Hypergravity affects cell cycle progression and caveolin-1 expression of in vitro cultured human primary endothelial cells

In hypogravity conditions unloading of skeletal muscle fibres causes alterations in skeletal muscle structure and functions including growth, gene expression, cell differentiation, cytoskeletal organization, contractility and plasticity. Recent studies have identified sphingosine I -phosphate (SPP) as a lipid mediator capable of eliciting intracellular Ca2+ transients, cell proliferation, differentiation, suppression of apoptosis, as well as cell injury repair. The aim of this research is to evaluate a possible involvement of SPP in skeletal muscle cells differentiation and repair from space-flight damage. Particularly, we investigated the Ca2+ sources and the changes on the cytoskeletal rearrangement induced by SPP in a mouse skeletal (C2C12) myoblastic cell line. Confocal fluorescence imaging revealed that SPP elicited Ca2+ transients which propagated throughout the cytosol and nucleus. This response required extracellular and intracellular Ca2+ mobilization. SPP also induced cell contraction through a Ca2(+)- independent/Rho-dependent pathway. The nuclear Ca2+ transients are suggestive for an action of SPP in the differentiation program and damage repair.     

The pattern of dihydrofolate reductase expression through the cell cycle in rodent and human cultured cells

We have examined the pattern of dihydrofolate reductase (DHFR) enzyme and mRNA levels in cell cycle stage-specific populations obtained by centrifugal elutriation in Chinese hamster ovary cells and in a derivative line in which the dihydrofolate reductase gene is amplified approximately 50-fold. On a per cell basis, we observed a 2-fold increase in DHFR activity as cells progressed from G1 to G2/M with a concomitant 2-fold increase in the rate of protein synthesis and steady state level of mRNA. Analysis of DHFR mRNA levels in cell cycle stage-specific mouse 3T6 and human 143 tk- cells gave a similar pattern. We also demonstrate that simple alterations in growth conditions prior to elutriations can dramatically increase the levels of DHFR mRNA in all cell cycle states, thereby indicating that growth response associated with the DHFR gene functions independent of the cell cycle. We conclude that during periods of exponential growth the increases in dihydrofolate reductase activity, rate of protein synthesis, and steady state levels of mRNA parallel the general increases in cell volume and protein content associated with normal progression through the cell cycle, and therefore DHFR cannot be considered a cell cycle-regulated enzyme.     

Lethal effects of fluorodeoxyuridine on cultured mammalian cells at various stages of the cell cycle

The lethal damage induced by the exposure of synchronized Chinese hamster cells to various concentrations of 5-fluoro-2′deoxyuridine (FUdR) was not selectively restricted to cells exposed during the period of DNA synthesis S. The colony survival fraction observed after treatment for one hour with 5 × 10^?5 M FUdR was very low (0.0001–0.0003) whether the drug was administered during early G₁, late G₁, early S or in middle S. The survival of cells treated with the same concentration of FUdR during mitosis, however, was significantly higher (0.62) showing that mitotic cells were less sensitive to FUdR. Administration of 10^?7M thymidine or “conditioned” medium for one hour reversed the lethal effect of FUdR or improved the survival, depending on the time after removal of the FUdR at which these substances were given.     

The relationship between stem cell seeding efficiency and position in cell cycle.

The seeding efficiency of colony-forming cells from normal, regenerating and velocity-sedimented cycling and non-cycling narrow preparations was compared. Colony-forming cells in cycle were found to exhibit a 50% reduction in splenic seeding when compared to normal marrow or sedimented non-cycling cells. The results of this study indicate that the spleen colony assay underestimates the total number of colony-forming cells by a fraction which is directly related to the number of cells in cycle.     

Decreased thrombin affinity of cell-surface thrombomodulin following treatment of cultured endothelial cells with beta-D-xyloside

Thrombomodulin, an endothelial cell-surface anticoagulant, has been postulated to contain a glycosaminoglycan. Thrombomodulin function was therefore studied in endothelial cells treated with beta-D-xyloside, an inhibitor of glycosaminoglycan attachment to proteoglycan core proteins. Beta-D-xyloside caused a reproducible 3 to 5-fold increase in the Km of thrombomodulin for thrombin and a 20-30% decrease in the rate of protein C activation by the thrombin-thrombomodulin complex. These results support a role for glycosaminoglycans in thrombomodulin function and suggest that beta-D-xylosides can be used to investigate both the anticoagulant mechanisms and the biosynthesis of cell-surface thrombomodulin.     

Regulatory mechanisms for thrombomodulin expression in human umbilical vein endothelial cells in vitro.

