bluuebunny/crossref_metadata_2025 huggingface dataset:
Hematopoietic progenitor cells of transgenic mice with increased copper/zinc‐superoxide dismutase activity are resistant to tumor necrosis factor
Authors: Sakashita Akiko , Koeffler H. Phillip , Epstein Charles J. , Carlson Elaine
Published: February 2005

The mechanism of growth inhibition mediated by tumor necrosis factor (TNF) is unclear. Since recent data strongly suggested that generation of superoxide is a key step in cytotoxicity of TNF, we reasoned that cells expressing high levels of enzymes that degrade superoxide radicals would be resistant to TNF. Therefore, we examined the TNF‐sensitivity of bone marrow progenitor cells of transgenic mice that expressed the gene for human copper zinc‐superoxide dismutase (CuZn‐SOD). The CuZn‐SOD is a key enzyme in the metabolism of superoxide radicals. Heterozygous and homozygous transgenic mice had 3‐ and 5‐fold increased levels of CuZn‐SOD activity, respectively. Bone marrow cells of transgenic and nontransgenic mice were plated in soft gel culture with TNF (0.01–100 ng/ml). TNF inhibited myeloid colony formation supported by either granulocyte‐macrophage colony‐stimulating factor (GM‐CSF) or G‐CSF from nontransgenic mice in a dose‐dependent manner. In contrast, the myeloid clonal growth of homozygote transgenic mice was not inhibited by TNF at concentrations up to 100 ng/ml. As expected, the effects of TNF on erythroid clonogenic cells, which do not produce superoxide, and the action of transforming growth factor‐β on myeloid progenitor cells, were similar in both transgenic and nontransgenic mice. These results suggest that the mechanism of TNF‐mediated growth inhibition of hematopoietic cells occurs through production of superoxide. © 1994 Wiley‐Liss, Inc.
Binding and degradation of hyaluronan by human breast cancer cell lines expressing different forms of CD44: Correlation with invasive potential
Authors: Culty Martine , Shizari Mehran , Thompson Erik W. , Underhill Charles B.
Published: February 2005

In the present study, we examined a panel of human breast cancer cell lines with regard to their expression of CD44 and ability to bind and degrade hyaluronan. The cell lines expressed varying amounts of different molecular weight forms of CD44 (85–200 kDa) and, in general, those that expressed the greatest amounts of CD44 were the most invasive as judged by in vitro assays. In addition, the ability to bind and degrade hyaluronan was restricted to the cell lines expressing high levels of CD44, and both these functions were blocked by an antibody to CD44 (Hermes‐1). Moreover, the rate of [3H]hyaluronan degradation was highly correlated with the amount of CD44 (r = 0.951, P < 0.0001), as well as with the invasive potential of the cells. Scatchard analysis of the [3H]hyaluronan binding of these cells revealed the existence of significant differences in both their binding capacity and their dissociation constant. To determine the source of this deviation, the different molecular weight forms of CD44 were partially separated by gel filtration chromatography. In all cell lines, the 85 kDa form was able to bind hyaluronan, although with different affinities. In contrast, not all of the high molecular weight forms of CD44 had this ability. These results illustrate the diversity of CD44 molecules in invasive tumor cells, and suggest that one of their major functions is to degrade hyaluronan. © 1994 Wiley‐Liss, Inc.
Hormonal regulation of some steps of thyroglobulin synthesis and secretion in bicameral cell culture
Authors: Desruisseau S. , Alquier C. , Depetris D. , Gruffat D. , Chabaud O.
Published: February 2005

Porcine thyroid cells were cultured for 15 days on porous bottom chambers with or without different mixtures of hormones added to serum‐free basal medium. Assays with 10% serum were also performed for comparison with previously published results. The effects of the hormones, particularly insulin, TSH and hydrocortisone, were studied on total RNA content, thyroglobulin mRNA level, the amount of thyroglobulin secreted into the apical medium and on glycosylation. Insulin and TSH similarly increased the total RNA content, and their effects were additive. Thyroglobulin mRNA content was increased twofold by insulin and threefold by TSH. When they were added simultaneously, the maximal level of thyroglobulin mRNA was reached, showing that TSH and insulin effects on thyroglobulin gene expression were additive. Hydrocortisone alone did not modify total RNA or thyroglobulin mRNA content but the hormone amplified total RNA when insulin and TSH were present together. The basal level of thyroglobulin secreted into the apical medium was increased threefold by insulin and fourfold by TSH. The effects of these two hormones added together appeared to be additive. Hydrocortisone had no effect alone or even when combined with insulin or TSH. However, when the three hormones were added together, the hormonal response was amplified. TSH effect and insulin effect on the incorporation of 3H‐mannose into thyroglobulin as well as on the anionic residue content of the molecule were additive. © 1994 Wiley‐Liss, Inc.
Changes in cytoskeletal proteins and their mRNAs during maturation of human erythroid progenitor cells
Authors: Wickrema Amittha , Dai Chun‐Hua , Krantz Sanford B. , Koury Stephen T.
Published: February 2005

