bluuebunny/crossref_metadata_2025 huggingface dataset:
Mechanism and regulation of neutrophil priming by platelet‐activating factor
Authors: Gay James C.
Published: February 2005

Although a weak direct stimulus of superoxide anion (O2−) production, platelet‐activating factor (PAF) markedly enhances responses to chemotactic peptides (such as n‐formyl‐met‐leu‐phe, FMLP) and phorbol esters (such as phorbol myristate acetate, PMA) in human neutrophils. The mechanism of priming was explored first through inhibition of steps in the signal transduction pathway at and following PAF receptor occupation. Priming was not altered by pertussis toxin or intracellular calcium chelation, but the PAF receptor antagonist WEB 2086 and the protein kinase C (PKC) inhibitors sphinganine and staurosporine significantly inhibited the primed response. In order to study the regulation of PAF priming, the effect of PAF alone was desensitized by exposure to escalating doses of PAF prior to exposure to the secondary stimuli. The priming effect of PAF was not desensitized under these conditions. The role of PKC in desensitization was also studied. Prior exposure to PAF also desensitized the increase in membrane PKC activity evoked by a single concentration of PAF. However, when the PAF response was desensitized, PKC priming of the response to FMLP or PMA still occurred, suggesting that PKC activity may play a role in the maintenance of the primed state despite PAF desensitization. These data suggest that: (1) PAF priming is receptor‐ and PKC‐mediated but is independent of pertussis toxin‐inhibitable G‐proteins or intracellular calcium, (2) during migration in vivo, neutrophils may be desensitized to the direct effects of PAF but maintain the capacity for enhanced responses to other stimuli, (3) desensitization of PAF‐induced particulate PKC activity also occurs, but PAF primes PKC activity despite PAF desensitization, and (4) distinct mechanisms govern the direct and priming effects of PAF on oxidative metabolism. © 1993 Wiley‐Liss, Inc.
Transforming growth factor β1 selectively regulates ornithine decarboxylase gene expression in malignant H‐ras transformed fibrosarcoma cell lines
Authors: Hurta Robert A. R. , Greenberg Arnold H. , Wright Jim A.
Published: February 2005

Negative growth regulators such as the transforming growth factor beta (TGF‐β) family appear to be important inhibitors in most tissue types. However, inhibition of DNA synthesis and cell proliferation is frequently lost during malignant transformation, and in some cases, tumor cell proliferation is actually stimulated by TGF‐β. The present study demonstrates a novel link between alterations in TGF‐β regulation during malignant conversion, and the expression of ornithine decarboxylase, a key rate‐limiting activity in the biosynthesis of polyamines, and an enzyme that plays an important role in cell growth and differentiation. A panel of radiation and H‐ras transformed mouse 10T1/2 cell lines exhibiting increasing malignant potential was investigated for possible TGF‐β1 mediated changes in ornithine decarboxylase gene expression. Selective induction of gene expression was observed since only H‐ras transformed cell lines with malignant potential exhibited marked elevations in ornithine decarboxylase message levels. Ornithine decarboxylase gene expression in nontransformed 10T1/2 cells and cell lines capable of only benign tumor formation was unaffected by TGF‐β1 treatment. H‐ras transformed cells were transfected with a plasmid placing the TGF‐β1 coding region under the control of a zinc sensitive metallothionein promoter. When these cells were cultured in the presence of zinc an elevation of TGF‐β1 mRNA was observed within 30 min. This increase in TGF‐β1 message closely coincided with an elevation in ornithine decarboxylase message, and preceded an induction of jun‐B, an early response gene in cells sensitive to TGF‐β1 stimulation. Evidence for regulation of ornithine decarboxylase gene expression by TGF‐β1 at both transcription and posttranscription was found. Actinomycin D pretreatment of malignant cells prior to TGF‐β1 exposure prevented the increase in ornithine decarboxylase message. Marked differences in the rates of ornithine decarboxylase message decay were observed when cells treated with TGF‐β1 were compared to untreated controls, with the half‐life of ornithine decarboxylase mRNA increasing from 2.5 h in untreated cells to 17.5 h in cells exposed to TGF‐β1. In addition, evidence was obtained for a cycloheximide sensitive regulator of ornithine decarboxylase gene expression, since the presence of this protein synthesis inhibitor increased the levels of ornithine decarboxylase message, and this effect was synergistically augmented by exposure of cells to cycloheximide and induction of TGF‐β1 gene expression together. These results show for the first time that TGF‐β1 can regulate ornithine decarboxylase expression in malignant H‐ras transformed cells, and suggest a mechanism of growth factor stimulation of malignant cells, in which early alterations in the control of ornithine decarboxylase gene expression are important. © 1993 Wiley‐Liss, Inc.
Rapid apoptotic cell death of B‐cell hybridomas in absence of gene expression
Authors: Perreault Josée , Lemieux Réal
Published: February 2005

