Georgii P. Georgiev

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

The involvement of Mts1(S100A4), a small Ca2+-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.

Non-random cleavage of SV40 DNA in the compact minichromosome and free in solution by micrococcal nuclease

Abstract The distribution of the primary cleavage sites produced by micrococcal nuclease on SV40 DNA in the compact minichromosome was analysed. Minichromosomes purified by improved method were digested in mild conditions in order to minimize possible changes in nucleosomal and supranucleosomal arrangement. The primary cuts were found to be non-randomly and unevenly distributed through the whole SV40 genome and those located in the “early” half of SV40 DNA were mapped. Some of them were also preferentially attacked on the naked DNA treated under the same conditions. Possible relation of the results to the nucleosomal organization of the compact minichromosome is discussed.

Effect of mts1 (S100A4) expression on the progression of human breast cancer cells

The mts1 (S100A4) gene, encoding a Ca2+‐binding protein of the S‐100 subfamily, is involved in the control of tumor metastasis in some murine tumor cell lines. To further analyze its role, we transfected hormone‐responsive human breast cancer MCF‐7 cells with the mts1 gene under the control of a strong constitutive promoter. All of the 3 tested clones (MCF‐7/mts1) producing Mts1 protein acquired an ability for hormone‐independent growth in nude mice. Tumors derived from mts1 transfectants revealed local invasiveness into surrounding muscle and adipose tissues and metastasized to regional lymph nodes and lungs, characteristics which are rarely observed with parental MCF‐7 cells. Electron‐microscopic analysis of MCF‐7/mts1 cells demonstrated structural changes in anchoring junctions, particularly in intermediate filament attachment sites (desmosomes). The mts1‐transfected clones expressed estrogen receptor, and their growth in tissue culture was both estrogen‐ and anti‐estrogen‐responsive. Changes in regulation of the estrogen‐dependent proteins progesterone receptor and cathepsin D were observed in some of the transfected clones. Our results indicate that mts1 expression in human breast cancer cells induces several changes characteristic of malignant phenotype and tumor progression.

Elastic torsional strain in DNA within a fraction of SV40 minichromosomes: relation to transcriptionally active chromatin.

After treatment of SV40 minichromosomes with DNA topoisomerase I, the superhelicity in the bulk of the DNA extracted from minichromosomes is known to remain unchanged. However, we found that the DNA extracted from a small fraction of SV40 minichromosomes (2‐5%), was almost completely relaxed, and covalently closed as shown by agarose gel electrophoresis. Thus, the DNA in these 2‐5% of SV40 minichromosomes was probably torsionally strained (TS). The proportion of such TS minichromosomes is close to the estimated proportion of transcriptionally active minichromosomes. The distribution of the TS minichromosomes in sucrose gradient coincided with the distribution of transcriptionally active complexes. Both sedimented faster than the majority of minichromosomes. Furthermore, after treatment with topoisomerase I the relaxed minichromosomes could be quantitatively separated from the bulk of material by recentrifugation in a sucrose gradient. A major part of the endogenous RNA polymerase activity was recovered in the relaxed fraction. These data suggest that TS‐minichromosomes correspond to transcriptionally active chromatin. After relaxation with topoisomerase I the TS minichromosomes lacked histones.

Proteins tightly bound to DNA in the regions of DNA attachment to the skeletal structures of interphase nuclei and metaphase chromosomes

Proteins tightly bound to the DNA of mouse L cells were examined both in the regions of DNA attachment to the protein skeleton and in the DNA loops. The interphase nuclear matrix and the metaphase chromosomal scaffold, containing from 2 to 15% of the original DNA, as well as the material released from the skeleton were isolated by mild nuclease treatment. At least six proteins resistant to sarcosyl-Cs2SO4 treatment are associated exclusively with skeleton-attached DNA both in interphase nuclei and metaphase chromosomes. Two (p52 and p60) can be dissociated from the complex by mercapto-ethanol-sarcosyl treatment. Both skeleton-attached and released DNA fractions contain a group of tightly bound low molecular weight polypeptides consisting of a major p18 band and one or two minor bands. They seem to be randomly distributed throughout the whole DNA loop. Some of the specific proteins that are tightly associated with DNA at the site of its interaction with the skeleton may directly be responsible for DNA attachment to the skeleton.

