Fra-1 induces morphological transformation and increases in vitro invasiveness and motility of epithelioid adenocarcinoma cells.

Two cell lines originating from a common ancestral tumor, CSML0 and CSML100, were used as a model to study AP-1 transcription factors at different steps of tumor progression. CSML0 cells have an epithelial morphology; they express epithelial but not mesenchymal markers and are invasive neither in vitro nor in vivo. CSML100 possesses all characteristics of a highly progressive carcinoma. These cells do not form tight contacts, are highly invasive in vitro, and are metastatic in vivo. AP-1 activity was considerably higher in CSML100 cells than in CSML0 cells. There was a common predominant Jun component, namely, JunD, detected in both cell lines. We found that the enhanced level of AP-1 in CSML100 cells was due to high expression of Fra-1 and Fra-2 proteins, which were undetectable in CSML0 nuclear extracts. Analysis of the transcription of different AP-1 members in various cell lines derived from tumors of epithelial origin revealed a correlation of fra-1 expression with mesenchymal characteristics of carcinoma cells. Moreover, we show here for the first time that the expression of exogenous Fra-1 in epithelioid cells results in morphological changes that resemble fibroblastoid conversion. Cells acquire an elongated shape and become more motile and invasive in vitro. Morphological alterations were accompanied by transcriptional activation of certain genes whose expression is often induced at late stages of tumor progression. These data suggest a critical role of the Fra-1 protein in the development of epithelial tumors.

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.

Transcription of a novel mouse semaphorin gene, M-semaH, correlates with the metastatic ability of mouse tumor cell lines.

In the attempt to identify genes associated with metastasis, we have compared gene expressions of two metastatic cell lines, 4T1 and 66cl4, and one noninvasive, nonmetastatic cell line, 67NR, which originate from the same mouse mammary adenocarcinoma. Using the technique of differential display, we identified a novel member of the semaphorin/collapsin family in the two metastatic cell lines. We have named it M-semaH. Northern hybridization to a panel of tumor cell lines revealed transcripts in 12 of 12 metastatic cell lines but in only 2 of 6 nonmetastatic cells and none in immortalized mouse fibroblasts. To our knowledge, this is the first time that the expression of a semaphorin gene has been shown to correlate positively with tumor progression. We have characterized two transcripts present in the tumor cells. One transcript, M-semaH-v, is a putative splice variant, which is less abundant in normal tissue and lacks 478 bp in the 3' untranslated region. Both transcripts encode the same 775 amino acids with the features of a secreted glycoprotein. Northern analysis suggests that the M-semaH gene is involved in embryonic development and in situ hybridization locates the M-semaH expression to the developing lungs, to developing skeletal elements, and to the ventral horns of the developing neural tube.

E-cadherin and metastasin (mts-1/S100A4) expression levels are inversely regulated in two tumor cell families.

Metastasin is putatively associated with cytoskeletal proteins and may influence cell motility, although its exact physiological role is not known. Because E-cadherin is an invasion suppressor molecule, and metastasin a metastasis-inducing molecule, we wondered which molecule was playing a dominant role in the balance that finally leads to noninvasiveness or invasiveness. The expression levels of E-cadherin and metastasin were monitored in two mouse tumor cell families and were found to be inversely regulated. Moreover, overexpression obtained via transfection of plasmids coding for either one of these two molecules abrogated expression of the other molecule as investigated via Northern and Western blotting experiments. Invasiveness was accordingly influenced. Expression levels of alpha- and beta-catenins were not influenced by up-regulated metastasin, but their intracellular distribution was disturbed. The present results suggest that metastasin-induced invasiveness of several malignant tumor cells is at least partially caused by down-regulation of E-cadherin.

Reversal of the in vivo metastatic phenotype of human tumor cells by an anti-CAPL (mts1) ribozyme.

The putative role of the CAPL gene in enhancing the development of human cancer metastasis was examined by transfecting human high-expressing osteosarcoma cells with a hammerhead ribozyme directed against the gene transcript. The ability of the ribozyme to cleave target mRNA in intact cells was demonstrated in a 5'-rapid amplification of cDNA ends assay. In transfected cells, a suppression of the capacity to give skeletal metastases upon intracardial injection into nude rats was observed in cell clones with reduced expression of CAPL mRNA and protein, whereas in vitro and in vivo cell proliferation and tumorigenicity were unchanged. The results provide direct evidence that the expression level of the CAPL-encoded protein can determine the metastatic potential of osteosarcoma cells, and they demonstrate an association between reduced gene expression and proliferation-independent inhibition of the metastatic capacity of human tumor cells. The effects of the specific cleavage of CAPL mRNA indicate that the gene product is involved in key cellular functions associated with the metastatic process and suggest that therapeutic modulation of the protein function may represent a novel approach for inhibiting the metastatic spread of cancer cells.

