bluuebunny/crossref_metadata_2025 huggingface dataset:
Okadaic acid‐induced actin assembly in neutrophils: Role of protein phosphatases
Authors: Downey Gregory P. , Takai Akira , Zamel Ricardo , Grinstein Sergio , Chan Chi Kin
Published: February 2005

Activation of neutrophils results in morphological and functional alterations including changes in cell shape and initiation of motile behavior that depend on assembly and reorganization of the actin cytoskeleton. Phosphoproteins are thought to be key intermediates in the regulation of cytoskeletal alterations and whereas much attention has been directed at the role of protein kinases, relatively little information is available on the importance of phosphatases. To elucidate the role of protein phosphatases, we studied the effects of the phosphatase inhibitors okadaic acid and calyculin A on the actin cytoskeleton of human neutrophils. Exposure of cells to okadaic acid resulted in assembly and spatial redistribution of actin, which peaked at 25 min and returned to baseline levels by 45 min, as assessed by flow cytometric analysis of NBD‐phallacidin stained cells and confocal fluorescence microscopy, respectively. These effects correlated with an increase in protein phosphorylation, determined by incorporation of 32P into cellular proteins using SDS‐PAGE and autoradiography. Similar but more rapid responses were observed in electropermeabilized cells treated with okadaic acid or calyculin A. The dose dependence of these effects was compatible with a role for phosphatase type 1 as the target enzyme. These findings also suggested the presence of constitutively active protein kinases capable of effecting actin polymerization. Phosphorylation of myosin light chain (MLC) has been postulated to promote actin assembly, but myosin light chain kinase (MLCK) appeared not to be involved because: (1) the effect of okadaic acid was not inhibited by the MLCK inhibitor KT5926 and (2) in permeabilized cells suspended in medium with free calcium [Ca2+] < 10 nM (conditions under which MLCK is inactive), the effect of okadaic acid persisted. The role of phosphatases in stimulus‐induced actin assembly was assessed in cells preincubated with okadaic acid for 45 min, after F‐actin levels had returned to baseline. Under these conditions, okadaic acid completely abrogated actin assembly induced by phorbol myristate acetate, platelet activating factor, and leukotriene B4, whereas the effects of the chemotactic peptide fMLP and opsonized zymosan (OpZ) were unaffected. We conclude that serine and threonine phosphatases exert a tonic negative influence on actin assembly and organization. Furthermore, divergent pathways seem to mediate the response to lipidic stimuli, on one hand, and fMLP and OpZ, on the other, as evidenced by the differential susceptibility to inhibition by okadaic acid. © 1993 Wiley‐Liss, Inc.
Mitogen‐activated Ca++ channels in human B lymphocytes
Authors: Brent Lawrence H. , Gong Qihua , Ross James M. , Wieland Steven J.
Published: February 2005

Two complementary experimental methods have been used to examine mitogen‐induced transmembrane conductances in human B cells using the Daudi cell line as a model for human B cell activation. Spectrofluorometry was used to investigate mitogen‐induced changes in [Ca++]i and transmembrane potential. Activation of human B cells with anti‐μ antibodies resulted in a biphasic rise in [Ca++]i, the second phase being mediated by the influx of extracellular Ca++. Ca++ influx was inhibited by high [K+]e, suggesting that this influx was transmembrane potential sensitive. Membrane currents of Daudi cells were investigated using voltage clamp techniques. Before mitogenic stimulation, the cells were electrically quiet. Within several minutes of the addition of anti‐μ antibodies to the bath solution, inward currents were observed at negative voltages. Whole‐cell currents changed instantly with voltage steps and were transmembrane potential sensitive in that at potentials more positive than −40 mV no currents were detectable. A similar conductance was also activated by the introduction of IP3 into the intracellular solution, suggesting that IP3 generation after surface IgM crosslinking is involved in the activation of this conductance. Both anti‐μ and IP3 induced currents were blocked by 1 mM La+++, which is known to block Ca++ channels. These results strongly support the presence of membrane Ca++ channels in human B cells that function in the early stages of activation. Changes in transmembrane potential appear to be important in regulating Ca++ influx. These mechanisms work in concert to regulate the level of [Ca++]i during the early phases of human B cell activation. © 1993 Wiley‐Liss, Inc.
Regulation of insulin‐like growth factor‐binding‐protein‐1, 2, 3, 4, 5, and 6: Synthesis, secretion, and gene expression in estrogen receptor‐negative human breast carcinoma cells
Authors: Sheikh M. Saeed , Shao Zhi‐Ming , Hussain Arif , Clemmons David R. , Chen Jian‐Chyi , Roberts Charles T. , Leroith Derek , Fontana Joseph A.
Published: February 2005

