Antiproliferative Effects of Mesenchymal Stem Cells and Epithelial Cells on Lymphocytes.

We analyzed the interactions between peripheral blood lymphocytes from heterologous donors with mesenchymal stem cells obtained from the tooth pulp and trophoblast. In mixed cultures, proliferation of both lymphocytes and mesenchymal stem cells was suppressed. Similar suppressive effects were observed in lymphocyte cultures mixed with epithelial cells (hepatocytes HeG2 and renal epithelial cells HEK293). This suppression can be determined by impairment of normal adhesion contacts between cells of different origin.

Interaction of Lymphocytes with Mesenchymal Stem Cells.

We studied the interaction of neural stem cells and dental pulp-derived mesenchymal stem cells with lymphocytes from autologous and heterologous donors. Flow cytometry analysis with the use of CFSE-labeled lymphocytes demonstrated an increase in the content of proliferating CD8, CD16 and CD56 cells, but not CD4 cells in cultures of HLA-DR-negative mesenchymal stromal cells from the dental pulp co-cultured with lymphocytes. In neural cultures expressing HLA-DR, all subpopulations of T cells and NK cells were activated. No differences between the autologous and heterologous cultures were revealed.

[Study of the quality of aminated substrates used for hybridization analysis].

A set of methods for analysis of the quality of aminated substrates that could be a basis for the large-scale manufacturing of biological microchips is suggested. The analysis includes the determination of the number of amino groups, their availability for the immobilization of phosphorylated oligonucleotides, and the characterization of surface properties of the substrates in respect to the nonspecific sorption of reagents during hybridization. A simple procedure was suggested for determination of the density/number of amino groups. It is based on the use of dimethoxytrityl chloride with the subsequent spectrophotometric determination of dimethoxytrityl cation. The availability of amino groups was estimated by covalent attachment of an oligonucleotide probe containing a fluorescently labeled group to the aminated surface and the subsequent comparison of the intensity of fluorescing zones formed on the chip. The sorption properties of the surface were investigated with the help of a model hybridization reaction. A comparative analysis of aminated glasses manufactured by various firms and in our laboratory showed that the glasses with the amino group density from 0.7 to 2.0 groups/nm2 prepared by our procedure have the best properties for the hybridization analysis.

[Inactivation and sensitization of the tumor cells by the gene Bax transfection].

After the transfection of the gene Bax into the cultured tumor cells of human ovary adenocarcinoma SKOV3 and uterus carcinoma HeLa in vitro the high sensitivity of the cells SKOV3 to the protein Bax produced after the gene Bax transfection was found. The sensitivity of the cells HeLa to the gene Bax transfection was much smaller. The hyperexpression of gene Bax and hypersensitivity to doxorubicin were seen in HeLa cells received as a result of the gene Bax transfection and subsequent selection. All cells of the line SKOV3 with the increased expression of the transfected gene Bax died. In the cell line SKOV3 the mutation in a gene Bax was found which has a genotype G7/G9 against a native type of a gene Bax--G8/G8. It was concluded that the found in the exone 3 of the gene Bax mutation G7/G9 in cells SKOV3 results in an inactivation of proapoptotic activity of the protein Bax.

[Dimethyl suberimidate as a specific inductor of apoptosis in transformed cells]. / Dimetilsuberimidat kak spetsificheskii induktor apoptoza transformirovannykh kletochnykh kul'tur.

A modification of protein-protein interactions can be considered to be a way to regulate cell death. Chemical cross-linking agents have been traditionally used for protein complexing. This study has been undertaken to test a possibility to induce and(or) to modify cell death by a homobifunctional cross-linker dimethyl suberimidate (DMS). It was shown that the protein cross-linking by DMS resulted in a death of transformed cells by apoptosis. DMS-induced apoptosis was accompanied by cell cycle perturbations and down-regulation of p21/Waf1 mRNA expression. The RT-PCR analysis of bcl-2 family genes revealed the engagement of mitochondria in DMS-induced cytotoxicity. Then, the influence of DMS treatment on TNF-dependent and Fas-mediated apoptosis was investigated. Cell pre-incubation with DMS resulted in their increasing sensitivity for the TNF cytotoxic effect, though activities of anti-Fas cytotoxic antibodies were inhibited. The effects observed are probably due to cross-linking of TNF-receptors. Thus, this study first demonstrated that a chemical cross-linker DMS in capable of inducing apoptosis in transformed cells and modifying TNF-dependent and Fas-mediated apoptosis.

