[Mesenchymal stem cells expressing cytosine deaminase inhibit growth of murine melanoma B16F10 in vivo].

The aim of this study was to estimate the efficacy of mesenchymal stem cell-based suicide gene therapy in mice bearing murine melanoma B16F10. Adipose mesenchymal stem cells (MSCs) were transfected with plasmid constructs expressing cytosine deaminase fused with uracil phosphoribosyltransferase (CDA/UPRT) or CDA/UPRT fused with HSV-1 tegument protein VP22 (CDA/UPRT/VP22). In this study, we demonstrate that direct intratumoral transplantation of MSCs expressing CDA/UPRT or CDA/UPRT/VP22 followed by systemic administration of 5-fluorocytosine (5-FC) results in a significant inhibition of tumor growth. There was a 53% reduction in tumor volume in mice treated with CDA/UPRT-MSCs and 58% reduction in tumor volume in mice treated with CDA/UPRT/VP22-MSCs as compared with control animals transplanted with B16F10 melanoma alone. Injection of CDA/UPRT-MSC and CDA/UPRT/VP22-MSC prolonged the life span of mice bearing B16F10 melanoma by 15 and 26%, respectively. The data indicate that in murine B16F10 melanoma model, MSCs encoding CDA/UPRT suicide gene have a significant antitumor effect.

Peripherally applied synthetic peptide isoAsp7-Aß(1-42) triggers cerebral ß-amyloidosis.

Intracerebral and intraperitoneal inoculation with ß-amyloid-rich brain extracts originating from patients with Alzheimer's disease as well as intracerebral injection of aggregates composed of synthetic Aß can induce cerebral ß-amyloidosis, and associated cognitive dysfunctions in susceptible animal hosts. We have found that repetitive intravenous administration of 100 µg of synthetic peptide corresponding to isoAsp7-containing Aß(1-42), an abundant age-dependent Aß isoform present both in the pathological brain and in synthetic Aß preparations, robustly accelerates formation of classic dense-core congophilic amyloid plaques in the brain of ß-amyloid precursor protein transgenic mice. Our findings indicate this peptide as an inductive agent of cerebral ß-amyloidosis in vivo.

Comparative analysis of expression of human telomerase catalytic subunit at the transcription level in cell cultures of different origin.

The expression of human telomerase catalytic subunit in HL-60 and HT-1080 malignant transformed cells and telomerized fibroblasts was studied by quantitative PCR. It was found that the number of transcripts of human telomerase catalytic subunit per cell in telomerized fibroblasts could be hundreds of times higher than in HL-60 and HT-1080 cells. Telomerized fibroblast cultures are suggested as experimental systems for selection of basal compounds for creation of anticancer drug prototypes, the molecular target of which is human telomerase catalytic subunit. The effects of human telomerase catalytic subunit expression on the fibroblast proteome are analyzed.

Mechanisms of positive effects of transplantation of human placental mesenchymal stem cells on recovery of rats after experimental ischemic stroke.

Mesenchymal stem cells isolated from human placenta and in vitro labeled with fluorescent magnetic microparticles were intravenously injected to rats 2 days after induction of focal cerebral ischemia (endovascular model). According to MRT findings, transplantation of mesenchymal stem cells led to an appreciable reduction of the volume of ischemic focus in the brain. Two or three weeks after transplantation, labeled cells accumulated near and inside the ischemic focus, in the hippocampus, and in the subventricular zone of both hemispheres. Only few human mesenchymal stem cells populating the zone adjacent to the ischemic focus started expressing astroglial and neuronal markers. On the other hand, transplantation of mesenchymal stem cells stimulated proliferation of stem and progenitor cells in the subventricular zone and migration of these cells into the ischemic zone. Positive effects of transplantation of these cells to rats with experimental ischemic stroke are presumably explained by stimulation of proliferation of resident stem and progenitor cells of animal brain and their migration into the ischemic tissue and adjacent areas. Replacement of damaged rat neurons and glial cells by transplanted human cells, if it does take place, is quite negligible.