Insulin‐like growth‐factors I and II (IGF‐I, II) are potent mitogens for breast carcinoma proliferation. In extracellular fluids, most of the IGF‐I and II is associated with specific IGF‐binding proteins (IGFBPs). The role of these IGFBPs in IGF action is still not clear, but it has been demonstrated that these proteins may either enhance or inhibit IGF‐mediated cellular effects. Synthesis and secretion of IGFBPs have been demonstrated in breast carcinoma cells. In this study, we examined retinoic acid (RA) and IGF‐I modulation of IGFBP mRNA and IGFBP levels in two ER‐negative human breast carcinoma cell lines. Treatment of MDAMB‐231 and MDA‐MB‐468 cells with RA increased the levels in conditioned media of a Mr 42‐46‐kDa IGFBP, which was immunoprecipitated by an IGFBP‐3 antibody. IGF‐I also increased the accumulated levels of IGFBP‐3 in the conditioned media of both cell lines. Both cell lines expressed high basal levels of IGFBP‐3 mRNA; the addition of RA increased IGFBP‐3 mRNA levels by 1.5‐fold, whereas the addition of IGFI had no effect on IGFBP‐3 mRNA levels in either cell line. The difference in the magnitude of the RA enhancement of IGFBP‐3 mRNA levels (1.5‐fold) and RA stimulation of IGFBP‐3 levels in conditioned media (3.5–4‐fold) suggests that some of the effect of RA is at a posttranscriptional level. IGF‐I increased the levels of IGFBP‐2 and IGFBP‐5 in conditioned media by greater than tenfold but had no effect on IGFBP‐2 and IGFBP‐5 mRNA levels, again suggesting the involvement of posttranscriptional controls. Pretreatment of MDA‐MB‐468 and MDA‐MB‐231 cells with IGF‐I receptor antibody (αIR3) blocked the IGF‐I effect on IGFBP‐3 levels in the media in both cell lines and IGFBP‐2 and IGFBP‐5 secreted levels in MDA‐MB‐468 cell conditioned media. The addition of RA also blocked IGF‐I stimulation of IGFBP2 and IGFBP‐5 levels. Cycloheximide treatment completely blocked the RA and/or IGF‐I‐mediated modulation of these binding proteins, suggesting that these agents enhance IGFBP‐3, IGFBP‐2, and IGFBP‐5 synthesis and consequent secretion. MDA‐MB‐468 cells expressed IGFBP‐5 mRNA, whereas both MDA‐MB‐231 and MDA‐MB‐468 expressed IGFBP‐6 mRNA. RA enhanced IGFBP‐6 gene expression by threefold in MDA‐MB‐231 cells, whereas IGF‐1 had no effect on IGFBP‐6 gene expression in either cell line. These results demonstrate that RA and IGF‐I modulate IGFBP levels in a number of breast carcinoma cell lines. Such modulation of IGFBPs may in turn affect IGF mediated cellular responses and thus the biologic behavior of these malignant cells.
Isolation of a temperature‐sensitive variant Chinese hamster ovary cell line with a morphologically altered endocytic recycling compartment
Authors: McGraw Timothy E. , Dunn Kenneth W. , Maxfield Frederick R.
Published: February 2005

We have enriched a mutagenized population of Chinese hamster ovary (CHO) cells for those defective in endocytosis by selection for survival to treatment with transferrin (Tf)‐ricin and Tf‐diphtheria toxin conjugates. Surviving cells were screened with a fluorescently labeled Tf uptake assay to identify cells with mor‐phologically aberrant endocytic phenotypes. One of the cell lines identified, B104‐5, has a striking temperature‐induced alteration in the morphology of its endocytic receptor recycling compartment. In parental cells the tightly clustered endocytic recycling compartment is located near the Golgi complex. In the mutant cells, following incubation at 40°C, this compartment appears fragmented and widely dispersed. Surprisingly, this alteration in the morphology of the recycling compartment has no effect on the kinetics of Tf internationalization and recycling. The wild‐type endocytic compartment is closely aligned with the microtubule‐organizing center and the Golgi apparatus, and like the Golgi, its clustered appearance is dependent upon intact microtubules. Although the disruption of the B104‐5 receptor recycling compartment morphology can be phenocopied in wild‐type cells by microtubule depolymerizing drugs, the microtubule cytoskeleton in B104‐5 cells appears normal in immunofluorescent staining. B104‐5 cells, unlike the parental cells, do not proliferate at 40°C. The mutation in B104‐5 cells is recessive, as fusion with wild‐type cells results in a reversion of the B104‐5 phenotype. The finding that the morphology of the recycling compartment in CHO cells can be altered without affecting recycling of endocytosed Tf is consistent with the variety of recycling compartment morphologies observed among different cell lines. An interpretation of this result is that the lesion in B104‐5 cells is in a gene that is involved in determining the endocytic compartment morphologies observed in different cell lines. © 1993 Wiley‐Liss, Inc.
Transforming growth factor‐β1 stimulates macrophage urokinase expression and release of matrix‐bound basic fibroblast growth factor
Authors: Falcone Domenick J. , McCaffrey Timothy A. , Haimovitz‐Friedman Adriana , Garcia Michelle
Published: February 2005

Macrophage expression of urokinase‐type plasminogen activator (uPA) appears to play a role in their release of matrix‐bound basic fibroblast growth factor (bFGF) and transforming growth factor‐β (TGF‐β). In experiments reported here, we have examined the potential regulatory effects of bFGF and TGF‐β1 on macrophage uPA expression. TGF‐β1 stimulated in a dose‐ and time‐dependent manner the expression of secreted membrane and intracellular uPA activities by a macrophage cell line (RAW264.7). When examined at similar concentrations, bFGF had little effect, and interleukin‐1α, tumor necrosis factor‐α, and monocyte colony stimulating factor had no effect on macrophage uPA expression. Exposure of macrophages to TGF‐β1 led to a rapid and sustained increase in the steady‐state levels of uPA mRNA that was independent of de novo protein synthesis and was completely inhibited by actinomycin D. However, the TGF‐β1‐induced increase in uPA mRNA was largely unaffected by subsequent incubation of cells with actinomycin D. The protein kinase C inhibitior H7 markedly reduced the ability of TGF‐β1 to stimulate expression of uPA activity. Likewise, okadaic acid and microcystin, inhibitors of serine/threonine phosphatases, potentiated the ability of TGF‐β1 to upregulate macrophage uPA expression. TGF‐β1 primed cells converted nearly all added plasminogen to plasmin and expressed sixfold more membrane‐bound plasmin than control cells. Preincubation of TGF‐β1 with either serum or methylamine‐modified α2‐macroglobulin did not affect its ability to induce macrophage uPA expression. When control and TGF‐β1‐primed macrophages were cultured on matrices containing bound125I‐bFGF, their release of 125I‐bFGF was increased five and tenfold, respectively, in the presence of plasminogen. The ability of TGF‐β to induce macrophage uPA expression and the plasmin‐dependent release of matrix‐bound bFGF may provide an indirect mechanism by which TGF‐β stimulates angiogenesis. © 1993 Wiley‐Liss, Inc.
Cellular resistance to oxidative stress is accompanied by resistance to cisplatin: The significance of increased catalase activity and total glutathione in hydrogen peroxide‐resistant fibroblasts
Authors: Spitz Douglas R. , Phillips John W. , Adams Donna T. , Sherman C. Michael , Deen Dennis F. , Li Gloria C.
Published: February 2005