Apoptosis is an active form of cell death which plays an important role in different biological processes. The induction of apoptosis usually requires de novo gene expression but has also been observed in certain types of cells in absence of gene expression or following a block of gene expression. We show here that inhibition of macromolecular synthesis induced the rapid apoptotic death of most antibody‐secreting B‐cell hybridomas. The effect was observed in presence of both protein synthesis (cycloheximide [CHX]) and transcription (actinomycin D [Act D]) inhibitors and was characterized by extensive degradation of nucleic acids (DNA and RNA) within 2–3 h of treatment. The CHX treatment not only severely impaired the proliferation of the cells but also resulted in the loss of cell viability (MTT assay) without the need of de novo gene expression. The susceptibility to apoptosis varied among different B‐cell hybridomas and was inherited from the SP2/0 myeloma cell fusion partner. These results indicate the constitutive activation of a death program in B‐cell hybridomas and its inhibition at a late stage by the continuous expression of gene(s) coding for short‐lived protein(s). The occurrence of this phenomenon may well be related to the abundant and deregulated (trans‐location) expression, in this type of cells, of the c‐myc gene which has recently been shown to be a potent inducer of apoptosis in growth‐arrested fibroblasts. © 1993 Wiley‐Liss, Inc.
Platelet‐activating factor stimulates phosphatidic acid formation in cultured rat mesangial cells: Roles of phospholipase D, diglyceride kinase, and de novo phospholipid synthesis
Authors: Kester Mark
Published: February 2005

Platelet‐activating factor (PAF) stimulates phospholipase C (PLC)‐induced hydrolysis of phosphatidylinositol‐4,5‐bisphosphate (Ptdlns‐4,5‐P2). Yet, PAF‐stimulated diglycerides (DG) are still elevated at time points where inositol polyphosphates have returned to basal levels. Thus, other signal transduction pathways that hydrolyze phosphatidylcholine (PtdCho) or phosphatidylethanolamine (PtdEth) and form DG and phosphatidic acids (PA) through either PLC or phospholipase D (PLD) may also mediate PAF‐stimulated cellular responses. Initially the effects of PAF upon 32P‐PA generation in mesangial cells (MC) were assessed. PA formation may be indicative of several metabolic pathways including PLD and DG kinase activities as well as de novo phospholipid synthesis. PAF (10−7 M) increased 32P‐PA formation as early as 5 seconds and this elevation persisted up to 15 minutes. When MC were pretreated with the DG kinase inhibitor‐R59022, PAF‐induced 32P‐PA formation was diminished at early but not late time points, demonstrating that the initial component of PA formation may be due, in part, to PLC activation and subsequent phosphorylation of DG. The reciprocal reaction, PA phosphohydrolase, which dephosphorylates PA to from DG was not stimulated by PAF, suggesting that the sustained elevation of DG induced by PAF is primarily a reflection of PLC. 3H‐glycerol pulse‐labeling experiments suggest that PAF also stimulates de novo phospholipid synthesis which also contributes to PA formation. Conclusive proof for PLD in the generation of PA was obtained by assessing the formation of 3H‐phosphatidylethanol (PEt) from 3H‐alkyl‐lyso‐glycero phosphocholine (GPC) and exogenous ethanol. PAF stimulated alkyl‐PEt generation in the presence but not the absence of 0.5% ethanol. Also, PAF induced a concomitant elevation of alkyl‐PA at 15 minutes and this elevation of alkyl‐PA was reduced when the cells were exposed to exogenous ethanol, reflecting the formation of PEt. Corroborating evidence suggests that PAF stimulates 3H‐choline and 3H‐ethanolamine release, suggesting that PtdCho and PtdEth are substrates for PLD. Thus, these data demonstrate that MC respond to PAF with elevated PLD and DG kinase activities as well as with an increased rate of de novo lipid synthesis which increases PA, a potential intracellular signal. © 1993 Wiley‐Liss, Inc.
Homologous desensitization of bombesin‐induced increases in intracellular Ca2+ in quiescent swiss 3T3 cells involves a protein kinase C‐independent mechanism
Authors: Walsh John H. , Bouzyk Mark , Rozengurt Enrique
Published: February 2005