We have used highly purified human early erythroid progenitors to study changes in cytoskeletal proteins during their maturation and terminal differentiation. When erythroid progenitors at the burst‐forming unit‐erythroid (BFU‐E) stage of development are grown in the presence of erythropoietin, the cells mature and terminally differentiate into reticulocytes during a 14–15‐day culture period. We have shown by immunofluorescence that spectrin is present in day 3 BFU‐E, at which time proteins band 3, ankyrin, and band 4.1 cannot be detected. Ankyrin and band 4.1 were detected in the majority of the cells by day 7 of culture, at the colony‐forming unit (CFU)‐E stage, whereas only 15% of the cells were positive for band 3 protein on day 7 of culture. The mRNA level for spectrin was already at its maximum on day 8 whereas the mRNAs for band 3, ankyrin, and band 4.1 were just beginning to accumulate. After enucleation, spectrin, band 3, ankyrin, and band 4.1 fluorescence were all associated with the reticulocytes. Actin was localized at the constriction between the extruding nucleus and the incipient reticulocyte in enucleating erythroblasts suggesting a key role for actin in the enucleation of human erythroblasts. Our investigations have also shown that purified human erythroid progenitors cultured in serum‐free suspension media are capable of enucleating without the requirement of an extracellular matrix. These results demonstrate that the synthesis and expression of major cytoskeletal proteins in the human erythrocyte membrane occur in an asynchronous manner and that the remodeling of the membrane skeleton begins at a very early stage during erythrocyte development. © 1994 Wiley‐Liss, Inc.
Stimulation of in vitro hematopoiesis by a murine fetal hepatocyte clone through cell—cell contact
Authors: Nanno Masanobu , Hata Masahiro , Yagi Hideki , Itoh Tsunetoshi , Doi Hideyuki , Satomi Susumu , Sakata Tsuneaki , Suzuki Ryuji
Published: February 2005

We have previously shown that a fetal liver‐derived epithelial cell clone, FHC‐4D2, could support hematopoiesis in vitro through its colony‐stimulating factor (CSF) activities in a short‐term culture. In this study, since FHC‐4D2 cells were found capable of maintaining hematopoietic progenitors in the coculture for a long time, we examined how FHC‐4D2 could exert hematopoietic supporting activity in a long‐term culture by coculturing adult bone marrow (BM) cells or fetal liver (FL) cells on a monolayer of FHC‐4D2 cells. This clone could maintain the colony‐forming unit of granulocytes and macrophages (CFU‐GM) of BM for ≥ 12 weeks under the coculture condition, but the fibroblastic cell clone from the fetal liver, FHC‐4A3, could not support the survival of CFU‐GM, even for 1 week. In addition to BM CFU‐GM, the FHC‐4D2 clone also supported the survival of FL CFU‐GM, burst‐forming unit of erythroid cells (BFUe), and colony‐forming unit of mixed progenitors (CFU‐Mix) for longer than 4 weeks. When BM cells were separated by a membrane filter from the FHC‐4D2 cells in the coculture, the comparable number of CFU‐GM was maintained at day 3, but virtually no hematopoietic progenitors were detected at the end of the first week. CFU‐GM were present in both nonadherent and adherent cells to the FHC‐4D2 cells at day 3 of the coculture, but at day 7, the adherent population contained greater number of CFU‐GM. CFU‐GM derived from the adherent cells formed larger colonies and contained more bipotential CFU‐GM than the nonadherent population. When BM cells from mice given 5‐fluorouracil were cocultured with FHC‐4D2 cells under the limiting dilution condition, interleukin‐3 (IL‐3)‐responsive CFU‐GM were induced from immature hematopoietic progenitor cells that were otherwise unresponsive to IL‐3. From these data we conclude that the FHC‐4D2 clone could generate and maintain IL‐3‐responsive hematopoietic progenitors via close contact and that, in the fetal liver, the contact between hepatocytes and hematopoietic cells may be critically important in inducing the differentiation of resting, IL‐3‐unresponsive immature hematopoietic cells into CFU‐GM (progenitors responsive to IL‐3) and in triggering the self‐renewal of CFU‐GM. © 1994 Wiley‐Liss, Inc.
Effect of tyrosine kinase inhibition on basal and epidermal growth factor‐stimulated human Caco‐2 enterocyte sheet migration and proliferation
Authors: Basson Marc D. , Turowski Gregory , Zarif Abdul , Modlin Irvin M. , Beidler David R. , Jena Bhanu P. , Madri Joseph A.
Published: February 2005

Mucosal healing requires enterocyte migration (restitution) supplemented by proliferation. Proliferation and migration may be studied independently by thymidine uptake and proliferation‐blocked cell migration using human Caco‐2 enterocyte monolayers in culture. Since epidermal growth factor (EGF) promotes mucosal healing and the EGF receptor is a tyrosine kinase, we hypothesized that tyrosine kinases might therefore modulate enterocyte migration and proliferation. The tyrosine kinase inhibitors genistein and 2, 5‐dihydroxymethylcinnamate, which block kinase ATP‐binding and substrate‐binding sites, respectively, were studied alone and with EGF. Proliferation was blocked with mitomycin. Although each inhibitor decreased basal and EGF‐stimulated monolayer expansion when cell proliferation occurred, neither genistein nor 2, 5‐dihydroxymethylcinnamate decreased migration when proliferation was blocked. However, each inhibitor prevented EGF stimulation of proliferation‐blocked migration and thymidine uptake. More substantial inhibition of basal proliferation by genistein correlated with increased protein‐linked DNA breaks, which may reflect nonspecific inhibition of DNA topoisomerase activity by genistein. The more specific 2,5‐dihydroxymeth‐ylcinnamate blocked changes in the α2 integrin subunit organization which may modulate EGF‐stimulated migration. Antiproliferative effects of tyrosine kinase inhibitors decrease basal monolayer expansion but true basal enterocyte migration appears independent of tyrosine kinase regulation. However, a specific tyrosine kinase‐dependent modulation of cell‐matrix interaction inhibits EGF‐stimulated migration. © 1994 Wiley‐Liss, Inc.
Synergistic stimulation of DNA synthesis by bradykinin and vasopressin in swiss 3T3 cells
Authors: Kiehne Karlheinz , Rozengurt Enrique
Published: February 2005