Studies designed to better understand the involvement of cellular resistance to oxidative stress in mechanisms of cellular resistance to cisplatin were undertaken using H2O2‐resistant variants of the HA 1 Chinese hamster fibroblast cell line. H2O2‐resistant cell lines were resistant to clonogenic inactivation mediated by cisplatin with dose modifying factors at 10% survival of 1.5–3.0, relative to HA 1 cells. The most cisplatin resistant of these cell lines (OC5) also demonstrated fewer DNA–DNA crosslinks induced by cisplatin, relative to HA 1. Since H2O2‐resistant cells contained increased catalase activity as well as total glutathione (GSH) content, the involvement of these cellular antioxidants in the resistance to cisplatin toxicity was evaluated. Treatment of HA 1 and H2O2‐resistant cell lines (OC5, OC14) with 9 mM aminotriazole reduced catalase activity by 60–65% but had no effect on the cytotoxicity of cisplatin. In contrast, treatment with 5 mM buthionine sulfoximine reduced total GSH by 90% and sensitized the cells to cisplatin cytotoxicity. Furthermore, extracellular reaction of GSH with cisplatin prior to treating HA 1 cells reduced the toxicity of the compound, indicating that this reaction is capable of participating in the detoxification of cisplatin. These results indicate that cellular adaptation to oxidative stress renders cells resistant to DNA damage as well as to cytotoxicity associated with cisplatin treatment. Furthermore, increases in total GSH content (but not catalase activity) appear to partially account for cisplatin resistance demonstrated by H2O2‐resistant cells. © 1993 Wiley‐Liss, Inc.
Decrease in cellular replicative potential in “giant” mice transfected with the bovine growth hormone gene correlates to shortened life span
Authors: Pendergrass William R. , Li Yi , Jiang DeZhao , Wolf Norman S.
Published: February 2005

Adult mice, (C57BL/6 × Sjl)F1 hybrids, transfected with the bovine growth hormone gene (bGH) grow to twice normal size, but have a mean life span less than 50% that of control siblings without the transgene. The replicative potentials of cells from six different tissue sites (tail skin and ear skin dermal fibroblasts, tail subdermal connective tissue fibroblasts, kidney medulla epithelial cells, bone marrow myofibroblasts, and spleen myofibroblasts) were assayed in vitro using clone size distribution analysis. Cells from all of the above bGH+ tissues produced a smaller fraction of large clones, relative to age‐matched controls, in all of these cell types. The loss of replicative potential did not appear to be the result of negative conditioning of the cloning media by the bGH+ cells, and was tightly correlated to the period of accelerated growth in these animals (3–12 weeks), a time when additional GH receptors are expressed. © 1993 Wiley‐Liss, Inc.
Novel action of transforming growth factor β1 in functioning human pancreatic carcinoid cells
Authors: Ishizuka Jin , Beauchamp R. Daniel , Sato Kazuo , Townsend Courtney M. , Thompson James C.
Published: February 2005

We have shown recently that 5‐HT is an autocrine growth stimulatory factor for a cell line (BON) that is derived from a human pancreatic carcinoid tumor. This action is mediated by a 5‐HT receptor‐linked decrease of cyclic adenosine monophosphate (AMP) production, but not mediated by a 5‐HT receptor‐linked stimulation of phosphatidylinositol hydrolysis. The BON cells also express transforming growth factor betas (TGFβs) (1, 2, and 3) and release TGFβ into their medium. In this study, we examined the effects of TGFβ1 on the secretion of 5‐HT, on signal transduction pathways involved in 5‐HT secretion, and on growth of BON cells. TGFβ1 inhibited basal and acetylcholine‐stimulated release of 5‐HT, but did not inhibit isobutylmethylxanthine‐stimulated release of 5‐HT. TGFβ1 inhibited both basal and acetylcholine‐stimulated hydrolysis of phosphatidylinositol in a dose‐dependent manner, but did not affect cyclic AMP production. TGFβ1 inhibited growth of BON cells in culture; this effect was reversed by exogenously administered 5‐HT. Three different specific and saturable TGFβ1 binding sites were identified; binding assays performed after mild acid wash (0.1% acetic acid, pH 2.5) conditions uncovered TGFβ receptors that were apparently occupied by endogenously produced TGFβ species. Affinity cross‐linking assay showed that BON cells had three different TGFβ binding proteins. These results suggest that TGFβ1 can inhibit growth of BON cells by altering secretory responses of 5‐HT by means of receptor‐mediated inhibition of phosphatidylinositol hydrolysis. We conclude that growth of BON cells is regulated, at least in part, by the opposing receptor‐mediated autocrine actions of 5‐HT and TGFβ. © 1993 Wiley‐Liss, Inc.
Receptors for Escherichia coli heat stable enterotoxin in human intestine and in a human intestinal cell line (Caco‐2)
Authors: Cohen Mitchell B. , Jensen Nancy J. , Hawkins Jennifer A. , Mann Elizabeth A. , Thompson Michael R. , Lentze Michael J. , Giannella Ralph A.
Published: February 2005