Addition of bombesin to Swiss 3T3 cells causes a rapid and transient increase in the intracellular concentration of Ca2+ ([Ca2+]i), which is followed by desensitization to a subsequent addition of the peptide. The concentrations of bombesin used to study this acute cellular desensitization (0.1–0.5 nM) did not deplete the intracellular pool of Ca2+ released by inositol(1,4,5)trisphosphate, as shown by addition of vasopressin after consecutive additions of bombesin. Two lines of evidence support the conclusion that activation of protein kinase C (PKC) does not mediate the acute homologous desensitization of Ca2+ responses induced by bombesin. First, long‐term treatment (48 h) of Swiss 3T3 cells with phorbol 12, 13‐dibutyrate (PDB) to deplete PKC did not prevent homologous desensitization. The responses to second additions of bombesin at 0.1, 0.25, and 0.5 nM were 42%, 26% and 11% of the initial responses, respectively. Second, the PKC inhibitor GF 109203X did not alter homologous desensitization at concentrations that completely prevented the inhibition of Ca2+ mobilization induced by PDB and blocked PDB‐mediated phosphorylation of the prominent PKC substrate 80K/MARCKS. We conclude that acute homologous desensitization of Ca2+ responses induced by bombesin occurs through a PKC‐independent mechanism. © 1993 Wiley‐Liss, Inc.
Activation of potassium channels by hypoxia and reoxygenation in the human lung adenocarcinoma cell line A549
Authors: Koong A. C. , Giaccia A. J. , Hahn G. M. , Saad A. H.
Published: February 2005

Active oxygen species are generated in cells during pathophysiologic conditions such as illflammation and postischemic reperfusion. If oxygen radical scavengers are added before reperfusion, then the magnitude of injury is reduced. We inves‐tigated whether free radicals generated following exposure to hypoxia and reoxygenation activate voltage‐dependent K+ ion channels in tumor cells in vitro. Using the technique of whole cell voltage clamping, we recorded currents from two families of potassium (K+) channels that were activated following reoxygenation. One of these groups possessed the electrophysical characteristics of a tetraethylammonium (TEA)‐sensitive delayed rectifier channel and the other possessed characteristics of a Tea‐insensitive slow inactivating channel. We present evidence which suggests that K+ channels are activated following reoxygenation but not during the hypoxia phase. The K+ currents decayed with time following reoxygenation. The decay characteristics of the K+ currents depended on the duration and level of hypoxia to which the cells were exposed. To determine whether activation of K+ channels by reoxygenation was initiated by free radicals, we pretreated cells with N‐Acetyl L‐Cysteine (NAC), a free radical scavenger, and found that this pretreatment abolished the currents induced by reoxygenation. We also present evidence that free radicals do not directly act on the channel itself, but activate a protein kinase which, in turn, activates the K+ channels. Taken together, these results indicate that one of the early responses to oxidative stress is the activation of K+ currents. © 1993 Wiley‐Liss, Inc.
Estrogen enhances epidermal growth factor‐induced DNA synthesis in mammary epithelial cells
Authors: Vanderboom Russell J. , Sheffield Lewis G.
Published: February 2005

Estradiol (E2) priming (1 nM for 48 h) of normal murine mammary gland epithelial cells significantly increased the response of those cells to epidermal growth factor (EGF)‐induced DNA synthesis. The synergism between E2 and EGF was evident in two aspects: After serum‐free synchronization for 24 h, more cells entered the S‐phase of the cell cycle after E2 priming and when treated with 0.17 nM EGF (13%) than did control cells (1.3%) or cells treated with EGF (4%) or E2 (3.5%) alone; further, the dose of EGF required to elicit maximal response was reduced an order of magnitude in estrogen‐primed cells (0.17 nM) compared to controls (1.7 mM). Estrogen alone, however, did not increase DNA synthesis in these cells. Ligand binding studies indicate that these effects of estrogen on proliferating mammary epithelial cells may be explained, at least in part, by a 3.7‐fold increase in the number of high affinity EGF‐receptors observed in estrogen primed cells (7,300 receptors per cell) compared to estrogen deprived cells (1,960 receptors/cell). © 1993 Wiley‐Liss, Inc.
Pool of ligand‐bound platelet‐derived growth factor β‐receptors remain activated and tyrosine phosphorylated after internalization
Authors: Sorkin Alexander , Eriksson Anders , Heldin Carl‐Henrik , Westermark Bengt , Claesson‐Welsh Lena
Published: February 2005

