consensus sequence. Previously, this sequence was demonstrated to be involved in a complex regulatory element of the mouse mts1 gene. In comparison with the kB-like sequence, mouse, rat, and human sequences were found to involve one, two, and three nucleotide substitutions, respectively. Alterations in a spectrum of nuclear proteins bound to various kB-like sequences were analyzed. Possible effects of these alterations on mts1 transcription regulation were discussed. The possibility of the human kB-like sequence to function as positive regulator of transcription initiation was demonstrated in vivo.

[Isolation of cDNA clones which are specifically transcribed in metastatic and nonmetastatic murine tumors]. / Vydelenie klonov kDNK, spetsificheski transkribiruemykh v metastaziruiushchikh i nemetastaziruiushchikh opukholiakh myshi.

Gene cloning was used with a view to examine differences in gene expression in metastatic (CSML-100) and non-metastatic (CSML-0) cell lines. Differential screening of the cDNA libraries obtained was performed. Mts271 gene which is specifically expressed in metastatic cells was isolated.