Escherichia coli heat stable enterotoxin (STa) and the newly identified endogenous ligand guanylin bind to an intestinal receptor and activate membrane bound guanylate cyclase. We compared STa binding and affinity crosslinking of STa receptors in human small intestine to those in the Caco‐2 human colon carcinoma cell line. STa had similar kinetics of binding in human intestinal and Caco‐2 brush border membranes. In both human intestine and Caco‐2 brush border membranes, multiple specifically radiolabeled bands, including a 140–165 kDa band, were identified by affinity crosslinking. However, in human intestine the most prominent autoradiographic species was a 60 kDa band. A 60 kDa protein was also specifically immunoprecipitated from solubilized human brush border membranes using antisera raised against a cloned STa receptor fusion protein. Our observations of multiple crosslinked proteins in human intestine and Caco‐2 cells could be explained by the existence of several members of a family of STa receptors and/or the existence of smaller STa binding proteins generated by the protease cleavage of a larger complete STa receptor. © 1993 Wiley‐Liss, Inc.
Activation of phospholipase C by heat shock requires GTP analogs and is resistant to pertussis toxin
Authors: Calderwood Stuart K. , Stevenson Mary Ann , Price Brendan D.
Published: February 2005

The heat shock response in mammals consists of a complex array of intracellular reactions initiated by stress, although its regulation is poorly understood. We have investigated the role of transmembrane signal transduction in the response, examining mechanisms involved in the activation of phospholipase C (PLC) by heat shock. In rodent fibroblasts permeabilized with digitonin, heat shock and receptor‐mediated PLC activity exhibited a strict GTP analog dependency. This indicates that heat shock‐mediated phopholipase activation, in common with receptor mediated stimulation, does not involve direct effects on the phospholipases and suggests the participation of GTP binding (G) proteins in the activation process. When cells were treated with the inhibitor pertussis toxin (PTX), the phospholipases retained their inducibility by heat shock, but became refractory to thrombin treatment, indicating that heat shock may influence PLC activity through a distinct population of G proteins compared to thrombin. The data seem to exclude a role for PTX sensitive G proteins in the production of IP3 after heating and suggest a pathway involving the direct thermal activation of the Gq class of G proteins, which are coupled to the PLCβ1 isoform. © 1993 Wiley‐Liss, Inc.
Heat shock in human neutrophils: Superoxide generation is inhibited by a mechanism distinct from heat‐denaturation of NADPH oxidase and is protected by heat shock proteins in thermotolerant cells
Authors: Maridonneau‐Parini Isabelle , Malawista Stephen E. , Stubbe Hiltrud , Russo‐Marie Françloise , Polla Barbara S.
Published: February 2005

Independently of the stimulating agent used, generation of O2− by human neutrophils is transiently inhibited when the cells have been exposed to elevated temperatures. This phenomenon is concomitant with the synthesis of heat shock proteins (HSPs). We have investigated a possible relationship between HSPs and modulation of NADPH oxidase activity in human neutrophils exposed to heat. HSPs were not involved in the inhibition of O2− generation since 1) in enucleated cytoplasts, which are unable to synthesize proteins, the generation of O2− was inhibited after exposure to 43°C, 2) using actinomycin D (Act D) in intact cells, it was possible selectively to inhibit the synthesis of HSPs without modifying the inhibition of NADPH oxidase activity that followed HS. Furthermore, the recovery of NADPH oxidase activity was not under the control of HSPs because the enzyme recovered as well in Act D‐treated neutrophils. The NADPH oxidase activity was reconstituted in a cell‐free assay by combining the cytosol with the plasma membrane‐enriched fraction in the presence of arachidonic acid (AA) and NADPH. Subcellular fractions obtained from control or heated neutrophils exhibited similar oxidase activities suggesting that heat exposure did not induce denaturation of the oxidase components but rather altered the mechanisms of translocation and/or assembly of these components with the plasma membrane. This hypothesis was supported by the inhibition of the granule release in heated cells, a process which also requires translocation and association fusion with the plasma membrane. On the other hand, preexposure of neutrophils to HS prevented the inhibition of O2− generation during a second challenging HS. This acquired thermotolerance was abolished when the synthesis of HSPs was inhibited during the first HS with Act D, indicating a direct relationship between protection of O2− generation and synthesis of HSP. Here we demonstrate that synthesis of HSPs and inhibition or recovery of NADPH oxidase activity are concomitant but unrelated phenomena. In contrast, accumulation of HSPs in thermotolerant neutrophils appeared to play an important role in the prevention of NADPH oxidase inhibition. These results provide further insights into the behavior of human neutrophils and NADPH oxidase upon heat injury. © 1993 Wiley‐Liss, Inc.
Modulation of myogenic determination factor 1 expression by cell‐cell contact
Authors: Song An , Daub Raymond , Harrington Maureen A.
Published: February 2005

Myogenic determination factor 1 (MyoD1) expression is modulated by a variety of agents including growth factors and activated cellular proto‐oncogenes. However, little is known about the effect of cell‐cell contact, which increases as myoblasts terminally differentiate, on the control of MyoD1 expression. Steadystate levels of MyoD1 transcripts decline over a 6–12 hour time period when myoblasts plated at a single cell density are incubated in media supplemented with 0.2% serum; by 48 hours MyoD1 mRNA levels have returned to the initial basal level. The decline in MyoD1 transcripts is diminished, but not prevented in myoblasts which maintain cell‐cell contacts (at least 50% of cells with two or more sites of contact). MyoD1 transcript levels do not change if single cell cultures are maintained in 10% serum or are cocultured with fibroblasts. Analysis of conditioned media revealed that myoblasts plated at the single cell density or at a density which allowed multiple sites of cell‐cell contact are not producing an activity(s) responsible for modulating MyoD1 mRNA levels. The changes in MyoD1 expression are mediated at the transcriptional level. Thus changes in the degree of cell‐cell contact in cultures of myogenically determined cell lines effect changes in MyoD1 gene expression. Consequently when the influence of cytokines or other pharmacological agents on commitment to terminal myogenic differentiation is examined, the degree of cell‐cell contact within the culture system may affect the response elicited. © 1993 Wiley‐Liss, Inc.
Phenotypic effects of aldosterone and dexamethasone in a SV40‐transformed mammalian cortical ascending limb cell line exhibiting mineralocorticoid receptors
Authors: Vandewalle A. , Rafestin‐Oblin M.‐E. , Hagege J. , Géniteau‐Legendre M. , Cassingéna R. , Ronco P. M.
Published: February 2005

