[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.

[Comparative transcriptomic and proteomic analysis of the fetal and adult human liver].

The transcriptome and proteome comparative analysis of the fetal liver (9.5-10.5 weeks of gestation) and normal adult liver was developed in the present investigation. We used 44k microarrays of Agilent company and 2D-GE MALDI-TOF in transcriptome and proteome approaches, respectively. The top lists of expression genes and proteins for fetal liver obtained by these methods were compared. Transcriptome analysis confirmed proteome data only partially, but these two semi-quantitative approaches established the interdependences between expression levels of genes and proteins. The discrepancies between data obtained by two approaches were discussed. The unique feature of the fetal liver cell content is the combination of hemopoietic and nonmaturated liver cells. Fetal liver changes its hemopoietic role during embryogenesis towards the detoxicative tissue being able to metabolize different substances and produce wide serum of proteins. The status of fetal liver as the hemopoietic tissue was entirely characterized by transcriptome analysis having registered embryonic and adult hemoglobins, insufficient cytochrome-dependent detoxification system and highly developed anti-apoptotic potential.

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.

[Enhanced control of proliferation in telomerized cells].

Clones of telomerized fibroblasts of adult human skin have earlier been obtained. It was shown that despite their fast growth in mass cultures, these cells poorly form colonies. Conditioned medium, antioxidants, and reduced partial oxygen pressure enhanced their colony formation, but not to the level characteristic of the initial cells. The conditioned medium of telomerized cells enhanced colony formation to a much greater extent than that of the initial cells. A study of proteome of the telomerized fibroblasts has revealed changes in the activities of tens of genes. A general trend consists in weakening and increased lability of the cytoskeleton and in activation of the mechanisms controlling protein degradation. However, these changes are not very pronounced. During the formation of immortal telomerized cells, selection takes place, which appears to determine changes in the expression of some genes. It was proposed that a decrease in the capacity of telomerized cells for colony formation is due to increased requirements of these cells to cell-cell contacts. The rate of cell growth reached that characteristic of mass cultures only in the largest colonies. In this respect, the telomerized fibroblasts resembled stem cells: they are capable of self-maintenance, but "escape" to differentiation in the absence of the corresponding microenvironment (niche), which is represented by other fibroblasts. Non-dividing cells in the test of colony formation should be regarded as differentiated cells, since they have no features of degradation, preserve their viability, actively move, grow, phagocytized debris, etc. It was also shown that telomerization did not prevent differentiation of myoblasts and human neural stem cells. Thus, the results obtained suggest the existence of normal mechanisms underlying the regulation of proliferation in the telomerized cells, which opens possibilities of their use in cell therapy, especially in the case of autotransplantation to senior people, when the cell proliferative potential is markedly reduced and accessibility of stem cells is significantly restricted.

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.

[Calcium antagonists nicardipine and verapamil suppress Ca(2+) entry and activate Na(+) entry into platelets]. / Kal'tsievye antagonisty nikardipin i verapamil podavliaiut vkhod Ca(2+) i usilivaiut vkhod Na(+) v trombotsity.

In micromolar concentrations both antagonists suppressed CA2+ entry and simultaneously elevate the agonist-induced plasma membrane depolarization due to Na+ inward current via these channels. Potentiation by nicardipine of the Na+ current induced by the platelet-activating factor, was revealed. Both antagonists caused plasma membrane depolarization suppressed by Na+ and Ca2+ ions. The depolarization disappeared after substitution of NaCl by an isotonic solution of choline chloride. The antagonists nicardipine and verapamil seem to modulate the platelet receptor-operated channels suppressing Ca2+ entry and elevating Na+ current via these channels.

A 59 amino acid insertion increases Ca(2+) sensitivity of rbslo1, a Ca2+ -activated K(+) channel in renal epithelia.