Vasopressin and bradykinin bind to receptors coupled to GTP‐binding proteins and rapidly induce polyphosphoinositide breakdown leading to Ca2+ mobilization and activation of protein kinase C. Both peptides are known to induce mitogenesis in the presence of growth factors that act through receptors with intrinsic tyrosine kinase activity. Surprisingly, addition of a combination of vaso‐pressin and bradykinin to Swiss 3T3 cells synergistically stimulates DNA synthesis in the absence of any other growth factors. This effect is induced at nanomolar concentrations of the peptides and could be inhibited by addition of specific receptor antagonists or broad spectrum neuropeptide antagonists. Bradykinin, which stimulates transient activation of protein kinase C, induces DNA synthesis in synergy with substances that cause long‐term activation of protein kinase C, like vasopression or phorbol 12, 13‐dibutyrate. Down‐regulation of protein kinase C inhibited the induction of mitogenesis by the combination of vasopressin and bradykinin, thus demonstrating the importance of long‐term activation of this enzyme for DNA synthesis. Analysis of tyrosine phosphorylated proteins of Mr = 110,000–130,000 and Mr = 70,000–80,000 revealed a biphasic response after stimulation with bradykinin, whereas the response induced by vasopressin declined after the initial maximum. The combination of bradykinin with vasopressin caused an enhanced and prolonged increase in tyrosine phosphorylation of these proteins as compared with the individual peptides. Inhibition of tyrosine phosphorylation by tyrphostin was paralleled by inhibition of DNA synthesis. Together, these results demonstrate synergistic stimulation of DNA synthesis by bradykinin and vasopressin via prolonged stimulation of multiple signaling pathways and imply that the interactive effects of Ca2+ ‐mobilizing peptides on mitogenesis may be more general than previously thought. © 1994 Wiley‐Liss, Inc.
Cytokine production and DNA synthesis by Human peripheral Lymphocytes in response to Palmitic, Stearic, Oleic, and Linoleic acid
Authors: Karsten S. , Schäfer G. , Schauder P.
Published: February 2005

Effects of palmitic, stearic, oleic, and linoleic acid on mitogen‐induced DNA synthesis, on production of IL‐1β, IL‐2, IFN‐gamma, and TNF‐α, and on IL‐2R expression were determined in human peripheral lymphocytes. Free fatty acids (FFA) were added over a wide range of concentrations to cells cultured under serum free conditions with fatty acid free albumin. DNA synthesis was stimulated by low and inhibited by high FFA concentrations. Physiologica concentrations were stimulatory, except for linoleic acid. Cytokine production became affected by all FFA tested. Palmitic acid enhanced the release of IFN‐gamma at concentrations that diminished TNF‐α production. Saturated fatty acids were significantly more potent than unsaturated fatty acids in affecting cytokine production. IFN‐gamma secretion was significantly more stimulated or inhibited by the various FFA compared with the other cytokines. IL‐2R expression correlated with the production of IL‐2. When tested in combination, stimulatory as well as inhibitory effects of the individual FFA became attenuated. It is suggested that palmitic, stearic, oleic, and linoleic acid are physiological regulators of DNA synthesis and cytokine release in human peripheral lymphocytes. Modulation of FFA ratios may be an effective means for the fine tuning of the immune system. As secretory mechanisms of cytokines appear to exhibit substrate specificity for FFA, the release of individual cytokines may be selectively influenced by FFA. © 1994 Wiley‐Liss, Inc.
Modulation of cardiac myocyte phenotype in vitro by the composition and orientation of the extracellular matrix
Authors: Simpson D. G. , Terracio L. , Terracio M. , Price R. L. , Turner D. C. , Borg T. K.
Published: February 2005

Cellular phenotype is the result of a dynamic interaction between a cell's intrinsic genetic program and the morphogenetic signals that serve to modulate the extent to which that program is expressed. In the present study we have examined how morphogenetic information might be stored in the extracellular matrix (ECM) and communicated to the neonatal heart cell (NHC) by the cardiac α1β1 integrin molecule. A thin film of type I collagen (T1C) was prepared with a defined orientation. This was achieved by applying T1C to the peripheral edge of a 100 mm culture dish. The T1C was then drawn across the surface of the dish in a continuous stroke with a sterile cell scraper and allowed to polymerize. When NHCs were cultured on this substrate, they spread, as a population, along a common axis in parallel with the gel lattice and expressed an in vivo‐like phenotype. Individual NHCs displayed an elongated, rod‐like shape and disclosed parallel arrays of myofibrils. These phenotypic characteristics were maintained for at least 4 weeks in primary culture. The evolution of this tissue‐like organizational pattern was dependant upon specific interactions between the NHCs and the collagen‐based matrix that were mediated by the cardiac α1β1 integrin complex. This conclusion was supported by a variety of expermental results. Altering the tertiary structure of the matrix or blocking the extracellular domains of either the cardiac α1, or β1 integrin chain inhibited the expression of the tissue‐like pattern of organization. Neither cell‐to‐cell contact or contractile function were necessary to induce the formation of the rod‐like cell shape. However, beating activity was necessary for the assembly of a well‐differentiated myofibrillar apparatus. These data suggest that the cardiac α1 β1 integrin complex serves to detect and transduce phenotypic information stored within the tertiary structure of the surrounding matrix. © 1994 Wiley‐Liss, Inc.
Slow oscillations of free intracellular calcium ion concentration in human fibroblasts responding to mechanical stretch
Authors: Arora Pamela D. , Bibby Kathryn J. , McCulloch Christopher A. G.
Published: February 2005