We have examined the state of tyrosine phosphorylation of ligand‐bound, internalized platelet‐derived growth factor (PDGF) β‐receptors. Analysis by immunofluorescence staining of cells stimulated with PDGF‐BB at 370C indicated colocalization of phosphotyrosine, PDGF β‐receptors, and PDGF‐BB in endosome‐like vesicles. Treatment of cells with an acidic buffer, which removed cell surfacebound PDGF‐BB, led to a considerable decrease in phosphorylation and kinase activity of cell surface localized PDGF β‐receptors, but not of internalized receptors. Immunoprecipitations using antisera against phosphotyrosine and the PDGF β‐receptor from metabolically labeled cells showed that a major fraction of the tyrosine‐phosphorylated pool of receptors were still accessible to the acid buffer treatment after 10 min of incubation of the cells at 370C. Under these conditions, about 20‐25% of the total pool of tyrosine‐phosphorylated, receptors were intratcellular, since they remained tyrosine phosphorylated after the acid buffer treatment. A considerable pool of tyrosine‐phosphorylated, internalized receptors, after 10 min of incubation of the cells at 370C, could also be detected by immunoblotting analysis, using antisera against the PDGF β‐receptor and phosphotyrosine. Analysis by in vitro kinase assays of immunoprecipitated PDGF β‐receptors, obtained from PDGF‐BB‐stimulated cells different times after acid wash, showed that the internalized receptors retained kinase activity. These data suggest that a pool of internalized PDGF β‐receptors remain active and may participate in signalling a considerable time after internalization. © 1993 Wiley‐Liss, Inc.
Packaging zinc, fibrinogen, and factor XIII in platelet α‐granules
Authors: Marx Gerard , Korner Gil , Mou Xiaode , Gorodetsky Raphael
Published: February 2005

Zirc(II) accumulated by platelets has profound effects on platelet activity. This study is focused on the distribution of Zn(II) between human platelet subcellular compartments. After incubation with 86Rb+ and platelet lysis, the organelles were separated by sucrose density gradient centrifugation. Fibrinogen served as a marker for α‐granules. 86Rb+ and factor XIII served as markers for the cytoplasmic fractions. Zn(II) was found to be distributed between the cytoplasm and the α‐granules, with variations between different individual units. The total platelet Zn concentration and its relative subcellular distribution were dependent on its extracellular level. Incubation of platelets with 100 μM Zn(II) resulted in a twofold increase of its level in the cytoplasm and by one order of magnitude in the α‐granules. In addition to the anticipated factor XIII activity in the cytoplasmic pool fraction, we found thrombin‐inducible factor XIII activity within the α‐granules. Immunoblotting confirmed the presence of both the a and b subunits of plasma factor XIII (a2b2 form) in the α‐granules. As fibrinogen is not synthesized in the platelet, we propose that by virtue of their mutual binding, fibrinogen, Zn(II) and plasma factor XIII‐a2b2 are simultaneously taken up into the α‐granules by endocytosis, presumably through the vehicle of the GPIIb/IIIa fibrinogen receptor. A rationale for copackaging these components within the α‐granules is that Zn(II) inhibits factor XIII activity and thereby prevents the premature cross‐linking of the concentrated fibrinogen prior to platelet activation and secretion. By contrast, cytoplasmic Zn(II) may increase platelet responsiveness to agonists due to its interaction with cytoplasmic modulators of platelet activity. © 1993 Wiley‐Liss, Inc.
Ligand‐dependent inhibition of myoblast differentiation by overexpression of the type‐1 insulin‐like growth factor receptor
Authors: Quinn Lebris S. , Ehsan Mitra , Steinmetz Brian , Kaleko Michael
Published: February 2005

The insulin‐like growth factors (IGFs) have paradoxical effects on skeletal myoblast differentiation. While low concentrations of IGF stimulate myoblast differentiation, high concentrations of IGF induce a progressive decrease in myoblast differentiation. The mechanism of this inhibition is unknown. Using a retroviral expression vector, we developed a subline of mouse P2 mouse myoblasts (P2‐LISN) which expressed 7.5 times higher levels of type‐1 IGF receptors than control (P2‐LNL6) myoblasts, which were infected with a virus lacking the type‐1 IGF receptor sequence. Overexpression of the type‐1 IGF receptor caused the IGF dose‐response curves of stimulation and progressive inhibition of differentiation to shift to the left. Additionally, at high insulin and IGF‐I concentrations, complete inhibition of P2‐LISN myoblast differentiation occurred. These results suggest that inhibition of differentiation at high ligand concentrations was not due to the primary involvement of other species of receptors for IGF. Type‐1 IGF receptor downregulation as a mechanism for inhibition of differentiation was also ruled out since P2‐LISN myoblasts constitutively expressed high levels of type‐1 IGF receptors. Additionally, inhibition of differentiation at high concentrations of IGF‐I was not correlated with overt stimulation of proliferation or with IGF binding protein (IGF‐BP) release into the culture medium. These results indicate that the type‐1 IGF receptor mediates two conflicting signal pathways in myogenic cells, differentiation‐inducing and differentiation‐inhibitory, which predominate at different ligand concentrations. © 1993 Wiley‐Liss, Inc.
Epidermal growth factor induces terminal differentiation in human epidermoid carcinoma cells
Authors: Konger Raymond L. , Chan Thomas C. K.
Published: February 2005