We have analyzed the functional and morphological effects of corticosteroid hormones in a SV40‐transformed rabbit cortical‐ascending‐limb (CAL) cell line (RC.SV2, Vandewalle et al., 1989) having mineralocorticoid (MR) and glucocorticoid (GR) receptors (Rafestin‐Oblin et al., 1993). Both aldosterone and dexamethasone (5 × 10−8M) induced a marked increase in (3H)ouabain binding (used to quantify membrane Na+ ‐K+ ATPase) detectable as early as 6 hours and maximal at 24 hours (+56‐57%) (due to a 1.6‐1.8‐fold increase in cell membrane binding sites without Kd alteration), and significantly augmented the ouabainsensitive component of Rb+ influx. Triiodothyronine (T3, 10−9 M) also stimulated ouabain binding by 21% but was not permissive for steroid action, whereas 5 μg/ml insulin had no effect. Both steroid hormones, T3 and insulin induced the formation of domes that was tightly correlated with ouabain binding (r = 0.949) except for insulin. The effects of aldosterone and dexamethasone on cell monolayers and cell ultrastructure were, however, strikingly different as aldosterone induced a marked amplification of basolateral areas with appearance of large intercellular spaces, reminiscent of the changes observed in deoxycorticosteronetreated rats, whereas dexamethasone predominantly influenced cell height. This discrepancy might be due to specific occupancy of MR and GR by aldosterone and dexamethasone, respectively, and/or to nongenomic effects of dexamethasone. We have thus characterized a cell culture model making it possible to analyze the actions of mineralocorticoid and glucocorticoid hormones in the mammalian kidney. © 1993 Wiley‐Liss, Inc.
High‐level expression of cytokine‐induced neutrophil chemoattractant (CINC) by a metastatic rat cell line: Purification and production of blocking antibodies
Authors: Wittwer Arthur J. , Carr Linda S. , Zagorski John , Dolecki Gregory J. , Crippes Barbara A. , De Larco Joseph E.
Published: February 2005

Significant levels of cytokine‐induced neutrophil chemoattractant (CINC) were found in serum‐free medium conditioned by a highly metastatic rat cell line, RC20. To study CINC's role in inflammation and metastasis, CINC was purified from this source for use in in vitro assays and for antibody production in goats and rabbits. CINC was a potent chemoattractant for rat neutrophils (EC‐50 0.5 nM). A fusion protein of glutathione‐S‐transferase and CINC (GST‐CINC) was produced in E. coli. Anti‐CINC polyclonal IgG was purified from immune goat and rabbit sera by protein A and GST‐CINC affinity chromatography. Both goat and rabbit anti‐CINC antibody preparations at 4 μg/mL (an 11‐fold molar excess) were found to completely block the activity of 2.5 nM CINC in a rat neutrophil chemotaxis assay. These antibodies have been used to develop a sensitive immunoassay for CINC. The availability of large amounts of affinity‐purified blocking anti‐CINC antibody will allow investigations into the role played by CINC in rodent inflammation models and in the metastasis of RC20 cells. © 1993 Wiley‐Liss, Inc.
Dibutyryl cyclic adenosine monophosphate differentially regulates cell proliferation in low and high alkaline phosphatase SaOS‐2 human osteosarcoma cells: Evidence for mediation by the insulin‐like growth factor‐II system
Authors: Mohan S. , Strong D. D. , Hilliker S. , Malpe R. , Lee K. , Farley J. , Baylink D. J.
Published: February 2005

In the present study, we have sought to determine whether a given signal transduction pathway can have diverse effects on subpopulations of cells of a lineage depending upon the stage of differentiation. To test this hypothesis, we selected the cyclic adenosine monophosphate (cAMP) signal transduction pathway because of its recognized importance in mediating the actions of many hormones, e.g., parathyroid hormone which acts on the bone‐forming cells, the osteoblasts. Subpopulations of human osteosarcoma SaOS‐2 cells with low (LSaOS) and high (HSaOS) alkaline phosphatase (ALP) content were chosen as model systems for preosteoblasts (pre‐OB) and osteoblasts (OB), respectively. Dibutyryl cyclic AMP (DBcAMP) treatment of serum free cultures produced a differential effect on the proliferation of LSaOS cells (40 ± 5% of control at 1 mM DBcAMP, P < 0.001) compared with HSaOS cells (no statistically significant effect). The finding supports the hypothesis. Next, we sought evidence for mediation, at least in part, by the insulin‐like growth factor (IGF)‐II regulatory system. We report that the basal expression of IGF‐II, IGF binding protein (IGFBP)‐3, and IGFBP‐4 was higher in LSaOS cells than in HSaOS cells with the opposite true for type I IGF receptor. DBcAMP treatment of LSaOS cells decreased IGF‐II and IGFBP‐3 but increased IGFBP‐4 and type I IGF receptor; no effect was observed for the type II IGF receptors. DBcAMP treatment of HSaOS cells had no detectable effect on IGF‐II; IGFBP‐3, or type I and type II IGF receptor expression; only IGFBP‐4 expression increased with DBcAMP. These observations suggest that the differential regulation of cell proliferation by the cAMP signal transduction pathway may be mediated, at least in part, by the IGF‐II regulatory system. © 1993 Wiley‐Liss, Inc.
Cell surface expression of DBA binding sites in LLC‐PK1 cells increases at confluence and is enhanced by PKA activation
Authors: Amsler Kurt
Published: February 2005