Calcium transients in single, human gingival fibroblasts were studied after mechanical stretching of flexible culture substrates. A model system was developed to reproducibly stretch and rapidly (< 1 sec) refocus cells in the same focal plane so that changes in the concentration of free intracellular calcium ions ([Ca2+]i) were monitored without delay. Attached cells were grown on flexible bottom Petriperm dishes, loaded with fura‐2/AM, and stretched by 1% or 2.8% of substrate area. The stretch caused no significant cell detachment or membrane lesions. A 1% stretch induced no calcium response, but a 2.8% stretch stimulated an initial calcium transient and the subsequent generation of [Ca2+]i oscillations of up to 2,000 sec. At 1% stretch, there was no calcium response. Cell shape and plating time were important determinants in the calcium response to mechanical stimulation: the responder cells were small and round without long processes. Major calcium transients were inhibited completely by 5 mM EGTA or by 10 μM gadolinium ions, by 50 μM nifedipine, or 250 μM verapamil, suggesting an influx of calcium through stretch‐activated (SA) channels and L‐type calcium channels. Depolarization by high KCl (144 mM) in the extracellular medium enhanced the amplitude of calcium transients by 54%. Calcium oscillations were not inhibited by preincubation with thapsigargin, caffeine, cholera toxin, staurosporine or 1‐(5‐isoquinolinesulfonyl)‐2‐methylpiperazine (H‐7), indicating that IP3 sensitive pools, IP3 insensitive pools, G5α subunits, and protein kinase C, respectively, were not involved in the generation of calcium oscillations. Pretreatment with genistein, a specific tyrosine kinase inhibitor or cytochalasin D, an inhibitor of actin polymerization, or pertussis toxin, an inhibitor of Giα and Goα subunits, completely abolished calcium transients and oscillations. These results indicate that Ca2+ flux due to mechanical stretching is likely mediated through SA ion channe s and is dependent on tyrosine kinases, pertussis toxin‐sensitive subunits of G‐proteins, and actin filaments. © 1994 Wiley‐Liss, Inc.
Mechanisms of enhanced angiotensin II–stimulated signal transduction in vascular smooth muscle by aldosterone
Authors: Ullian Michael E. , Fine Jana J.
Published: February 2005

We tested the hypothesis that mineralocorticoids potentiate angiotensin II–stimulated phospholipase C activation through an increased number of angiotensin II receptors in cultured rat aortic vascular smooth muscle cells. Exposure of cells to aldosterone for 24 h resulted in concentration‐dependent increases in angiotensin II receptor binding. Via studies of angiotensin II displacement by non‐peptide receptor antagonists, both basal and upregulated angiotensin II receptors were found to be of the AT1, subtype. Incubation with 1 μM aldosterone resulted in 50%–100% enhancement of angiotensin II (100 nM)–stimulated diacylglycerol formation and intracellular calcium mobilization. Exposure to 100 nM 1,25‐(OH)2 VitD3, which did not upregulate angiotensin II receptors, did not potentiate stimulated inositol phosphate formation. Incubation with aldosterone resulted in potentiation of inositol phosphate formation upon receptor occupation (100 nM angiotensin II) but not upon post‐receptor stimulation (25 mM NaF/10 μM AlCl3). Aldosterone did not increase basal phospholipase C activity or content of the inositol trisphosphate precursor phosphatidylinositol‐4,5‐bisphosphate. These data are consistent with the hypothesis that aldosterone potentiates angiotensin II–stimulated, phospholipase C‐dependent intracellular signals solely by coupling to an increased number of angiotensin II receptors. This mechanism may contribute to the sensitized vascular responses to angiotensin II observed in states of mineralocorticoid excess. © 1994 Wiley‐Liss, Inc.
High resolution analysis of cell cycle‐correlated vimentin expression in asynchronously grown, TPA‐treated MPC‐11 cells by the novel flow cytometric multiparameter BrdU‐hoechst/PI and immunolabeling technique
Authors: Giese Günter , Kubbies Manfred , Traub Peter
Published: February 2005

High resolution, multiparameter analysis using the flow cytometric BrdU/Hoechst quenching technique has been applied to study cell cycle kinetics and vimentin expression in individual cells of asynchronously grown MPC‐11 mouse plasmacytoma cell cultures treated with 12‐O‐tetradecanoylphorbol‐13‐acetate (TPA) to induce in vitro differentiation. BrdU treatment up to 16 h in the absence or presence of TPA did not affect either cell cycle progression or the kinetics or quantity of vimentin expression. TPA‐treated cells became arrested in G1 phase of the second cell cycle; however, this G1 phase arrest was transient only. In addition, G1 phase cells located prior to a putative transition point at the beginning of TPA treatment were completely blocked in cell cycle progression. There is also evidence that cells located in G1 or G2/M phase at the beginning of TPA treatment finally expressed low levels of vimentin. On the contrary, cells located in S phase at TPA exposure showed high vimentin levels after treatment. The results presented here show that, with the flow cytometric BrdU/Hoechst quenching technique, one can correlate time‐dependent protein expression at the single cell level in asynchronously grown cultures not only with the actual cell cycle state, but also with the history of cell replication. © 1994 Wiley‐Liss, Inc.
Redistribution of plasma membrane proteins by electroosmosis elicits cytosolic calcium response in tumor mast cells
Authors: Feder Toni J. , Webb Watt W.
Published: February 2005

Activation of mast cells and basophils by binding of ligands that crosslink and micro‐aggregate cell surface receptors leads to a series of responses including a phosphoinositide cascade, elevation of intracellular free calcium ([Ca2+]i), morphological changes in the cell plasma membrane, and ultimately, exocytosis of granules containing histamine and other mediators of the allergic response. In rat basophilic leukemia (RBL) cells, a tumor mast cell line, stimulation by immunoglobulin E receptor crosslinking induces these responses. In order to determine whether redistribution or aggregation of cell surface proteins is sufficient to induce a response in these cells without extrinsic crosslinking, we have redistributed cell surface proteins by electroosmotic segregation and looked for second messenger [Ca2+]i responses. Video imaging of calcium ion activity using the fluorescent calcium sensitive dye fura‐2 revealed the effects of receptor motion and aggregation induced by application of small (10 V/cm) electric fields. A synchronous, monotonic rise in [Ca2+]i generally occurs within a few minutes after a steady field has been applied, while the redistribution of surface proteins is still in progress. The oscillations in [Ca2+]i characteristic of antigen‐stimulated cells are not seen, nor are any effects observed in weak alternating fields (0.02, 60 Hz). The observed rise in [Ca2+]i induced by static electric fields is attributed to perturbation of [Ca2+]i regulation by the large‐scale redistribution of membrane constituents induced by surface electroosmosis. © 1994 Wiley‐Liss, Inc.
Influence of nidogen complexed or not with laminin on attachment, spreading, and albumin and laminin B2 mRNA levels of rat hepatocytes
Authors: Levavasseur Françoise , Mayer Ulríke , Guillouzo André , Clement Bruno
Published: February 2005