Previous studies have reported that the proliferation of A431 cells, a human squamous cell carcinoma cell line, was stimulated by picomolar epidermal growth factor (EGF) but inhibited by nanomolar EGF. This biphasic dose‐response phenomenon is not observed in normal human epithelial cells where nanomolar EGF is usually mitogenic. We have examined the effects of inhibitory and stimulatory concentrations of EGF on the growth and differentiation of A431 cells. In the presence of 100 pM EGF, A431 cells showed a mild increase in growth rate (129% of control) compared to cells grown in the absence of EGF. At 10 nM EGF, growth inhibition to 63% of control was observed. EGF at 10 nM stimulates a twofold increase both in cornified envelope formation and in epidermal transglutaminase activity, suggesting that high concentrations of EGF induce terminal differentiation in A431 cells. Mitogenic concentrations of EGF (100 pM) had no significant effect on these differentiation markers. Chronic exposure of A431 cells to 20 or 50 nM EGF resulted in EGF‐resistant A431 variants that are neither growth arrested nor induced to terminally differentiate by 10 nM EGF. Removal of EGF from the growth medium of the EGF‐resistant cells resulted in the reversion of these cells back to the wild‐type A431 biphasic response pattern within 2 weeks. Our results suggest that A431 cells have the capacity to non‐mutatively alter their response pattern to EGF in vitro to maintain themselves in a state of optimum proliferation and away from terminal differentiation. © 1993 Wiley‐Liss, Inc.
Characterization of a γ‐glutamyl transpeptidase positive subpopulation of endothelial cells in a spontaneous tube‐forming clone of rat cerebral resistance‐vessel endothelium
Authors: Wang Bao‐Le , Grammas Paula , De Bault Lawrence
Published: February 2005

A spontaneous tube‐forming clone of rat cerebral resistance‐vessel endothelium was characterized in long‐term serial culture. In this study, a clone, RV‐150 ECT, of cerebral resistance vessel endothelial cells in long‐term culture has been shown to have a subpopulation of γ‐GTP positive cells that are present in all cultures regardless of confluency status or tube‐forming stages. In pre‐confluent and confluent cultures, the γ‐GTP positive cells are few in number, stain weakly, and are randomly distributed in the monolayers. In monolayer post‐confluent cultures, γ‐GTP positive cells increase in number, stain strongly, and begin to show signs of non‐random distributions. In early post‐confluent cultures that have become a mixture of monolayer and multilayer cells, there is a further increase in γ‐GTP positive cells which begin to form distinct groupings. In mid post‐confluent fultures, the multilayered areas of the culture have begun clustering to form clear multicellular aggregates. The γ‐GTP positive cells at this stage are reduced in number and are predominately associated with the cell clusters. In late postconfluent cultures, the multicellular clusters develop clear cell cords between/among the clusters. At this stage the γ‐GTP positive cells are associated exclusively with cell clustters. With cord development, the γ‐GTP positive cells are associated with bothe clusters and cords, and are reduced in number apparently because of selective degeneration of these cells. The results of this study demonstrate that a phenotypically distinct subpopulation of endothelial cells exhibits characteristic features of the blood‐brain barrier, namely γ‐GTP. The ability of these cells to express this property in long‐term serial culture suggests that this may represent a useful in vitro model to study the growth and differentiation of bloodbrain barrier vessels. © 1993 Wiley‐Liss, Inc.
Thrombin receptor activating peptides induce Ca2+ mobilization, barrier dysfunction, prostaglandin synthesis, and platelet‐derived growth factor mRNA expression in cultured endothelium
Authors: Garcia Joe G. N. , Patterson Carolyn , Bahler Chris , Aschner Judy , Hart C. Michael , English Denis
Published: February 2005

Endothelial cell activation by thrombin is a key event in wound healing, Inflammation, and hemostasis. To better define thrombin‐endothelial cell interactions we synthesized several peptides of varying length corresponding to the initial 14 amino acid sequence of the cloned human platelet thrombin receptor after cleavage at an arginine41 site (R/SFLLRNPNDKYEPF). Thrombin receptor activating peptides (TRAPs) as short as 5 amino acids induced significant levels of PGl2 synthesis and expression of PDGF mRNA in human endothelium and produced dose‐dependent cellular contraction and permeability of confluent human umbilical vein and bovine pulmonary artery endothelial monolayers. To explore whether TRAPs utilized similar signal transducing pathways as α‐thrombin to accomplish endothelial cell activation, phospholipase C production of the Ca2+ secretagogue IP3 was measured and detected 10 seconds after either TRAP 7 or α‐thrombin. Furthermore, TRAPs ranging from 5‐14 residues induced significant dose‐dependent incsreases in Fura‐2 fluorescence indicative of Ca2+ 1 mobilization. These results indicate that thrombin‐mediated proteolytic cleavage of the human and bovine thrombin receptor initiates stimulus/coupling respones such phospholipase C activation, Ca2+ mobilization, and protein kinase C activation. The functional consequence of this cellular activation via the cleaved receptor is enhanced cellular contraction, barrier dysfunction, PGI2 synthesis, and expression of PDGF mRNA. © 1993 Wiley‐Liss, Inc.
Modulation of endothelial autacoid release by protein kinase C: Feedback inhition or non‐specific attenuation of receptor‐dependent cell activation?
Authors: Hecker Markus , Lückhoff Andreas , Busse Rudi
Published: February 2005