Vital dye dil and fluorescent magnetic microparticles do not affect the phenotype of mesenchymal stem cells from human amnion and their differentiation capacity.

We present a method of labeling of mesenchymal stem cells from human amnion with a fluorescent dye Dil and microspheres (Bangs Laboratories). The possibility of administration of loaded cell culture was verified and comparative analysis of the phenotype of mesenchymal stem cells by the expression of fibronectin, nestin, CD13, CD29, CD34, CD44, CD54, CD90, CD105, CD106, HLA-ABC, HLA-DR, and PCNA was carried out. The labeled cells retained osteogenic differentiation capacity. The results suggest that fluorescent dye Dil and microspheres from Bangs Laboratories can be used for monitoring of mesenchymal stem cells from human amnion in in vivo experiments.

Development and introduction of production standards for cell products of mesenchymal origin.

The results of the development and introduction of universal production standards for cell products of mesenchymal origin are presented: technology for obtaining and culturing of primary cell cultures from human postnatal organs and tissues, cell product quality and safety control procedures, methods for cell product storage and transportation, and the necessary files.

In vitro comparison of immunological properties of cultured human mesenchymal cells from various sources.

Cell-mediated cytotoxicity was studied in vitro during the interaction of bone marrow mesenchymal stem cells, fibroblast-like cells from newborn umbilical cord, and skin fibroblasts of an adult donor with peripheral blood mononuclear cells. Independently on the origin, mesenchymal cells were not lysed with allogeneic natural killer cells and cytotoxic lymphocytes. Mixed cultures of mesenchymal cells had no cytotoxic effect on peripheral blood mononuclear cells and did not activate proliferation of T and B lymphocytes, natural killer cells, and CD14+ lymphocytes. In vitro experiments showed that mesenchymal cells of different origin and allogeneic immunocompetent cells are tolerant to each other.

Use of bone marrow mesenchymal (stromal) stem cells in experimental ischemic stroke in rats.

The effects of human mesenchymal stem cells on neurological functions and behavioral reactions of animals and on damaged brain tissue were studied on the model of focal cerebral ischemia in rats. Homing and differentiation of transplanted mesenchymal stem cells were also studied. Significant regression of neurological disorders after cell transplantation was noted, no appreciable shifts were detected by magnetic resonance tomography. Homing of transplanted cells was detected mainly in the zone of focal ischemia. Some cells died, others exhibited signs of differentiation into neurons and glia.

Implication of alpha5beta1 integrin in invasion of drug-resistant MCF-7/ADR breast carcinoma cells: a role for MMP-2 collagenase.

Expression of alpha5beta1 integrin in the drug-resistant MCF-7/ADR breast carcinoma cells was inhibited by treatment of these cells with alpha5-specific siRNA. The decrease of alpha5beta1 expression resulted in a sharp decrease of expression of MMP-2 collagenase and inhibition of invasion activity of these cells in vitro. Similar decrease of invasion was also observed during inhibition of MMP-2 expression by treatment of these cells with MMP-2-specific siRNA. Inhibition of alpha5beta1 expression was also accompanied by significant decrease in cell content of active (phosphorylated) forms of signal protein kinases Akt and Erk1/2. Inhibition of activity of these kinases by treatment of cells with PI-3K/Akt-specific inhibitor LY294002 or Erk-specific inhibitor PD98059 resulted in inhibition of MMP-2 expression and the decrease of invasion in vitro. These data suggest that alpha5beta1 controls invasion ability of these cells by regulating expression of MMP-2, which involves PI-3K and Erk1/2 protein kinase signaling.

STAT1 gene expression in cervical carcinomas.

