[Neurogenic potential of human mesenchymal stem cells isolated from bone marrow, adipose tissue and endometrium: a comparative study].

Mesenchymal stem cells (MSCs) can be isolated from many adult tissue sources. These cells are a valuable substrate in cell therapy for many diseases and injuries. Different types of MSCs vary in plasticity. We performed a comparative study of the neurogenic potential of three types of human MSCs derived from bone marrow (BMSCs), subcutaneous adipose tissue (ADSCs) and endometrium (isolated from the menstrual blood) (eMSCs). It was shown that all three types of MSC cultures demonstrate multipotent plasticity and predisposition to neurogenesis, based on the expression of pluripotency markers SSEA-4 and neuronal precursors' markers nestin and beta-III-tubulin. Further analysis revealed the transcription of the neuronal marker MAP2 and neurotrophin-3 in undifferentiated BMSCs and ADSCs. Additionally, a significant basal level of synthesis of brain-derived neurotrophic factor (BDNF) in eMSC culture was also observed. Stimulation of neural induction with such agents as 5-azacytidine, recombinant human basic fibroblast growth factor (bFGF), recombinant human epidermal growth factor (EGF), a recombinant human fibroblast growth factor 8 (FGF8), morphogen SHH (sonic hedgehog), retinoic acid (RA) and isobutyl-methyl-xanthine (IBMX), showed further differences in the neurogenic potential of the MSCs. The components of the extracellular matrix, such as Matrigel and laminin, were also the important inducers of differentiation. The most effective neural induction in BMSCs proceeded without the RA participation while the cells pretreated with 5-azacytidine. In contrary, in the case of eMSCs RA was a necessary agent of neural differentiation as it stimulated the transcription of neurotrophin-4 and the elevation of secretion level of BDNF. The use of laminin as the substrate in eMSCs appeared to be critical, though an incubation of the cells with 5-azacytidine was optional. As far as ADSCs, RA in combination with 5-azacytidine caused the elevation of expression of MAP2, but reduced the secretion of BDNF. Thus, the effect of RA on neural differentiation of ADSCs in ambiguous and, together with the study of its signaling pathways in the MSCs, requires further research. The therapeutic effect of transplanted MSCs is commonly explained by their paracrine activity. The high basal level of BDNF synthesis in the eMSCs, along with their high proliferative rate, non-invasive extraction and neural predisposition, is a powerful argument for the use of the intact eMSCs as a substrate in cell therapy to repair nerve tissue.

[E-cadherin affects MAP activation by growth factors stimulation in human carcinoma cells].

Met and EGF receptor (EGFR) activation is correlated with dissociation of cell-cell adhesion and with increase in mobility of cancer cells. E-cadherin is a major protein of adhesion junctions. Using different approaches we have shown that EGF receptors intracellular localization depends of E-cadherin function. It was found that EGFR localized on the membrane in HT-29 cells which formed mature cell-cell contacts. Moreover, EGFR was colocalized with E-cadherin at the site of cell-cell adhesion in Triton-insoluble fraction. EGFR was accumulated preliminary in cytosol in E-cadherin negative HBL-100 cells. Study of signal transduction mediated by EGF and HGF in cells with different state of cell adhesion demonstrated that E-cadherin could affect ERK-signal-duration. Our preliminary studies proposed that mislocalization of Met and EGFR in E-cadherin negative cells altered receptors downstream signaling.

Affinity purification of Alu-DNA-repeat-binding proteins from human somatic cells.

A 66-kD Alu-DNA-repeat binding protein was identified in human somatic cell nucleoplasm. Gel shift assay, southwestern blotting, and affinity purification on DNA attached to a carrier were used. A 60-kD protein copurified with the 66-kD protein during affinity purification, probably due to protein--protein interactions. The gel shift assay reveals multiple complexes with exponential dependence of their relative mobility. The short binding site of the 66-kD protein was defined with the help of synthetic oligonucleotides. It is localized between the A and B boxes of RNA polymerase III promotor and is the same as that reported for the Alu-binding protein from human spermatozoids. The same short binding site, the similarity of the isolation procedure from germ and somatic cells, and similar binding properties and molecular masses suggest homology of the two proteins. The relationship of the proteins we studied and the Alu-DNA-binding proteins described in the literature is discussed.

[Dependence of the process leading to phosphorylation of retinoblastoma protein (pRb) on the status of the actin cytoskeleton]. / Zavisimost' protsessa, vedushchego k fosforilirovaniiu belka retinoblastomy (pRb), ot sostaiianiia aktinovogo tsitoskeleta.