Receptor‐mediated elevations of intracellular Ca2+ in endothelial cells may be controlled by a negative feedback mechanism through activation of protein kinase C (PKC). To test this hypothesis, we studied the effects of an activation or inhibition of PKC on the release of nitric oxide (NO) and prostacyclin (PGI2) from cultured bovine and porcine aortic endothelial cells (EC). Preincubation with the PKC activators phorbol‐12‐myristate‐13‐acetate (PMA) (3‐300 nM) or 1‐oleyl‐2‐acetyl‐glycerol (OAG) (30 μM) significantly attenuated the release of NO and PGI2 from EC stimulated with bradykinin (0.3–30 nM), whereas phorbol‐12, 13‐didecanoate (PDD) (30–300 nM), which does not activate PKC, had no effect. UCN‐01 (10 nM), a specific PKC inhibitor, significantly augmented the bradykinin‐stimulated release of NO from EC. These effects were correlated with a reduced (PMA) or enhanced (UCN‐01) elevation of intracellular Ca2+ in response to bradykinin in both types of EC. Neither the PKC activators nor the inhibitor had any effect on resting intracellular Ca2+ or basal endothelial autacoid release. Several isoforms of PKC (namely PKCα, PKCδ, PKCϵ, and PKCζ) were detected in bovine, human, and porcine EC by immunoblotting analysis with isotype‐specific anti‐PKC antibodies, which, except PKCϵ, were predominantly located in the cytosol. Incubation of bovine EC with PMA elicited a significant increase in membrane‐bound PKCα immunoreactivity, whereas there was no translocation of PKCα from the cytosolic to the membrane fraction with bradykinin. As determined by histone phosphorylation, PKC activity was similarly reduced in the cytosol, but increased in the membrane fraction of bovine EC exposed to PMA, whereas bradykinin had no significant effect. These findings indicate that endothelial autacoid release can be modulated by activators and inhibitors of PKC. However, stimulation of EC with bradykinin does not lead to a detectable activation of PKC, suggesting that PKC does not exert a negative feedback in the signal transduction pathway of this receptor‐dependent agonist. © 1993 Wiley‐Liss, Inc.
Substance P: A competence factor for human fibroblast proliferation that induces the release of growth‐regulatory arachidonic acid metabolites
Authors: Kähler Christian M. , Herold Manfred , Wiedermann Christian J.
Published: February 2005

Substance P (SP) was recognized to stimulate cell growth. The mechanisms of growth control by SP are unknown. We, therefore, investigated mechanisms of the effect of SP on proliferation of human skin fibroblasts. SP did not stimulate proliferation of fibroblasts growth arrested by serum starvation over 48 hours. However, in the presence of acetylsalicylic acid SP potently stimulated fibroblast growth. A bell‐shaped dose‐response curve with maximal stimulation at picomolar concentrations was found. Specificity of the mitogenic effect was analyzed by use of synthetic SP analogs. Only neurokinin‐1 receptor agonists were active, whereas a specific neurokinin‐2 receptor analog did not exhibit mitogenicity. Analyzing the supernatants of growth‐arrested fibroblasts treated with SP indicated that SP provokes release of the arachidonic acid metabolites, prostaglandin E2, and prostacyclin but not thromboxane B2 or leukotriene B4. Since similar response patterns in proliferation and arachidonic acid metabolite release have been described for several proinflammatory cytokines, some of which are known to act as competence factors in proliferation, we characterized the mitogenic effect of SP. Results established that SP stimulates fibroblast growth in a manner typical of competence factors. We conclude that arachidonic acid metabolites are involved in the cell cycle‐dependent mitogenic action of SP on human skin fibroblasts. © 1993 Wiley‐Liss, Inc.
Early induction of LDL receptor gene during rat liver regeneration
Authors: Bocchetta M. , Bruscalupi G. , Castellano F. , Trentalance A. , Komaromy M. , Fong L. G. , Cooper A. D.
Published: February 2005