It has been reported that thrombomodulin (TM) expression in endothelial cells is modulated by various agents. We investigated cellular regulatory mechanisms for TM expression in human umbilical vein endothelial cells (HUVECs), incubated with agents, by measuring the time course changes in surface TM activity, total TM antigen in cell lysates, and TM mRNA levels. While dibutyryl cAMP (3 mM) increased TM mRNA levels in HUVECs and was followed by increased TM activity, dibutyryl cGMP had no effect on TM activity. Phorbol myristate acetate (PMA) induced rapid loss of surface TM activity (approximately 8 h) and later increased TM mRNA levels between 4 h and 40 h (maximum at 24 h), resulting in biphasic effects on TM activity. Tumor necrosis factor or interleukin-1 beta suppressed surface TM activity and TM mRNA levels. Internalization/degradation of TM in HUVECs incubated with PMA or cytokines was suggested by co-culture with chloroquine. The decrease in surface TM activity observed was not caused by the release of TM molecules from the cells into the conditioned media. These results suggest that TM activity in HUVECs is modulated by independent mechanisms involving cytoplasmic TM mRNA levels and internalization/degradation of TM molecules. These regulatory mechanisms may involve protein kinase A and protein kinase C-dependent mechanisms but are independent of protein kinase G.     

Analysis of cell cycle phases and progression in cultured mammalian cells

Fluorescence activated cell sorting (FACS) analysis has become a standard tool to analyze cell cycle distributions in populations of cells. These methods require relatively large numbers of cells, and do not provide optimal resolution of the transitions between cell cycle phases. In this report we describe in detail complementary methods that utilize the incorporation of nucleotide analogs combined with microscopic examination. While often more time consuming, these protocols typically require far fewer cells, and allow accurate kinetic assessment of cell cycle progression. We also describe the use of a technique for the synchronization of adherent cells in mitosis by simple mechanical agitation (mitotic shake-off) that eliminates physiological perturbation associated with drug treatments.     

Adenosine regulates thrombomodulin and endothelial protein C receptor expression in folliculostellate cells of the pituitary gland

Adenosine stimulates the release of interleukin 6 (IL-6) and vascular endothelial growth factor from folliculostellate cells of the anterior pituitary gland indicating that such cells are also involved in the communication between the immune and endocrine systems during stress and inflammation. In order to understand the precise actions of adenosine on folliculostellate cells, DNA microarray analysis was used to determine global changes in gene expression. Hierarchical clusters revealed, of the genes that had altered expression, the majority were suppressed and many, such as B cell translocation gene 2 and cyclin-dependent kinase inhibitor 2b were related to cell cycle arrest or inhibition of proliferation. Several of the up-regulated genes were associated with cytokine signalling or membrane receptor activity. The most notable of these being IL-6, sulfiredoxin 1, endothelial protein C receptor (EPCR) and thrombomodulin (THBD) which can all play a role in controlling inflammation. The EPCR and THBD pathway is well known in anti-coagulation but also has anti-inflammatory and anti-apoptotic properties. Up-regulation of EPCR and THBD in folliculostellate cells was confirmed by qRT-PCR and western blotting analysis and their expression were also demonstrated in many of the hormone-secreting cells of the anterior pituitary gland. Our findings suggest that adenosine can stimulate expression of stress and inflammation related genes from folliculostellate cells of the anterior pituitary gland. These genes include EPCR and THBD, neither of which has been previously identified in the pituitary gland.     

Inverse relationship between connexin43 and desmin expression in cultured porcine aortic smooth muscle cells.

Our previous work has shown that in vascular tissues the elastic medial regions express high levels of the gap junctional protein, connexin43, but low levels of desmin, while the muscular medial regions express low levels of connexin43 but high levels of desmin. It is uncertain, however, whether this regional difference at the tissue level extends down to the level of the individual cell, or reflects an averaged relationship of groups of cells of different connexin43 and desmin expression. The present study has addressed this question using cultured porcine aortic smooth muscle cells. Immunoconfocal microscopic analysis of single-labeled cells showed that while smooth muscle alpha-actin, calponin and vimentin were positively labeled in the majority of medial smooth muscle cells both in intact porcine aorta and corresponding cultured cells, desmin and connexin43 labeling was highly heterogeneous. In the cultured cells, 0.3-0.5% of cells were found to be desmin-positive, and quantitative analysis after double labeling for desmin and connexin43 revealed that the desmin-positive cells were smaller, and contained significantly lower numbers and smaller sizes of connexin43 gap-junctional spots than did desmin-negative cells. Our findings demonstrate that an inverse expression pattern of connexin43 and desmin holds true at the level of the individual cell. This suggests a close relationship between intrinsic phenotypic control and the regulation of connexin43 expression in the arterial smooth muscle cell.     

Inhibition of proliferation of cultured rat liver epithelial cells at specific cell cycle stages by transforming growth factor-beta

Proliferation of early-passage propagable cultured rat liver epithelial cells derived from normal adult rats is markedly inhibited by transforming growth factor-beta (TGF-beta). Inhibition, which is completely reversible, is effected at two distinct points of the cell cycle, the G1/S border and the G0 or early G1 phase. With increasing passages in culture, hepatic epithelial cells progressively become less sensitive to the inhibitory effect of TGF-beta.     