Nidogen/entactin is a Mr = 150,000 glycoprotein which is present within basement membranes in a noncovalent stable complex with laminin. We have studied the effects of nidogen/entactin complexed or not with laminin on attachment, spreading, and functions of adult rat hepatocytes in primary culture. Freshly isolated hepatocytes attached on either recombinant or EHS‐derived nidogen, although to a lesser extent than on laminin/nidogen complex, laminin, and E8 and P1 fragments of laminin. Hepatocytes bound on a nidogen fragment bearing the N‐terminal and rod‐like domains but not on either the N‐terminal globules or the rod‐like domain which contains a RGD sequence. Attachment of hepatocytes on nidogen and laminin/nidogen complex was inhibited by anti‐β 1 integrin antibodies. Hepatocytes remained rounded on nidogen and laminin, whereas they rapidly spread on laminin/nidogen complex and collagen IV. Nidogen, laminin, and laminin/nidogen complex transiently maintained high steady‐state albumin mRNA levels in cultured hepatocytes, but a decrease in albumin mRNA content was observed after 24 h, independently of the substrates. Actinomycin D and cycloheximide treatment indicated that the transient effect of these substrates on albumin expression was related to post‐transcriptional mechanisms. Laminin B2 mRNAs were not detectable in freshly isolated hepatocytes but were expressed in 4 h hepatocyte cultures. After 24 h, a dramatic increase in the steady‐state level of laminin B2 mRNA was found in hepatocytes cultured on nidogen and laminin/nidogen complex. This effect was slightly prevented in hepatocytes plated on laminin. These results show that interactions of hepatocytes with nidogen/entactin in vitro result only in a transient modulation of hepatocyte functions. © 1994 Wiley‐Liss, Inc.
Human Co115 colon carcinoma cells potentiate the degradation of laminin mediated by tissue‐type plasminogen activator
Authors: Tran‐Thang C. , Vouillamoz D. , Kruithof E. K. O. , Sordat B.
Published: February 2005

The plasminogen activation (PA) system of human Co 115 colon carcinoma cells was investigated. Analysis at the levels of protein and mRNA of cultured cells and of histozymography of tumor xenografts in nude mice showed that Co 115 cells produce only tissue type PA (t‐PA) and no urokinase (u‐PA). Also, mRNA for the u‐PA receptor and for PA inhibitorr type 2 (PAI‐2), but not for PAI‐I, were detected. We developed a quantitative degradation assay using glutaraldehyde‐immobilized125I‐Iaminin to investigate the capacity of Co 115 cells to degrade laminin. Laminin degradation by Co 115 cells was completely inhibited by 100 μg/ml of polyclonal anti‐t‐PA IgG, by the plasmin inhibitors aprotinin (100μg/ml) or ε‐aminocaproic acid (EACA; at 0.3 M), but not by antibodies against u‐PA or u‐PAR nor by nonimmune IgG. Cycloheximide‐treated Co 115 cells were unable to degrade laminin but increased laminin degradation induced by conditioned medium of Co 115 cells or recombinant t‐PA. No potentiation was observed when Co 115 cells and laminin were kept separated by Transwell™ inserts. Our results suggest that Co 115 human colon carcinoma cells degrade laminin by potentiating t‐PA‐mediated plasminogen activaton at the cells surface which requires close contact between tumor cells and laminin substrate. © 1994 Wiley‐Liss, Inc.
Early cellular events couple covalent binding of reactive metabolites to cell killing by nephrotoxic cysteine conjugates
Authors: Chen Qin , Jones Thomas W. , Stevens James L.
Published: February 2005

Addition of the nephrotoxic cysteine conjugate, S‐(1, 2‐dichlorovinyl)‐L‐cysteine (DCVC), to the LLC‐PK1 line of renal epithelial cells leads to covalent binding of reactive intermediates followed by thiol depletion, lipid peroxidation, and cell death (Chen et al., 1990, J. Biol. Chem., 265:21603‐21611.) The present study was designed to determine if increased intracellular free calcium might play a role in this pathway of DCVC‐induced toxicity by comparing the temporal relationships among increased intracellular free calcium, lipid peroxidation, and cytotoxicity. Intracellular free calcium increased 1 hr after DCVC treatment, long before LDH release occurred. The elevation of intracellular free calcium and cytotoxicity was prevented by inhibiting DCVC metabolism with AOA. The cell‐permeable chelators, Quin‐2AM and EGTA‐AM, prevented the toxicity. Pretreatment of cells with a nontoxic concentration of ionomycin increased intracellular free calcium and potentiated DCVC‐induced LDH release. However, the antioxidant, DPPD, which blocks lipid peroxidation and toxicity, did not affect the increase in intracellular free calcium, whereas buffering intracellular calcium with Quin‐2AM or EGTA‐AM blocked both lipid peroxidation and toxicity without preventing the depletion of nonprotein sulfhydryls by DCVC. Ruthenium red, an inhibitor of mitochondrial calcium uptake, also blocked cell death. We hypothesize that covalent binding of the reactive fragment from DCVC metabolism leads to deregulation of intracellular calcium homeostasis and elevation of intracellular free calcium. Increased intracellular free calcium may in turn be coupled to mitochondrial damage and the accumulation of endogenous oxidants which cause lipid peroxidation and cell death. © 1994 Wiley‐Liss, Inc.
Capping and receptor‐mediated endocytosis of cell‐bound verotoxin (shiga‐like toxin) 1: Chemical identification of an amino acid in the B subunit necessary for efficient receptor glycolipid binding and cellular internalization
Authors: Khine A. A. , Lingwood C. A.
Published: February 2005