After partial hepatectomy in the rat, there is substantial lipid accumulation in the liver. No information is available on the possible role of receptor‐mediated endocytosis in this process. Since the low density lipoprotein (LDL) receptor is stimulated as a part of an early growth response in cell culture (Ellsworth et al.: Biochem. J. 279:175–187, 1991), the metabolism of this receptor during liver regeneration was studied. The mRNA and membrane protein level of the receptor were measured in the liver and in the adrenal glands at different times after partial hepatectomy, corresponding to different phases of the cell cycle. A discontinuous pattern of receptor expression is detectable in the regenerating liver; a large increase of mRNA and membrane protein occurs at an early time (2–4 h), suggesting that there is induction of LDL receptor gene transcription shortly after partial hepatectomy. This response seems specific for the liver following injury since the adrenal receptor does not show a different pattern in partially hepatectomized rats and sham‐operated controls. After returning to control levels, the LDL receptor again increases slightly above control at 24 h, a time when cell replication begins. © 1993 Wiley‐Liss, Inc.
Bradykinin‐stimulated transient modulation of epidermal growth factor receptors in A‐431 human epidermoid carcinoma cells
Authors: Hosoi Kazuo , Kurihara Kinji , Ueha Takao
Published: February 2005

Of nine biological factors (ATP, bradykinin, vasopressin, substance P, angiotensin II, norepinephrine, epinephrine, 12‐tetradecanoylphorbol 13‐acetate (TPA), and A23187 calcium ionophore) examined, bradykinin, as well as ATP, TPA, and A23187, significantly increased the phosphorylation of epidermal growth factor (EGF) receptors and reduced the binding of EGF to their high‐affinity site. The reduction in EGF binding by bradykinin, ATP, and TPA was similarly reversed by concomitant incubation with staurosporine, a protein kinase C inhibitor, implying that the phosphorylation of EGF receptors was catalyzed probably by a protein kinase C of the same or similar type in each case. This possibility was confirmed by the fact that the major phosphorylation site of EGF receptors by the stimulation with either bradykinin, ATP or TPA was the same (Thi‐654). Different from the stimulations with ATP and TPA, the effect of bradykinin of decreasing the high‐affinity EGF binding was transient (a minimum binding at 2.5 min); the reduced EGF binding was, however, sustained for up to 30 min in the presence of calyculin A, a phosphoprotein phosphatase inhibitor. Moreover, the homogenate prepared from bradykinin‐stimulated A‐431 cells had stronger dephosphorylation activity for phosphorylated EGF receptors than that from control cells. These results suggest that bradykinin stimulates both the protein kinase C system and a phosphoprotein phosphatase(s) activity in A‐431 cells. Such biphasic effects of bradykinin to phosphorylate and dephosphorylate EGF receptors via protein kinase C and a phosphoprotein phosphatase, respectively, imply a homeostatic control of receptor function in regulating phosphorylation level by the same bioactive factor. © 1993 Wiley‐Liss, Inc.
Vascular smooth muscle cells synthesize two forms of insulin‐like growth factor binding proteins which are regulated differently by the insulin‐like growth factors
Authors: Cohick Wendie S. , Gockerman Amy , Clemmons David R.
Published: February 2005