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.

Characterization of a positive regulatory element in the mts1 gene.

The first intron of the mts1 gene, a gene that is selectively expressed in metastatic cells and in normal cells that are motile, was found to be highly homologous to the CD3 delta enhancer element. Because of the homology between the CD3 delta enhancer and the first intron of mts1, we analysed the first intron of the mts1 gene to determine whether it functions as a transcriptional regulatory element. Highly metastatic CSML-100 cells transfected with chloramphenicol acetyl transferase-containing plasmids demonstrated the ability of the mts1 first intron to function as a positive regulatory element. In vitro footprinting analysis using extracts from CSML-0 cells (which express mts1 at low levels) or CSML-100 cells (which express mts1 at high levels) identified a protected 16-nucleotide element in the first intron of mts1, regardless of the extract used. However, in vivo footprinting analysis of the same region identified the protected 16-nucleotide fragment only in the mts1 intron from CSML-100 cells, not from CSML-0 cells. Differences in the methylation pattern of the mts1 gene in CSML-100 cells and CSML-0 cells are known to exist, and may in part be responsible for the mts1 footprinting differences observed in vivo from the different cell lines.

Metastasis of mammary carcinomas in GRS/A hybrid mice transgenic for the mts1 gene.

Transgenic mice, carrying the mts1 gene, one of the genes involved in the acquisition of the metastatic phenotype, were generated. The mts1 gene was placed under the control of the mouse mammary tumor virus (MMTV) long terminal repeat (LTR) promoter leading to overexpression in the lactating mammary gland of transgenic animals. Animals bearing the transgene appear phenotypically normal. Animals of two transgenic lines (Tg463 and Tg507) were crossed with the GRS/A mice. The GRS/A strain is characterized by high incidence of mammary tumors which rarely metastasize. 40% of the tumor bearing hybrid GRS/A mts1 females were found to develop secondary tumors in the lungs. The Mts1 protein was detected in the transgene primary tumor cells as well as in the corresponding metastases. Nontransgenic littermates expressed the Mts1 protein only in the stromal cells surrounding the tumor but not in the tumor cells by itself. Taken together these observations indicate that overexpression of the mts1 gene in the mouse mammary carcinoma cells gives rise to more aggressive tumors which are able to metastasize.

The metastasis-associated Mts1(S100A4) protein could act as an angiogenic factor.

The involvement of Mts1(S100A4), a small Ca(2+)-binding protein in tumor progression and metastasis had been demonstrated. However, the mechanism by which mts1(S100A4) promoted metastasis had not been identified. Here we demonstrated that Mts1(S100A4) had significant stimulatory effect on the angiogenesis. We detected high incidence of hemangiomas--benign tumors of vascular origin in aged transgenic mice ubiquitously expressing the mts1(S100A4) gene. Furthermore, the serum level of the Mts1(S100A4) protein increased with ageing. Tumors developed in Mts1-transgenic mice revealed an enhanced vascular density. We showed that an oligomeric, but not a dimeric form of the Mts1(S100A4) protein was capable of enhancing the endothelial cell motility in vitro and stimulate the corneal neovascularization in vivo. An oligomeric fraction of the protein was detected in the conditioned media as well as in human serum. The data obtained allowed us to conclude that mts1(S100A4) might induce tumor progression via stimulation of angiogenesis.

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.

Transcriptional regulation of the mts1 gene in human lymphoma cells: the role of DNA-methylation.

The transcription of the mts1 gene putatively involved in the control of tumor metastasis was studied in three human lymphoma cell lines: MOLT-4, CEM and Jurkat. The level of the mts1 gene transcription is high in MOLT-4 cells, lower in CEM cells and hardly detectable in Jurkat cells. This correlates with the hypomethylation of DNA in the first exon and the first intron of the mts1 gene in the analyzed culture cells. This area was also found to be undermethylated in human peripheral blood cells--macrophages, neutrophils and lymphocytes where the mts 1 gene is highly expressed. 5-Azadeoxycytidine (AzadC)--an inhibitor of the eukaryotic DNA-methylase--significantly induces the expression of the mts1 gene in CEM and Jurkat cells and has little effect on mts1 gene transcription in MOLT-4 cells. The drug does not influence mts1 transcription in cultivated peripheral blood lymphocytes. These data indicate the possible involvement of the methylation of the first exon/first intron sequences in the transcriptional repression of the mts1 gene. The finding of two DNAaseI hypersensitivity sites (DHSs) mapped in the first intron of the mts1 gene supports this suggestion.