The glycolipid globotriaosylceramide (Gb3) is the plasma membrane receptor that mediates the internalization of verotoxin (VT1) into susceptible cells by capping and receptor‐mediated endocytosis (RME). Internalization of fluorescein isothiocyanate‐conjugated holotoxin into Daudi lymphoma cells was found to be slower than the pentameric receptor binding B subunit alone, suggesting that the A subunit may interact with the membrane to compromise the lateral mobility of the receptor bound B subunit. 3‐D reconstruction of fluorescent images by confocal microscopy confirmed the complete internalization of holotoxin. VT1 internalization and cytotoxicity was inhibited by monodansyl cadavarine, which supports a role for clathrin coated pits in the RME of VT1. Biotinylation of the B subunit (in contrast to fluorescein labelling) was found to prevent toxin internalization. This effect correlated with reduced binding of Gb3 and reduced cytotoxicity in vitro. By cleavage of the B subunit at the single tryptophan residue, the reduced Gb3 binding and lack of cellular internalization was shown to be due to the biotinylation of lysine 53 in the VT1 B subunit. This residue was not labelled with fluorescein isothiocyanate in the native protein. This conclusion was confirmed by the finding that biotinylation of VT2c (which contains lys 53) prevented glycolipid receptor binding, whereas biotinylation of VT2e (in which lys 53 is substituted by ile) had no effect. © 1994 Wiley‐Liss, Inc.
Differential activation by platelet‐derived growth factor‐BB of mitogen activated protein kinases in starved or nonstarved AKR‐2B fibroblasts
Authors: Hoppe Jürgen , Hoppe Viviane , Karenberg Thomas‐Andreas , Fenn Anke , Simm Andreas , Sachinidis Agapios
Published: February 2005

More than 90% of serum‐deprived (starved) AKR‐2B mouse fibroblasts are stimulated to divide by the addition of platelet‐derived growth factor (PDGF)‐BB. In density‐arrested (nonstarved) cells, PDGF‐BB affords protection from cell death without stimulation of cell division. In both cultivation conditions the cells express similar amounts of PDGF β‐receptors and the receptor kinase activity was identical as judged by its autophosphorylation capacity. Three signaling pathways were studied in detail: (1) Phospholipase C‐γ (PLC‐γ) and [Ca2+]i increase, (2) activation of the phosphatidylinositol‐3 kinase (PI‐3 kinase), and (3) activation of mitogen activated kinases I and II (MAP kinases I and II). There was no difference in starved or nonstarved cells regarding PLC‐γ activation, increase of [Ca2+]i, and stimulation of PL‐3 kinase activity. But most remarkably the activation of MAP‐I was largely suppressed in nonstarved cells. The implications of these signaling pathways in cell protection or cell division are discussed. © 1994 Wiley‐Liss, Inc.
Second messenger signaling of c‐fos gene induction by parathyroid hormone (PTH) and PTH‐related peptide in osteoblastic osteosarcoma cells: Its role in osteoblast proliferation and osteoclast‐like cell formation
Authors: Kano Junichi , Sugimoto Toshitsugu , Kanatani Masanori , Kuroki Yasuo , Tsukamoto Tatsuo , Fukase Masaaki , Chihara Kazuo
Published: February 2005

The present study was performed to clarify second messenger signaling in parathyroid hormone (PTH)‐induced c‐fos gene expression, to characterize the participation of the c‐fos gene in the regulation of osteoblast proliferation and function as well as osteoclast‐like cell formation by PTH and to compare these effects of PTH with those of PTH‐related peptide (PTHrP). Both human (h) PTH‐(1‐34) and hPTHrP‐(1‐34) at 10‐8 M induced a transient c‐fos gene expression to a similar degree in osteoblastic osteosarcoma cells, UMR‐106. N6, O2' ‐dibutyryl adenosine 3′, 5′‐cyclic monophosphate (dbcAMP) as well as Sp‐diastereoisomer of adenosine cyclic 3′,5′‐phosphorothioate (Sp‐cAMPS), an activator of cAMP‐dependent protein kinase (PKA), induced a weak c‐fos gene expression. Although Rp‐diastereoisomer of adenosine cyclic 3′,5′‐phosphorothioate (Rp‐cAMPS), an inhibitor of PKA, almost completely antagonized dbcAMP‐and Sp‐cAMPS‐induced expression of c‐fos gene, it did not cause an obvious inhibition of PTH‐or PTHrP‐induced expression. Phorbol 12‐myristate 13‐acetate (PMA), an activator of protein kinase C (PKC), induced an intense expression of the c‐fos gene, while 4α‐phorbol 12, 13‐didecanoate (4αPDD), incapable of activating PKC, and calcium ionophores (A23187 and ionomycin) did not. Protein kinase C inhibitor (H‐7, 50 μM) completely blocked the expression of the c‐fos gene by PTH as well as by PTHrP. Antisense oligodeoxynucleotides (as‐ODN) complementary to c‐fos mRNA, which have been shown to inhibit its mRNA translation, at 1 μM significantly antagonized PTH‐and PTHrP‐induced inhibition of [3H] thymidine incorporation and stimulation of osteoclast‐like cell formation in the presence of osteoblasts, but not an increase in alkaline phosphatase activity, compared to control oligodeoxynucleotides with same nucleotides as as‐ODN but with a random sequence. The present study indicates the involvement of PKC system in c‐fos gene expression by PTH as well as PTHrP and also indicates the involvement of the c‐fos gene in the regulation of bone cell physiology by PTH and PTHrP. © 1994 Wiley‐Liss, Inc.
Adaptation of EGF receptor signal transduction to three‐dimensional culture conditions: Changes in surface receptor expression and protein tyrosine phosphorylation
Authors: Mansbridge J. N. , Ausserer W. A. , Knapp M. A. , Sutherland R. M.
Published: February 2005