Adhesion and the cell cycle in cultured L929 and CHO cells

The adhesiveness of L929 and CHO-K1 cells was monitored throughout their respective cell cycles. The cell cycle stages were identified by a combination of measuring, histochemical and autoradiographic techniques. Adhesiveness was shown to be maximal during the S and late G2 phases, and minimal at mitosis. S cells adhered selectively to other S cells, while late G2 phase cells adhered selectively to other late G2 cells. Mixing of the two cell lines, L929 and CHO-K1, resulted in the S phase cells adhering to each other irrespective of the cell line to which they belonged. The phases of increased adhesiveness and selectivity coincided with a decrease in cell surface negative charge, corresponding to well documented changes in the morphology of the cells.     

Oxygen-sensitive stages of the cell cycle of human diploid cells

We had established that growth of human diploid WI-38 cells is reversibly inhibited by elevated partial pressures of oxygen (PO2) and we were interested in determining where in the cell cycle growth was delayed. A technique combining cytospectrophotometry and autoradiography was used to determine cell cycle parameters. Confluent cells that were subcultivated and exposed to a PO2 of 365 +/- 8 mm Hg were delayed primarily after DNA synthesis but before metaphase. At a PO2 of 590 +/- 35 mm Hg, most cells did not initiate DNA synthesis, and the few that did, failed to complete the process. When exponentially growing cells that had already begun DNA synthesis were exposed to a PO2 of 590 p 35 mm Hg, they accumulated after completing DNA synthesis but before initiating mitosis. The rate at which (3H)thymidine was incorporated into DNA was inversely correlated with oxygen tension (PO2 of 135--590 mm Hg). These results suggest that the process most sensitive to oxygen causes cells to be delayed after DNA synthesis but before metaphase. Slightly higher PO2's were needed to inhibit the initiation of DNA synthesis. Further, the rate of DNA synthesis is decreased by elevated oxygen tensions.     

Relationship between anticoagulant activities and polyanionic properties of rabbit thrombomodulin

Rabbit thrombomodulin displays three distinct blood anticoagulant activities: it promotes the activation of protein C by thrombin (protein C activation cofactor activity); it promotes the inactivation of thrombin by thrombin (direct anticoagulant activity). The effects on these activities of mouse anti-thrombomodulin monoclonal antibodies and of the heparin-neutralizing proteins, platelet factor 4, histidine-rich glycoprotein, and S-protein, were investigated. One of the antibodies, which did not influence the functional properties of thrombomodulin, was used as an immunoaffinity ligand for purification of the protein. Two other antibodies, which were found to abrogate the protein C activation cofactor activity of the purified thrombomodulin, also abolished the antithrombin-dependent and the direct anticoagulant activities. The heparin-neutralizing proteins all inhibited the two latter activities, albeit to a varying extent, but did not appreciably affect the activation of protein C. These results are interpreted in relation to our previous finding that rabbit thrombomodulin contains an acidic domain, tentatively identified as a sulfated glycosaminoglycan (Bourin, M.-C., Boffa, M.-C., Bj?rk, I., and Lindahl, U. (1986) Proc. Natl. Acad. Sci. U.S.A. 83, 5924-5928). It is proposed that the acidic domain interacts with thrombin at the protein C activation site and that this interaction is a prerequisite to the expression of direct as well as antithrombin-dependent anticoagulant activity. The interaction is not essential to, but compatible with, the activation of protein C. Experiments involving treatment of thrombomodulin with various glycanases or with nitrous acid, followed by measurement of anticoagulant activities, indicated that the acidic domain is constituted by a sulfated galactosaminoglycan and not by a heparin-related polysaccharide as previously suggested.     

Altered rabbit endothelial cell phenotypic expression in response to human fibronectin

When rabbit corneal endothelial cells were cultured in excess concentrations of human fibronectin an altered phenotypic expression was observed. Cell appearance was changed radically and collagen synthesis was specifically inhibited in a dose-response fashion. This study provides further evidence that fibronectin may be one of the developmental signals which act a molecular level and is capable of interspecies activity.     

The relationship between electron transport capacity and cell cycle stage in a marine diatom

The proportion of the primary electron transport acceptor forphotosystem II(Q) in the oxidized state was estimated usingin vivo fluorescence from populations of the marine diatom Thalassiosirapseudonana. Under nutrient replete conditions, the transitionfrom light-dependent to light-independent development duringthe cell cycle was correlated with a 20% decrease in the amountof Q in the oxidized state. As a consequence, mean redox statuswas correlated with the fraction of the population in the pre-commitmentor light-dependent period of the cycle. Comparison with theresults of other investigators suggests that de-coupling ofphotosynthetic units from the electron transport system linkingphotosystems II and I during the committed or light-independentperiod of the cycle, may be the mechanism controlling the redoxstate of Q.