We previously cloned a MaxiK channel alpha-subunit isoform, rbslo1, from rabbit kidney with an amino acid sequence highly homologous to mslo but with a 59 amino acid insertion between S8 and S9 (Morita et al., 1997. Am. J. Physiol. 273:F615-F624). rbslo1 activation properties differed substantially from mslo with much greater Ca2+ sensitivity, half-activation potential of -49 mV in 1 micron m Ca2+. We now report single-channel analysis of rbslo1 and delA, a construct produced by removal of the 59 amino acid insertion at site A. delA is identical to mslo from upstream of S1 to downstream of S10 with the exception of 8 amino acids. Slope of the steady-state Boltzmann voltage activation curve was 8.1 mV per e-fold change in probability of opening for both rbslo1 and delA. The apparent [Ca2+](i) properties in delA were more like mslo but the voltage-activation properties remained distinctly rbslo1. Ca2+ affinity decreased and transmembrane voltage effects on apparent Ca2+ affinity increased in delA. The differences between rbslo1 and other cloned channels appear to be localized at insertion site A with both the insertion sequence and amino acid substitutions near site A being important. The steeper activation slope makes the channel more responsive to small changes in transmembrane voltage while the insertion sequence makes the channel functional at physiological low levels of [Ca2+](i).

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.

Inhibitory effect of lysophosphatidylcholine and phospholipase A2-treated low density lipoproteins on receptor-dependent regulation of {Ca2+}i in platelets.

Treatment of LDL with phospholipase A from bee venom resulted in formation of lipid-protein particles (pl2 LDL) with increased content of lysophosphatidylcholine (LPC). At the same time, composition of other lipids and protein structure were unaffected. Both pl-LDL and LPC abolished PAF-, ADP- and thrombin-induced Ca2+ elevation in platelets and platelet aggregation, while LDL had no effect on hormone-stimulated increase in the intracellular Ca2+ content ({Ca2+}i). The effect persisted in Ca2+-free medium, indicating that pl-LDL and LPC also abolish Ca2+ mobilisation from intracellular stores. Neither LPC nor pl-LDL changed platelet Ca2+ levels and inhibited platelet aggregation induced by thapsigargin, a specific inhibitor of the endoplasmic reticulum Ca2+ ATPase. The inhibitory effect depended on LPC concentration, incubation time and the structure of LPC: lysophosphatidylethanolamine and phosphatidylcholine produced no inhibitory effect. The half-maximum-effective concentrations were the same for LPC and pl-LDL (2-4 microM). The results obtained indicate that LPC and pl-LDL inhibit the receptor-dependent increase in Ca2+. It can be suggested that the effect of LPC is mediated by redistribution of the plasma membrane integral proteins, which leads to disintegration of the intracellular signalling systems.

Blockade of receptor-operated calcium channels by mibefradil (Ro 40-5967): effects on intracellular calcium and platelet aggregation.

The goal of the present study was to evaluate if mibefradil, a novel nondihydropyridine Ca2+ antagonist, could block receptor-operated calcium channels (ROCC) present in human platelets and to determine the functional consequences of this blockade. Therefore, the effect of mibefradil on increases in intracellular Ca2+ concentrations and aggregation of human platelets induced by platelet activating factor (PAF) was examined. In order to differentiate effects on Ca2+ mobilization from intracellular stores from those on Ca2+ influx through ROCC, intracellular Ca2+ concentrations were measured either in fura-2-loaded platelets or in cells loaded with both BAPTA and fura-2. Mibefradil totally and dose dependently inhibited PAF-induced Ca2+ influx with a maximal effective concentration of 10 microM, but at this concentration only reduced Ca2+ mobilization from intracellular stores. A similar effect was observed when platelets were stimulated with ADP, suggesting that mibefradil was indeed interfering with ROCC and not specifically with PAF receptors. In the same range of concentrations, mibefradil inhibited Ca(2+)-dependent platelet aggregation induced by PAF. This effect was most likely due to the inhibition of ROCC, as Ca(2+)-independent aggregation induced by phorbol-myristyl-acetate (PMA) was insensitive to mibefradil. We conclude that mibefradil, which has previously been described to be an antagonist for L- and T-Type Ca2+ channels, also blocks receptor-operated Ca2+ channels. This blockade seems to be functionally relevant for platelet aggregation.