Metastasis-associated protein Mts1 (S100A4) inhibits CK2-mediated phosphorylation and self-assembly of the heavy chain of nonmuscle myosin.

A role for EF-hand calcium-binding protein Mts1 (S100A4) in the phosphorylation and the assembly of myosin filaments was studied. The nonmuscle myosin molecules form bipolar filaments, which interact with actin filaments to produce a contractile force. Phosphorylation of the myosin plays a regulatory role in the myosin assembly. In the presence of calcium, Mts1 binds at the C-terminal end of the myosin heavy chain close to the site of phosphorylation by protein kinase CK2 (Ser1944). In the present study, we have shown that interaction of Mts1 with the human platelet myosin or C-terminal fragment of the myosin heavy chain inhibits phosphorylation of the myosin heavy chain by protein kinase CK2 in vitro. Mts1 might also bind directly the beta subunit of protein kinase CK2, thereby modifying the enzyme activity. Our results indicate that myosin oligomers were disassembled in the presence of Mts1. The short C-terminal fragment of the myosin heavy chain was totally soluble in the presence of an equimolar amount of Mts1 at low ionic conditions (50 mM NaCl). Depolymerization was found to be calcium-dependent and could be blocked by EGTA. Our data suggest that Mts1 can increase myosin solubility and therefore suppress its assembly.

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.

A link between inflammation and metastasis: serum amyloid A1 and A3 induce metastasis, and are targets of metastasis-inducing S100A4.

S100A4 is implicated in metastasis and chronic inflammation, but its function remains uncertain. Here we establish an S100A4-dependent link between inflammation and metastatic tumor progression. We found that the acute-phase response proteins serum amyloid A (SAA) 1 and SAA3 are transcriptional targets of S100A4 via Toll-like receptor 4 (TLR4)/nuclear factor-κB signaling. SAA proteins stimulated the transcription of RANTES (regulated upon activation normal T-cell expressed and presumably secreted), G-CSF (granulocyte-colony-stimulating factor) and MMP2 (matrix metalloproteinase 2), MMP3, MMP9 and MMP13. We have also shown for the first time that SAA stimulate their own transcription as well as that of proinflammatory S100A8 and S100A9 proteins. Moreover, they strongly enhanced tumor cell adhesion to fibronectin, and stimulated migration and invasion of human and mouse tumor cells. Intravenously injected S100A4 protein induced expression of SAA proteins and cytokines in an organ-specific manner. In a breast cancer animal model, ectopic expression of SAA1 or SAA3 in tumor cells potently promoted widespread metastasis formation accompanied by a massive infiltration of immune cells. Furthermore, coordinate expression of S100A4 and SAA in tumor samples from colorectal carcinoma patients significantly correlated with reduced overall survival. These data show that SAA proteins are effectors for the metastasis-promoting functions of S100A4, and serve as a link between inflammation and tumor progression.

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.

Characterization of two splice variants of metastasis-associated human mts1 gene.

The mts1 gene is one of the genes specifically expressed in mouse metastatic tumors and tumor cell lines. In this paper, we present data on cloning and sequencing of two variants of human mts1 cDNAs (hu-mts1 and hu-mts1 (var)), as well as of the corresponding region in the human genome. Comparison of the genomic sequence with the sequence of the mts1 cDNAs demonstrates presence of two alternatively spliced variants of the mts1 in the human osteosarcoma cell line (OHS). The alternative splicing occurs within the 5'-untranslated region (UTR) of human mts1 pre-mRNA. Both splice variants, hu-mts1 and hu-mts1 (var), retain similar stability in the cells, contain one open reading frame coding for the MTS1 protein and differ only slightly in their translational capacity. The splice variants demonstrate dramatic variations in the level of expression in different human tissues and in human tumor cell lines. Although we have not revealed substantial differences in the mode of action of the two splice variants in the cells, the observed tissue specificity of expression supports the notion that it plays an important role in determining the activity of mts1 in different tissues.

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.

Enhancement of the polycation-mediated DNA uptake and cell transfection with Pluronic P85 block copolymer.

Polyelectrolyte complexes formed between DNA and poly(N-ethyl4-vinylpyridinium) cations were shown to effectively transfect mammalian cells [7]. This work suggests that the polycation-mediated uptake of the plasmid DNA and cell transfection are significantly enhanced when these complexes are administered simultaneously with a poly(ethylene oxide)-block-poly(propylene oxide)-block-poly(ethylene oxide) copolymer, Pluronic P85. The uptake studies were performed using radioactively labeled pRSV CAT plasmid on NIH 3T3, MDCK, and Jurkat cell lines. The transfection was investigated by chloramphenicol acetyltransferase assay using 3T3 cells as a model. The effects reported may be useful for the enhancement of the polycation-mediated cell transfection.

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.