Post‐confluent populations of LLC‐PK1 cells express many glycoproteins at the surface, including glycoproteins which bind the lectin Dolichos biflorus agglutinin (DBA). DBA‐binding glycoproteins are localized preferentially to the apical cell surface and exhibit molecular weights ranging from less than 30 kDa to greater than 200 kDa. Subconfluent cell populations exhibit little surface DBA binding. Upon attaining confluence DBA binding capacity increases progressively over several days. This correlates roughly with increasing activities of several differentiated apical membrane functions. Expression of DBA binding sites at the surface occurs on a cell‐by‐cell basis. An increasing level of surface DBA binding in the cell population corresponds to an increasing proportion of the cell population expressing binding sites. Activation of cAMP‐dependent protein kinase in post‐confluent cell populations, and to a lesser extent in subconfluent populations, increases surface DBA binding sites by a protein synthesis‐dependent mechanism after a lag period of at least 24 h. This results from increases in both the proportion of cells expressing surface binding sites and the level of binding sites on individual cells. © 1993 Wiley‐Liss, Inc.
Importance of cell aggregation for expression of liver functions and regeneration demonstrated with primary cultured hepatocytes
Authors: Yuasa Chie , Tomita Yumiko , Shono Masayuki , Ishimura Kazunori , Ichihara Akira
Published: February 2005

Adult rat hepatocytes aggregated to form floating multicellular spheroids when cultured in Primaria dishes, which have a positively charged surface, in serum‐free Williams' medium E (WE) supplemented with insulin and epidermal growth factor (EGF). These hormones were essential for maintenance of the spheroids, whereas the size of the spheroids depended on the inoculum cell density. The spheroids retained in vivo levels of expressions of albumin and glucokinase and synthesized scarcely any DNA even in the presence of insulin and EGF. On transfer to type I collagen‐coated dishes, the spheroids gradually disaggregated and the cells formed monolayers, in which the expressions of albumin and glucokinase were suppressed and DNA synthesis and hexokinase activity were increased. DNA synthesis of hepatocytes in monolayer culture was maximal 24 hr after transfer of the spheroids, ∼80% of the hepatocyte nuclei were labelled with bromodeoxyuridine during culture for 48 hr, and the mitotic index was ∼70% after 60 hr. These results suggest that, in spheroids, hepatocytes remained in the G0 phase, but that when they formed monolayers, they progressed to the G1 phase and proceeded through the cell cycle in the presence of insulin and EGF. This work shows that the cell cycle of hepatocytes in culture can be manipulated by providing conditions for quiescence as spheroids or growth as monolayers and that the shape of hepatocytes is important for regulating their growth and liver‐specific functions. © 1993 Wiley‐Liss, Inc.
Thrombospondin mediates migration and potentiates platelet‐derived growth factor‐dependent migration of calf pulmonary artery smooth muscle cells
Authors: Yabkowitz Rachel , Mansfield Pamela J. , Ryan Una S. , Suchard Suzanne J.
Published: February 2005

A precipitating factor in the development of atherosclerotic lesions is the inappropriate migration and proliferation of vascular smooth muscle cells (SMC) within the intima of the vessel wall. Focusing on the role of extracellular matrix proteins in SMC migration, we have demonstrated that thrombospondin (TSP) itself is a potent modulator of SMC motility and acts to potentiate platelet‐derived growth factor (PDGF)‐mediated SMC migration as well. Migration of SMC to TSP was dose dependent. Interestingly, maximal SMC migration to TSP exceeded that to either PDGF or basic fibroblast growth factor (bFGF). The distal COOH terminus of TSP was shown to mediate SMC migration as demonstrated by complete inhibition of the response by monoclonal antibody (mAb) C6.7. Nevertheless, proteolytic fragments of TSP were not as potent as intact TSP in mediating SMC migration. Only by combining the heparin‐binding domain (HBD) with the 140 kD COOH terminal fragment was SMC migration restored to levels seen with intact TSP. Based on antibody inhibition studies, an αv‐containing integrin receptor, but not αvβ1 or αvβ3, appeared to be involved in SMC migration to TSP. The coincidental expression of PDGF and TSP at sites of vascular injury and inflammation led us to evaluate the effect of suboptimal levels of TSP on SMC responsiveness to PDGF. SMC migration in response to PDGF was enhanced nearly 60% in the presence of suboptimal concentrations of TSP. This effect was specific for PDGF and dependent on the concentration of TSP with maximal potentiation obtained between 50–100 nM TSP, concentrations tenfold lower than those necessary for SMC migration to TSP itself. mAb C6.7 completely inhibited enhancement but, as with SMC migration to TSP alone, TSP proteolytic fragments did not possess the effectiveness of the intact molecule. Additional experiments assessing SMC migration to PDGF demonstrated that PDGF stimulated SMC motility indirectly by inducing TSP synthesis. These studies suggested that TSP functions as an autocrine motility factor to modulate SMC migration, which in conjunction with PDGF could serve to aggravate and accelerate development of atherosclerotic lesions at sites of vascular injury or inflammation. © 1993 Wiley‐Liss, Inc.
Cell adhesion markers are expressed by a stable human endothelial cell line transformed by the SV40 large T antigen under vimentin promoter control
Authors: Vicart Patrick , Testut Patrice , Schwartz Bertrand , Llorens‐Cortes Catherine , Perdomo Juana J. , Paulin Denise
Published: February 2005