A431 cells grown as three‐dimensional spheroids show growth stimulation in response to nanomolar concentrations of EGF in contrast to monolayer cultures that show inhibition. In investigating the alterations in EGF signal transduction that underlie this modification of the proliferative response, we have compared the expression of EGF receptors on A431 cells under these conditions and related our findings to tyrosine phosphorylation and the growth response. EGF receptors were measured by 125I‐EGF binding to trypsin‐dispersed cells. Unexpectedly, dispersion of the monolayers caused an 80% decrease in surface EGF receptor, although, after dispersion, EGF receptor was digested by trypsin with a half‐life of 69 ± 32 min. No evidence for a comparable loss of cellular EGF receptor was seen on trypsin dispersion of spheroids. After allowing for this effect, we found that the receptor density on nondispersed monolayers (5 × 106 per cell) was twentyfold greater than that on spheroids (0.25 × 106 per cell). EGF‐induced tyrosine phosphorylation was confined to the outermost cells of the spheroid, although the presence of surface‐expressed EGF pinding sites could be demonstrated throughout the structure and the number of EGF receptors/cell on dispersed spheroid cells showed a single distribution peak by flow cytometry, with no evidence for more than one population. Using RCM‐lysozyme as a substrate, tyrosine phosphatase activity in spheroids lay within the range observed in monolayer cultures. Autophosphorylation of the EGF receptor following EGF stimulation in monolayer cultures of A431 cells rose rapidly in the first 10 seconds and then slowly increased for at least 3 h. In spheroids, it reached a maximum within 10 seconds and then declined over 3 h. Since the microenvironment within a tumor resembles that in a spheroid, a similar reduction in surface EGF receptor expression may be expected in tumors relative to monolayer cultures, together with corresponding growth stimulation in response to EGF. © 1994 Wiley‐Liss, Inc.
Effects of P‐glycoprotein expression on cyclic AMP and volume‐activated ion fluxes and conductances in HT‐29 colon adenocarcinoma cells
Authors: Kunzelmann K. , Slotki I. N. , Klein P. , Koslowsky T. , Ausiello D. A. , Greger R. , Cabantchik Z. I.
Published: February 2005

The tissue distribution of P‐glycoprotein (Pgp) and the structurally related cystic fibrosis transmembrane conductance regulator (CFTR) is apparently mutually exclusive, particularly in epithelia; where one protein is expressed the other is not. To study the possible function(s) of Pgp and its potential effects on CFTR expression in epithelia, HT‐29 colon adenocarcinoma cells, which constitutively express CFTR, were pharmacologically adapted to express the classical multidrug resistance (MDR) phenotype (Pgp+). Concomitant with the appearance of Pgp and MDR phenotype (drug resistance, reduced drug accumulation and increased drug efflux), CFTR levels and cAMP‐stimulated Cl conductances were markedly decreased compared to wild‐type HT‐29 (Pgp−) cells (as shown using the whole cell patch clamp technique). Removal of drug pressure led to the gradual decrease in Pgp levels and MDR phenotype, as evidenced by increased rhodamine 123 accumulation (Pgp‐Rev). Concomitantly, CFTR levels and cAMP‐stimulated Cl− conductances incresed. The cell responses of Pgp/Rev cells were heterogeneous with respect to both Pgp and CFTR functions. We also studied the possible contribution of Pgp to hypotonically activated (HCS) ion conductances. K+ and Cl− effluxes from Pgp− cells were markedly increased by HCS. This increase was twice as high as that induced by the cation ionophore gramicidin; it was blocked by the Cl− channel blocker DIDS (4,4′‐disothiocyano‐2,2′‐disulfonic stilbene) and required extracellular Ca2+. In Pgp+ cells, the HCS‐induced fluxes were not significantly different from those of Pgp− cells. Verapamil (10 μM), which caused 80% reversal of Pgp‐associated drug extrusion, failed to inhibit the HCS‐evoked Cl− efflux of Pgp+ cells. Similarly, HCS increased Cl− conductance to the same extent in Pgp−, Pgp+ and Pgp‐Rev cells. Verapamil (100 μM), but not 1,9‐dideoxyforskolin (50 and 100 μM), partially inhibited the HCS‐evoked whole cell current (WCC) in all three lines. Since the inhibition by verapamil was not detected in the presence of the K+ channel blocker Ba2+ (3 mM), it is suggested that verapamil affects K+ and not Cl− conductance. We conclude that hypotonically activated Cl− and K+ conductances are similar in HT‐29 cells irrespective of Pgp expression. Expression of high levels of Pgp in HT‐29 cells confers no physiologically significant capacity for cell volume regulation. © 1994 Wiley‐Liss, Inc.
Enhanced expression of granulosa cell low density lipoprotein receptor activity in response to in vitro culture conditions
Authors: Reaven Eve , Tsai Louisa , Spicher Marjorie , Shilo Lotan , Philip Mohan , Cooper Allen D. , Azhar Salman
Published: February 2005

Previous studies have shown that the B/E (low density lipoprotein [LDL]) receptor pathway plays a minor role in cholesterol uptake in the intact rat ovary, but when granulosa cells are isolated and maintained in culture, the cells develop a fully functional B/E receptor system. In the current study we examined the development of the B/E receptor over time (96 h) in culture and compared its physiological function, expression of mRNA and protein levels, and morphological events to the upregulation induced in 24 h by hormone (human chorionic gonadotropin [hCG] or Bt2cAMP). With both protocols, increased progestin production occurs and is associated with elevated binding, uptake, and degradation of LDL in the medium although the impact of Bt2cAMP stimulation on all these measurements is several times that observed with time alone. Only the hormone‐stimulated LDL receptor response was associated with an increase in receptor protein (Western blot) or mRNA levels (RNase protection assay). We conclude that unstimulated granulosa cells show posttranslational increase in B/E receptor activity with time in culture, but transcriptional changes in B/E receptor follow stimulation with trophic hormone or its second messenger, cAMP. © 1994 Wiley‐Liss, Inc.
Properties of single potassium channels in guinea pig hepatocytes
Authors: Takanashi Hideo , Sawanobori Tohru , Kamisaka Kazuaki , Maezawa Hidenori , Hiraoka Masayasu
Published: February 2005