[Apoptosis of L929 cells induced by tumor necrosis factor]. / Apoptoz kletok L929 pod deistviem faktora nekroza opukholei.

We investigated the mode of TNF-dependent death of L929 murine fibroblasts and the influence of overexpression of bcl-2 family genes on this process. Based on morphological and biochemical data it has been shown that L929 cells died after TNF treatment by apoptosis irrespective of TNF dose and protein synthesis inhibition. Analysis of bcl-2 family gene transfectants revealed a down-regulation of TNF-induced apoptosis by bcl-2 and bclX overexpression, and an up-regulation by bax gene.

[Comparative study of Ca/Mg-dependent nucleases from the mammalian thymus]. / Sravnitel'noe issledovanie Ca/Mg-zavisimykh nukleaz timusa mlekopitaiushchikh.

Ca/Mg-dependent nuclease is a possible key enzyme of apoptosis. We isolated and purified nucleases from the human and rat thymus to a homogeneous state and compared some properties of the obtained preparations with those of the earlier isolated Ca/Mg-dependent nuclease from the calf thymus. The activity of the nuclease from the human thymus in the presence of bivalent ions decreases in a sequence:(Ca + Mg) = (Ca + Mn) > Mn, that from the rat thymus: (Ca + Mn) > Mn > (Ca + Mg), and that from the calf thymus: (Ca + Mn) > (Ca + Mg) > Mn. Nuclease are not active in a medium containing only Mg, Ca, Co, and Zn ions. The preparations proved to be unstable during their isolation and storage. If the relative molecular mass of the purifies preparations was according to electrophoresis in 12% DS-Na-polyacrylamide gel 28, 29, and 18.4 and 21 kDa for the calf, human, and rat, respectively, after storage at -20 degrees C for two to six months, the molecular mass of native proteins decreases to 17-14 kDa. Some other properties of the enzymes have been described.

[Cell sensitivity to Fas-mediated apoptosis depends on three forms of Fas-antigen]. / Kletochnaia chuvstvitel'nost' k Fas-oposredovannomu apoptozu zavisit ot trekh form Fas-antigena.

It is known that binding of specific anti-Fas antibodies to Fas-receptor induces apoptosis in various cell lines. But the mechanisms of functional regulation and realization of apoptosis remain so far unknown. We obtained HeLa cells transfected with cDNA of Fas-antigen, whose expression was located under the promoter controlled by isopropoyl beta-D-thiogalactopiranoside. Analysis of transfectants has shown that expression of Fas above a certain critical level leads to redistribution of Fas in the cells and is accompanied by changes in the cell cycles and cell sensitivity to the cytotoxic effects of anti-Fas and tumor necrosis factor (TNF). We propose that the sensitivity of cells to Fas-mediated apoptosis depends on the ratio of transmembrane, intracellular and soluble Fas-antigen in the cells.

[The programmed cell death of murine BW5147 thymoma under the action of dexamethasone]. / Programmiruemaia gibel' kletok timomy myshi BW5147 pod deistviem deksametazona.

By flow cytometry, imitation modelling and biochemical analysis, the mode and kinetics of dexamethasone-treated T-lymphoma cell death were studied. The hormone was shown to induce delays in pre- and postsynthetic phases of the cell cycle and the death of part of cells. A short exposure to dexamethasone reveals its cytostatic rather than cytolytic effect. Following G2/M delay and cytokinesis, part of cells dies. A reduced serum concentration (2%) causes shorter delays in the cell cycle and a more rapid cell death. Dexamethasone stimulates apoptosis which is indicated by internucleosomal DNA fragmentation, and by a coincidence in time of the processes of DNA degradation and increase in the other membrane permeability. These results are discussed in relation to the cell death and proliferation.

[The cellular apoptosis of murine BW5147 thymoma during cold shock]. / Apoptoz kletok timomy myshi BW5147 pri kholodovom shoke.