Markers of endothelium have been studied in a new endothelial cell line derived from human umbilical cord vein cells by microinjection of a recombinant gene that includes a deletion mutant of the human vimentin gene regulatory region controlling the large T and small t antigen coding region of the SV40 virus. In culture, this immortalized venous endothelial cell line (IVEC) demonstrated morphological characteristics of endothelium; uptake of acetylated low density lipoprotein and presence of the Factor VIII‐related antigen. Treatment of IVEC cells with Interleukin‐1β (IL‐1 β) at 10 U.ml−1 activates the expression of cell adhesion molecules such as endothelial leucocyte adhesion molecule (ELAM‐1), intercellular adhesion molecule‐1 (ICAM‐1), and vascular cell adhesion molecule‐1 (VCAM‐1), as observed in primary culture. Prostacyclin secretion was induced in the IVEC cells by 100 nM PMA treatment and thrombin at 0.5 U/ml. Angiotensin converting enzyme (ACE) activity detected in IVEC cells was present but lower than ACE activity in primary endothelial cells and was completely blocked by enalaprilat (1 μM), a specific ACE inhibitor. The presence of ACE mRNA was also demonstrated in IVEC cells by RT‐PCR amplification. Our data demonstrate that endothelial cells immortalized by use of this recombinant gene retain the morphological organization and numerous differentiated properties of endothelium. © 1993 Wiley‐Liss, Inc.
Adenine nucleotides and adenosine metabolism in pig kidney proximal tubule membranes
Authors: Blanco Julià , Canela Enric I. , Sayós Joan , Mallol Josefa , Lluis Carme , Franco Rafael
Published: February 2005

Exogenous adenosine triphosphate (ATP) added to brush‐border membrane vesicles was rapidly degraded mainly to inosine according to the high ecto‐nucleotidase activities in these vesicles. In the absence of phosphate, inosine was slowly transformed into hypoxanthine, and xanthine oxidase and dehydrogenase activities were not detected. The presence of ecto‐adenosine deaminase and ecto‐adenosine monophosphate (AMP) nucleotidase was shown. The ecto‐adenosine deaminase was inhibited by deoxycoformycin and was also detected in rat renal brush‐border membrane vesicles. Using orthovanadate, levamisole, and α, β‐methylene adenosine diphosphate as possible inhibitors, alkaline phosphatase was shown to be the main agent responsible for ecto‐AMP nucleotidase activity. In pig renal basolateral membrane vesicles and in whole cell extracts from pig renal cortex, ecto‐AMP nucleotidase was the limiting factor in ATP degradation. Comparing the ATP catabolism in the whole cell cortical extract with the catabolism in the same sample precleared of membranes, it was shown that ectonucleotidase activity is mainly bound to the membranous components. It is also shown that the whole cell extract of pig renal cortex has hypoxanthine phosphoribosyl transferase activity, and it seems probable that the rapid and specific formation of luminal inosine and its transport into the cell in competition with adenosine may start the purine salvage pathway through the synthesis of IMP from hypoxanthine. © Wiley‐Liss, Inc.
Use of p112‐deficient myoblasts to determine the temporal order of the in vitro expression of myogenic components
Authors: Chen Xiao Yong , Lo Theodore C. Y.
Published: February 2005

The present investigation examines the function and site(s) of involvement of an ecto‐protein kinase and its substrate protein (a cell surface 112 kDa protein) in the in vitro myogenic pathway. The phosphorylated 112 kDa protein (p112) has recently been shown to be involved in myogenesis. Not much information is currently available on the role of the ecto‐protein kinase and the 112 kDa protein in modulating the expression of the myogenic factors and various muscle‐specific proteins. Five different p112‐deficient rat myoblasts were used to examine the temporal order of the in vitro expression of the myogenic components namely, L6 myoblasts treated with BrdUrd or phloretin, a conditional p112‐defective mutant (clone D1), an ecto‐protein kinase‐deficient mutant (clone F72), and a mutant defective in the 112 kDa protein (clone D1/S4). All these p112‐deficient myoblasts were also impaired in myogenesis. The absence of p112, ecto‐protein kinase, and/or the 112 kDa protein was found to have no effect on the Myf‐5 mRNA level. However, the expected increase in NCAM and Myf‐4 mRNAs was not observed in any of the p112‐deficient myoblasts examined. This suggests that the p112 site of action is probably located upstream of the Myf‐4 and NCAM sites in the myogenic pathway. While 7–28 fold increases in the MLC, MHC, and TnT transcripts were observed during myogenesis, such increases were not observed in the p112‐deficient myoblasts. However, when mutant D1/S4 was transfected with the myf‐4 cDNA, expression of Myf‐4 in the transfectant resulted in increased level of the MLC, MHC, and TnT mRNAs, and in myotube formation, even though the Myf‐5 and NCAM mRNA levels and p112 were not altered. This suggests that p112 may function by activating transcription of Myf‐4, which will subsequently promote the expression of muscle‐specific proteins and myotube formation. In the absence of p112, Myf‐5 cannot activate the expression of Myf‐4, NCAM, MLC, MHC, TnT, and myotube formation. If all these components are involved in the same myogenic pathway, then p112 may be acting downstream from Myf‐5, and upstream from NCAM and Myf‐4. © 1993 Wiley‐Liss, Inc.
Anti‐integrin antibodies induce type IV collagenase expression in keratinocytes
Authors: Larjava Hannu , Lyons J. Guy , Salo Tuula , Mäkelä Marja , Koivisto Leeni , Birkedal‐Hansen Henning , Akiyama Steven K. , Yamada Kenneth M. , Heino Jyrki
Published: February 2005