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.

On the structure of transcriptional unit in mammalian cells

Abstract The end-analysis of nuclear dRNA fractions, combined with competitive hybridization, was developed to check the predictions following from the model of the structure of the transcriptional unit in Eukaryotes ( G. P. Georgiev , J. Theor. Biol., 25 (1969) 473). The following main results have been obtained: 1. (1) Heavy nuclear dRNA completely inhibits the hybridization of polysomal mRNA with DNA. 2. (2) In the conditions of actinomycin D chase, the molecular weight of giant dRNA decreases proportionally to the decay of dRNA, the specific activity of purified dRNA remains unchanged, and the content of true mRNA (as determined by competition with polysomal RNA in hybridization experiments) is significantly increased. 3. (3) Only giant nuclear dRNA contains triphosphate groups at the 5′ end, which may be considered as the markers of the nascent molecules. 4. (4) The 5′-end sequences of giant dRNA, containing triphosphate groups, are hybridized very efficiently with homologous DNA, indicating that they are represented by the replica from repiterated DNA base sequences. 5. (5) The 3′ ends in giant, as well as in low-molecular-weight, dRNA are re-represented by the replica from reiterated DNA base sequences. 6. (6) The 3′ end sequences are hybridized with DNA more effectively than the total molecule indicating that some of them are also transcribed from reiterated DNA sequences. 7. (7) The polysomal mRNA only slightly inhibits the hybridization between DNA and giant dRNA but very strongly competes with 3′-end sequences of giant dRNA. The latter fact shows that the ends of operons (transcribed as 3′-end sequences of giant dRNA) encode a true mRNA, transferred into polysomes. These results are in good agreement with the model, previously described.

Molecular cloning and characterization of the mouse tag7 gene encoding a novel cytokine.

Cloning of the mouse tag7 gene encoding a novel cytokine is described. The Tag7 protein consists of 182 amino acids. Genomic organization of the tag7 gene and its promoter region remind those of the genes of the tumor necrosis factor locus, although the tag7 gene is not linked to this locus. The gene is located on chromosome 7 at the area that corresponds to band 7A3, which has genetic linkage with lupus-like disease in mouse models. tag7 transcription is essential for lymphoid organs. It is also detected in certain areas of lungs, brain, and intestine and in some tumors. Tag7 protein is detectable in both cell-associated and soluble forms. The soluble form of Tag7 triggers apoptosis in mouse L929 cells in vitro and does not involve NF-κB activation. The relationship between Tag7 and tumor necrosis factor family of ligands is discussed.

Peptidoglycan Recognition Protein Tag7 Forms a Cytotoxic Complex with Heat Shock Protein 70 in Solution and in Lymphocytes

The peptidoglycan recognition protein Tag7 is shown to form a stable 1:1 complex with the major stress protein Hsp70. Neither protein is cytotoxic by itself, but their complex induces apoptotic death in several tumor-derived cell lines even at subnanomolar concentrations. The minimal part of Hsp70 needed to evoke cytotoxicity is residues 450–463 of its peptide-binding domain, but full cytotoxicity requires its ATPase activity; remarkably, Tag7 liberated from the complex at high ATP is not cytotoxic. The Tag7-Hsp70 complex is produced by tag7-transfected cells and by lymphokine-activated killers, being assembled within the cell and released into the medium through the Golgi apparatus by a mechanism different from the commonly known granule exocytosis. Thus, we demonstrate how a heat shock protein may perform functions clearly distinct from chaperoning or cell rescue and how peptidoglycan recognition proteins may be involved in innate immunity and anti-cancer defense.

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.