The mode of T-lymphoma cell death induced by cold shock was studied. The rewarming of cells at 37 degrees C following a brief period of cold (0 degrees C) resulted in internucleosomal DNA fragmentation. The cells underwent cold shock-mediated apoptosis only at a reduced (2%) serum concentration. The apoptosis was not blocked by macromolecular synthesis inhibitors such as cycloheximide and antinomycin D, or by Quin-2. EGTA per se was responsible for the initiation of cell death. Colchicine also induced internucleosomal fragmentation of DNA. Our findings suggest that cold shock induced apoptosis is associated with low temperature mediated disruption of microtubules. The role of Ca2+ and growth factors in cold shock induced cell death is discussed.

[Patterns in the degradation of individual genes of thymocytes in irradiated rats]. / Zakonomernosti degradatsii individual'nykh genov v timotsitakh obluchennykh krys.

Degradation of genes of actin, albumin, histones, heat shock protein, and ribosomal RNA within DNA of irradiated animal thymocytes has been investigated. It has been shown that single strand enzymatic breaks occurred in thymocyte DNA 2 h following irradiation are localized in linker sites of nucleosomes. All the transcribed genes under study degrade to fragments that correspond by their length to DNA of nucleosomes and their oligomers. The albumin gene nontranscribed in thymocytes also degrades; however, no low molecular weight fragments are found. The degree of gene degradation is invariable in time.

[The effect of gamma-irradiation on the poly(ADP-ribosylation) of nuclear proteins in rat thymocytes]. / Vliianie gamma-oblucheniia na poli(ADF-ribozilirovanie) iadernykh belkov timotsitov krys.

Differences in the spectra of modified nuclear proteins of thymocytes of control and irradiated rats were investigated using antibodies specific for poly(ADP-ribose) and incorporation of a label from 14C-NAD in vitro. Two classes of modified proteins were identified differing in the rate of the polymer metabolism and the degree of poly(ADP-ribosylation). No postirradiation changes were detected in poly(ADP-ribosylation) of the nuclear sap proteins and chromatin. A pronounced increase in modification of proteins with the molecular mass of 72 and 83 kD and a sharp decrease in poly(ADP-ribosylation) of a protein group with the molecular mass of 47 to 65 kD were detected within the nuclear matrix by the second hour following irradiation. A study was made of the localization of modified proteins in polydeoxynucleotide fractions of different sizes (mononucleosomes and their oligomers).

[Poly(ADP-ribosylation) of nuclear proteins in rat thymocytes]. / Poli(ADP-ribozilirovanie) iadernykh belkov timotsitov krys.

Specific antibodies to poly(ADP-ribose) were obtained and characterized. Using these antibodies, the tissue specificity of poly(ADP-ribose) modified nuclear proteins from rat thymocytes and hepatocytes was studied. The differences in the levels of poly(ADP-ribosylation) of nuclear proteins from both tissues were found to be quantitative rather than qualitative. Analysis of intranuclear distribution of poly(ADP-ribose) acceptor proteins revealed that the bulk of them is localized in the nuclear sap and matrix. A comparison of spectral properties of poly(ADP-ribosylated) proteins, using specific antibodies and label incorporation from [14C]NAD showed the existence of two protein groups. Some of those were modified in a great degree but exchange poly(ADP-ribose) at a slow rate, whereas others (e.g., histones and HMG proteins) modified in a small degree exchanged poly(ADP-ribose) at a much higher rate. The results obtained by different methods are discussed.

[Nature of breaks inducible in DNA at the early stages of interphase cell death]. / Priroda razryvov, indutsiruemykh v DNK na rannikh étapakh inerfaznoi gibeli kletok.

Dynamics of changes in 3'-OH- and 5'-OH-ends of DNA was determined by "nick"-translation and direct polynucleotide kinase reaction, respectively, in animal thymocytes after irradiation and administration of hydrocortisone. Breaks bearing both 3'-OH- and 5'-OH-ends were found in DNA after irradiation. In 40 min repair of single-strand breaks was almost completed, and enzymatic breaks were accumulated with 3'-OH-ends only. 60 min after the administration of hydrocortisone, the number of nuclear DNA breaks containing 3'-OH-ends, but not 5'-OH-ends, sharply increased. Upon DNA autolysis in isolated nuclei acid nuclease produced 5'-OH-ends, and Ca2+/Mg2+-dependent nuclease, 3'-OH-ends. No activity of Mg2+-dependent nuclease was registered either in the nuclei of control thymocytes or in the nuclei isolated from thymocytes of exposed rats.