During wound healing, pericellular proteolysis is thought to be essential for the detachment of keratinocytes from basement membrane and in their migration into the wound bed. We have characterized integrin‐type cell adhesion/migration receptors in human mucosal keratinocytes and examined their function in the regulation of type IV collagenase gene expression. Two major integrins of the β1 class, α2β1 and αβ1, were found to function as collagen and fibronectin receptors, respectively. Antibodies against β1 and α3 integrin subunits were found to stimulate the expression of the 92 kDa type IV collagenase severalfold in a dosedependent manner. Keratinocytes expressed also the 72 kDa type IV collagenase, the synthesis of which remained, however, unchanged in keratinocytes treated with anti‐integrin antibodies. Stimulation of 92 kDa enzyme was found to be caused directly by antibody binding to integrins, since Fab‐fragments of anti‐β1 antibodies alone were able to induce collagenase expression in the absence of secondary, clustering antibodies. Antibodies against α2β1 integrin caused no stimulation. Keratinocytes seeded on different substrata (plastic, collagen, fibronectin, laminin, or vitronectin) showed equal induction of type IV collagenase expression. Expression of 92 kDa type IV collagenase could not be induced by peptides (GRGDS, GRGES), proteins (fibronectin, laminin, fibrinogen., albumin), or antibodies to fibronectin. We suggest that proteolytic processes around keratinocytes can be regulated by extracellular factors signalling through integrin‐type receptors. © 1993 Wiley‐Liss, Inc.
Differential heat stress stability of epidermal growth factor receptor and erbB‐2 receptor tyrosine kinase activities
Authors: Liu Samuel M. , Carpenter Graham
Published: February 2005

The epidermal growth factor (EGF) and erbB‐2 receptors are structurally related membrane‐bound tyrosine kinases. While these proteins exhibit close sequence homology, 50% overall and 80% in the tyrosine kinase domains, they respond very differently to heat stress. In NIH‐3T3 or NR6 cells transfected with wild‐type EGF‐R and incubated at 37°C or heat shocked at 46°C, EGF binds to its receptor and stimulates receptor autophosphorylation to equivalent extents. At 46°C, however, the basal tyrosine kinase activity of the wild‐type erbB‐2 receptor is rapidly lost. When cells containing chimeric receptors composed of the EGF‐R extracellular domain and intracellular domain of erbB‐2 were heat stressed, 125I‐EGF bound to the receptors, but did not stimulate receptor autophosphorylation. The decline in EGF‐stimulated chimeric erbB‐2 receptor autophosphorylation is dependent on the length of heat shock, with nearly 100% of the kinase activity lost after 60 min at 46°C. The loss of chimeric receptor erbB‐2 kinase activity is not due to degradation of receptor protein, nor is it attributable to a specific transmembrane domain from either the EGF or erbB‐2 receptors. Sensitivity of erbB‐2 to heat stress is also not a result of denaturation of this receptor's carboxy‐terminal domain. Insertion of the erbB‐2 tyrosine kinase domain into the EGF‐R confers heat stress sensitivity to the resultant chimeric receptor. Thus, although the EGF‐R and erbB‐2 kinase domains show a high degree of homology, the secondary/tertiary structures of these domains would seem to be stabilized in distinct manners. © 1993 Wiley‐Liss, Inc.
Maturation‐dependent regulation of protein kinase C activity by vitamin D3 metabolites in chondrocyte cultures
Authors: Sylvia V. L. , Schwartz Z. , Schuman L. , Morgan R. T. , Mackey S. , Gomez R. , Boyan B. D.
Published: February 2005

Vitamin D3 metabolites regulate the differentiation of chondrocytes isolated from the growth zone or resting zone of rat costochondral cartilage. Since some of the direct membrane effects of vitamin D metabolites are nongenomic, we hypothesized that protein kinase C (PKC) plays a role in signal transduction for these chondrocyte differentiation factors and that the regulation of PKC by the vitamin D metabolites is cell maturation dependent. Confluent, fourth passage cultures of growth zone and resting zone chondrocytes were treated with vitamin D3 metabolites for up to 24 h, lysed, and cell extracts assayed for kinase activity using a specific PKC substrate peptide. The addition of 1,25‐(OH)2D3 to growth zone cell cultures resulted in a rapid dose‐dependent stimulation of PKC, significant at 10−9‐10−7 M, beginning at 3 min and sustained until 90 min; 1,25‐(OH)2D3 had no effect on PKC activity in resting zone chondrocyte cultures. The addition of 24,25‐(OH)2D3 to resting zone cultures showed a slower PKC activation, with significant stimulation seen at 90–360 min for 10−8‐10−7 M 24,25‐(OH)2D3. However, 24,25‐(OH)2D3 had no effect on PKC activity in growth zone cell cultures at all times and concentrations examined. The specificity of PKC stimulation by the vitamin D3 metabolites was verified using a specific pseudosubstrate region peptide inhibitor, which reduced PKC activity when included in the reaction mixture. Pretreatment of the cultures with U73,122, a phospholipase C inhibitor, decreased 1,25‐(OH)2D3—stimulated PKC activity but had no effect upon 24,25‐(OH)2D3–induced activity. The tyrosine kinase inhibitor, genistein, did not inhibit the PKC response in either vitamin D3 metabolites‐treated culture. Neither actinomycin D nor cycloheximide affected 1,25‐(OH)2D3–induced PKC activity in growth zone chondrocyte cultures, while both compounds inhibited 24,25‐(OH)2D3—indiuced activity in resting zone chondrocyte cultures. The results of this study indicate that vitamin D metabolites stimulate PKC activity in a metabolite‐ and cell‐maturation‐specific manner. Effects of 1,25‐(OH)2D3 appear to be nongenomic, whereas the effects of 24,25‐(OH)2D3 probably involve a genomic mechanism. © 1993 Wiley‐Liss, Inc.