Expression of the STAT1 gene belonging to the group of interferon-regulated genes was analyzed in cervical tumors and cell lines harboring the genome of human papilloma viruses (HPV) of so-called high risk group. Expression of this gene in invasive carcinomas was maintained on a definite level that was not significantly distinct from that in adjacent normal (control) tissue. Tumors from different patients differ from each other by expression level of the STAT1 gene. These variations can be attributed to the heterogeneity of tumor cell population and different ratio between normal and tumor cells, as well as to putative persistence of intra-individual variability of STAT1 expression in normal cell population. It was demonstrated that viral genome status (episomal or integrative) did not influence STAT1 gene transcription. In conclusion, these data demonstrate that the STAT1 gene is expressed in an individual and specific manner both in HPV-positive cervical tumors and cell lines harboring transforming genes of these viruses.

Comparative study of adult human skin fibroblasts and umbilical fibroblast-like cells.

Expression of markers, collagens, and HLA-1 by human skin fibroblasts and fibroblast-like cells isolated from the umbilical Wharton's jelly was compared. Skin fibroblasts express collagens (proteins characteristic of differentiated cells of this histogenetic series) and HLA-1, while umbilical cells express, in addition to collagens, juvenile surface markers and almost no HLA-1. This indicates that fibroblast-like cells isolated from different sources are different and can serve as sources for the creation of cell preparations with different characteristics in future.

Influence of mibefradil on Ca2+ influx induced by vasoactive hormones or depletion of intracellular calcium stores.

The influence of the new calcium antagonist mibefradil (CAS 116666-63-8, Ro 405967) on calcium influx into rabbit smooth muscle cells (VSMC) was studied. Angiotensin II-stimulated divalent cations entry was blocked by mibefradil at micromolar concentrations (1-10 mumol/l). In the same range of concentrations, the antagonist depressed capacitative calcium influx evoked by thapsigargin- and 2,5-di-tert-butylhydroquinone (BHQ)-dependent depletion of internal depots. The ability to block the receptor-mediated pathway of calcium current into VSMC may explain in part the difference between the mode of pharmacological action of mibefradil as compared to other calcium antagonists.

Effect of lysophosphatidylcholine on the structure and function of low density lipoproteins.

The treatment of LDL with bee-venom phospholipase A2 resulted in the formation of lipid-protein particles (phl-LDL) with an increased content of lysophosphatidylcholine (LPC). At the same time, the composition of other lipids and the protein structure remained unaffected. phl-LDL, as well as LPC, abolished the hormone-induced [Ca2+] increase in platelets and platelet aggregation induced by PAF, AMP and thrombin, whereas LDL produced no effect on the hormone-stimulated increase in the intracellular [Ca2+]. The effect persisted in a Ca(2+)-free medium, indicating that phl-LDL and LPC did not abolish the mobilization of intracellular stores with the above-mentioned inducers. Neither LPC no phl-LDL affected the [Ca2+]i level in platelets and suppressed the platelet aggregation evoked by tapsigargine, a specific inhibitor of endoplasmic reticulum Ca(2+)-ATPase, or by phorbol myristate acetate. The inhibitory effect depended on the LPC concentration and the time of platelet incubation with phl-LDL or LPC. The half-maximum efficient LPC concentrations were identical for LPC and phl-LDL (2-4 microM). The inhibitory effect was dependent on the LPC structure: lysophosphatidylethanolamine and phosphatidylcholine displayed no inhibitory effect. The results suggest that when added to washed platelets, free LPC and phl-LDL inhibit only the receptor-dependent increase of [Ca2+]i.

[Increase of calcium ion level in the cytoplasm of indo 1-loaded HeLa cells under the action of histamine]. / Povyshenie urovnia ionov kal'tsiia v tsitoplazme kletok HeLa, zagruzhennykh indo l, pod vliianiem gistamina.

The regulation of free cytoplasmic calcium in HeLa cells was studied using a fluorescent calcium indicator into 1. It is found that addition of histamine caused substantial increase in calcium level. In calcium-free medium the increase was much smaller, implicating both intra- and extracellular calcium as sources of observed Ca2(+)-rise. Protein kinase C activator, phorbol myristate acetate, completely blocked the effect of histamine. Agents causing rise in cellular cAMP level did not change the Ca2(+)-rise.