The patch‐clamp technique of cell‐attached and inside‐out configurations was used to study the single potassium channels in isolated guinea pig hepatocytes. The single potassium channels in isolated guinea pig hepatocytes were recorded at different K+ concentrations. A linear single‐channel current‐voltage relationship was obtained at the voltage range of ‐80 to ‐20 mV with slope conductance of 70 ± 6 pS (n = 10). Under symmetrical high K+ concentration of 148 mM in the cell‐attached patch membrane, the I‐V curve exhibited a mild inward rectification at potentials positive to +20 mV. The values of reversal potential was +5 ± 2 mV (n = 10). When the external potassium concentration ([K+]0) was decreased to 74 mM and 20 mM, the slope conductance was decreased to 48 ± 2 pS (n = 4) and 24 ± 3 pS (n = 3), respectively. The reversal potential was changed by 58 mV for a tenfold change in [K+]0, indicating that this channel was highly selective for K+. Open probabilities (P0) of the channel were 73‐93% without apparent voltage dependence. The distributions of open time of the channels were fitted to two exponentials, while those of closed time were fitted to three exponentials, exhibiting no voltage dependence. The success rate of K+ channel activity to be recorded was 28% at room temperature, and there were no increases in the success rate nor in the channel opening probabilities at a temperature of 34‐36°C. P0 in inside‐out patches was not changed by application of 1 μM Ca2+ nor 1 mM Mg2+ to the internal side of patch membranes. It is concluded that a novel type of the K+ channels in guinea pig hepatocytes had different properties of slope conductance, channel kinetics, and sensitivity to [Ca2+]i, from those in other species. © 1994 Wiley‐Liss, Inc.
Thyroid hormone suppresses the differentiation of osteoprogenitor cells to osteoblasts, but enhances functional activities of mature osteoblasts in cultured rat calvaria cells
Authors: Ohishi Keiji , Ishida Hiroshi , Nagata Toshihiko , Yamauchi Noriyuki , Tsurumi Chizuko , Nishikawa Seiji , Wakano Yoichi
Published: February 2005

The effects of thyroid hormone on osteoblastic differentiation and activity were studied in fetal rat calvaria (RC) cells cultured for up to 30 days in medium supplemented with thyroid hormone‐depleted serum. In this condition, the cells proliferated and differentiated to form mineralized bone nodules (BN) and expressed osteoblastic markers such as alkaline phosphatase (ALP), osteocalcin (OCN), and osteopontin (OPN). The continuous presence of triiodothyronine (T3) at 10‐9‐10‐8 M in the medium inhibited the osteoblastic differentiation: 34% decrease in ALP activity on day 12 and 60% decrease in BN formation on day 15 at 10‐8 M. T3 at these doses had no effect on the DNA content of RC cells at confluence (day 6). Short‐term (48‐h) exposure of T3 at 10‐9 M or higher decreased ALP activity when RC cells were differentiating (days 7–11). However, when BN formation by the cells had already reached a plateau (day 28), the activity was increased by treatment with T3 at 10‐7‐10‐6 M. OCN production was increased dose dependently by this treatment with T3 (2.1‐fold and 1.3‐fold of control at 10‐8 M on days 11 and 28, respectively). Similar increases were observed in the levels of OCN mRNA. In addition, increases in phosphorylated OPN in the medium (day 11) and mineralized matrix (day 28) were observed (1.5‐fold at 10‐8‐10‐6 M), while OPN synthesis and the level of its mRNA were depressed by T3 (60‐70% of control at 10‐8 M). These results suggest that T3 regulates osteoblastic differentiation and activity depending on the state of cell differentiation: T3 suppresses the differentiation of osteoprogenitor cells to osteoblasts, but enhances the functional activity of mature osteoblasts. © 1994 Wiley‐Liss, Inc.
Evidence that modulation of glucose transporter intrinsic activity is the mechanism involved in the allose‐mediated depression of hexose transport in mammalian cells
Authors: Pratt Susan E. , Colby‐Germinario Susan , Manuel Susannia , Germinario Ralph J.
Published: February 2005

In serum starved V79 Chinese hamster lung fibroblast cells, replacement of D‐glucose with D‐allose resulted in a significant 38 ± 18% (P < 0.05) reduction of 2‐deoxy‐D‐glucose (2‐DG) transport. Similarly, in a respiration‐deficient mutant cell line (V79‐G14), which has elevated 2‐DG transport activity, D‐allose reduced 2‐DG transport by 59 ± 18% (P < 0.05). [3H]D‐allose uptake by V79 cells occurred slowly and was not inhibited by cytochalasin B, suggesting diffusion as the mode of D‐allose entry. Western blot analysis using a rabbit polyclonal antibody to the human erythrocyte glucose transporter (GT) demonstrated that, in both cell lines, GT content and GT subcellular distribution were not significantly different in D‐glucose vs. D‐allose‐treated cells. δ‐Antibody, which has been shown to bind to exofacial epitopes of the GT (Harrison et al., 1990, J. Biol. Chem., 265:5793‐‐5801), did not demonstrate any differences in surface binding to D‐glucose vs. D‐allose‐treated intact V79 cells. D‐allose treatment of 3T3 fibroblasts resulted in a similar decrease (72%) of 2‐DG transport, however D‐allose had no apparent effect on basal sugar transport in 3T3 adipocytes. These results suggest that D‐allose reduces sugar transport through a modulation of the intrinsic activity of the GT, and that D‐allose may act in a tissue‐specific manner. © 1994 Wiley‐Liss, Inc.