It has been confirmed that the main actin-dependent period of G1 phase of the cell cycle is the middle of G1. As the critical points in G1 phase are associated with the synthesis of cycling D and E and with the formation of their active complexes with cyclin-dependent kinases (Cdk's), a study of a possible influence of actin filaments on these processes was performed. The activity of G1 kinases was estimated by the degree of phosphorylation of their specific substrate-retinoblastoma protein (pRb). Immunoblot analysis with specific antibodies to pRb revealed hypophosphorylated from of Rb in lysates of resting Swiss 3T3 cells and the appearance of hyperphosphorylated form after 12 h of EGF and serum stimulation. In was shown that actin filaments distruction by H2CB led to a decrease in hyper phosphorylated form appearance, depending on the depth of resting state of the cells and efficiency of their stimulation by growth factors. Thus, these data may suggest the involvement of actin cytoskeleton in functioning of the transcription chain cyclin/Cdk-R1-E2F.

[The role of the actin cytoskeleton in cell transition through the middle of the G1 phase of the mitotic cycle]. / Rol' aktinovogo tsitoskeleta v prokhozhdenii kletkami serediny fazy G1 mitoticheskogo tsikla.

The influence of dihydrocylochalasin B (H2CB), which selectively disrupts the actin cytoskeleton structure, on G1 phase progression after stimulation of quiescent Swiss 3T3 cells by epidermal growth factor (EGF) was investigated. H2CB was added to the culture medium (10 mg/ml) for different periods of time after cell cycle induction by EGF (10 ng/ml). Efficiency of mitogenic stimulation was estimated by 14C-thymidine uptake 21-23 h after EGF addition. It is shown that the main actin-mediated step is in the middle of G1, from 8 to 12 h after stimulation. Disorganization of actin cytoskeleton structure only during this time led to complete and irreversible block of cell entry into the S phase. On the contrary, the same effect of H2CB during the early period of G1 (first 6 h) led to the increase in 14C-thymidine incorporation in DNA. The specificity of actin-dependent period was proven in experiments with another inhibitor of cell proliferation--dancylcadaverine, whose effect was revealed at the earlier time--4-6 h after cell stimulation. Inhibition of protein synthesis during actin-dependent period (8-12 h) led to the same block of cell progression through G1 as it was seen after actin structure disruption. These data suggest that actin-dependent block is associated with the appearance and functioning of such specific regulators of G1 as cyclins (D, E) and their complexes with Cdk's (G1 kinases) which phosphorylate Rb protein (p1 10) associated with transcription factors E2F.

Epidermal growth factor induces tyrosine phosphorylation of epidermal growth factor receptors not occupied with ligand in intact A431 cells.

The mechanism of epidermal growth factor receptor (EGF-R) autophosphorylation in intact A431 cells was studied. We detected epidermal growth factor (EGF) induced tyrosine phosphorylation of EGF-R not occupied with ligand. Cell monolayers were subjected to irradiation after incubation with photoreactive derivative of EGF and uncoupled EGF was extracted by acidic treatment. Subsequent immunoprecipitation with antiphosphotyrosine antibodies resulted in precipitation of both EGF-R complexes with EGF and EGF-R with unoccupied ligand-binding site. The fact of precipitation of EGF-R with unoccupied ligand-binding site in conjunction with our finding of rapid dephosphorylation of EGF-R after EGF extraction by acidic treatment, strongly supports the interpretation that cross-phosphorylation of EGF-R may take place in intact cells.

[The phosphorylation and tyrosine kinase activity of an internalized complex of epidermal growth factor and its receptor]. / Fosforilirovanie i tirozinkinaznaia aktivnost' internalizovannogo kompleksa épidermal'nogo faktora rosta i ego retseptora.

A study was made of the functional state of the epidermal growth factor (EGF)--receptor complexes in A-431 cells. Conditions of surface bound EGF extraction were selected which allow to consider the intracellular EGF--receptor complexes only. A procedure of high efficient and specific immunoprecipitation of tyrosyl-phosphorylated EGF receptors was developed. It is shown that the dissociation of EGF--receptor complexes leads to receptor dephosphorylation due to a rapid and reversible inactivation of EGF receptor tyrosine kinase. The internalized receptor is found to be tyrosyl-phosphorylated and to retain tyrosine kinase for at least an hour after the internalization. The dynamics of dissociation, degradation and dephosphorylation of EGF--receptor complexes has been estimated. The rates of these processes prove to be almost negligible for the first 2.5 hours after internalization.

[The immunofluorescent detection of phosphorylated receptors of the epidermal growth factor during their internalization in A-431 cells]. / Immunofluorestsentnoe vyiavlenie fosforilirovannykh retseptorov épidermal'nogo faktora rosta v protsesse ikh internalizatsii v kletkakh A-431.

Phosphorylated receptors of the epidermal growth factor (EGF) were localized in the human epidermoid carcinoma cells using immunofluorescent staining with antibody to phosphotyrosine. The application of EGF at 4 degrees C was seen to induce a characteristic fluorescence of the cell margins, whereas no cell staining occurs in the absence of EGF. After a 1 hour incubation of cells at 37 degrees C, within which the internalized EGF receptor complexes are accumulated in the juxtanuclear compartment near the para-Golgi region, the staining with antiphosphotyrosine antibody reveals the receptors in this region. It is concluded that the internalized EGF-receptor complexes may remain in the phosphorylated state.