Vascular smooth muscle cells (SMC) synthesize insulin‐like growth factor‐I (IGF‐I), which is a mitogen for this cell type in vitro. Since IGF binding proteins (IGFBP) modulate IGF bioactivity, we determined which IGFBPs were secreted by porcine SMC. Porcine SMC secreted 34,000 and 24,000 Mr forms of IGFBPs which were identified as IGFBP‐2 and IGFBP‐4, respectively, by immunoblotting. Northern blot analysis showed single transcripts of 1.6 kb and 2.4 kb for IGFBP‐2 and IGFBP‐4, respectively. Secretion of IGFBP‐2 was not regulated to a significant degree, with insulin, IGF‐II, IGF‐I, forskolin, and dibutyryl cyclic adenosine monophosphate (cAMP) inducing minimal changes in IGFBP‐2 secretion of less than 30% by radioimmunoassay (RIA). Insulin increased (2.8 ± 0.1‐fold) the abundance of IGFBP‐4 protein in conditioned media (CM) and increased IGFBP‐4 mRNA levels. Growth factors for SMC such as platelet‐derived growth factor (PDGF), fibroblast growth factor (FGF), epidermal growth factor (EGF), and transforming growth factor beta‐1 (TGFβ‐1) were without effect on either IGFBP‐2 or −4. IGF‐I treatment decreased the amount of IGFBP‐4 present in CM, but a corresponding decrease in IGFBP‐4 mRNA levels was not observed. In order to determine if IGFBP‐4 could modulate IGF‐I bioactivity, IGFBP‐4 was added to pSMCs with and without IGF‐I. IGF‐I alone (20 ng/ml) induced a 1.6 to threefold increase in 3H‐thymidine incorporation. Addition of IGFBP‐4 (between 50 and 250 ng/ml) to cultures containing IGF‐I (20 ng/ml) had no effect on DNA synthesis compared to that observed with IGF‐I alone, while 500 ng/ml consistently caused a small decrease (15 ± 5%; mean ± SE). Immunoblotting of the CM obtained at the end of the 3H‐thymidine assay showed a loss of intact IGFBP‐4 in the cultures containing IGF‐I. This corresponded with an increase in the abundance of a 16,000 Mr immunoreactive fragment that did not bind IGF‐I. Coincubation with insulin had no effect on the amount of IGFBP‐4 that was converted to fragment, suggesting that the reaction was dependent upon IGF‐I binding to IGFBP‐4. In contrast, addition of IGFBP‐4 (500 ng/ml) to human fibroblast cultures with IGF‐I (20 ng/ml) almost completely inhibited the stimulatory effect of IGF‐I on DNA synthesis and no increase in fragment was detected in the CM. In summary, SMC secrete IGFBP‐2 and IGFBP‐4, both of which have been shown to regulate IGF‐mediated DNA synthesis. The factors that regulate the abundance of the two forms of IGFBPs differ suggesting that differential regulation of these substances may be an important mechanism by which SMC growth is regulated. © 1993 Wiley‐Liss, Inc.
Down‐regulation of retinoic acid receptor activity associated with decreased α and γ isoforms expression in F9 embryonal carcinoma cells differentiated by retinoic acid
Authors: Lazega David , Schenker Esther , Busso Nathalie , Zelent Arthur , Chen Alex , Waxman Samuel
Published: February 2005

F9 embryonal carcinoma cells differentiate in response to retinoic acid (RA). To investigate the regulation of RA receptors (RARs) expression during this process, cDNA probes specific for the major RAR isoforms were used. In contrast to the level of RARβ2 mRNA which was high in cells treated 5 days with RA and below detection in untreated cells, as previously described, the steady state levels of RARα1, α2, γl, and γ2 mRNAs were markedly decreased in the RA‐differentiated cells as compared to untreated cells. The down‐regulation of the RA‐responsive system in differentiated cells was also evident in gel shift assays as a marked decrease in binding capacity to a retinoid acid response element (βRARE), as well as in chloramphenicol acetyltransferase (CAT) assays as a sixfold decrease in RA‐mediated transacting activity via this element. The down‐regulation of RAR DNA‐binding and transacting activity coincided with the burst in tissue plasminogen activator secretion and thus, occurred at the hinge between early and late differentiation. The down‐regulation of RA responsiveness may constitute an important event in the transition between early and late differentiation stage in F9 cells. © 1993 Wiley‐Liss, Inc.
Increased epidermal growth factor receptor in an estrogen‐responsive, adriamycin‐resistant MCF‐7 cell line
Authors: Dickstein Bruce , Valverius Eva M. , Wosikowski Katja , Saceda Miguel , Pearson Jack W. , Martin Mary Beth , Bates Susan E.
Published: February 2005

We examined the expression of the estrogen and epidermal growth factor (EGF) receptors in a drug‐resistant subline of MCF‐7 cells in order to study potential alterations in hormone dependence or in the growth factor pathway that could be related to the development of drug resistance in human breast cancer. The drug‐resistant subline was derived from MCF‐7 cells by selection with Adriamycin in the presence of the P‐giycoprotein antagonist, verapamil, to prevent acquisition of the classical multidrug resistance phenotype. The Adriamycin‐resistant cells retain estrogen‐binding, estrogen‐responsive monolayer growth, and estrogen‐dependent tumorigenesis. Estrogen‐binding studies demonstrate 1.4 × 106 sites per cell with unaltered affinity when compared to parental MCF‐7 cells, which have 2.7 × 105 sites per cell. An increase in expression of EGF receptor, eight to 12‐fold, occurred early in the selection for drug resistance, and appears to be unrelated to verapamil exposure, since cells maintained in Adriamycin without verapamil also have increased EGF receptor expression. Partially drug‐sensitive revertants carried a verapamil, but out of Adriamycin, demonstrate a decline in EGF receptor expression. We postulate that activation of growth factor pathways in drug‐resistant cells may enhance mechanisms of drug resistance, or provide mitogenic stimuli for cells to recover after damage by drug exposure. © 1993 Wiley‐Liss, Inc.
Induction of the murine “W phenotype” in long‐term cultures of human cord blood cells by c‐kit antisense oligomers
Authors: Migliaccio Anna Rita , Migliaccio Giovanni , Mancini Giancarlo , Ratajczak Mariusz , Gewirtz Alan M. , Adamson John W.
Published: February 2005

The murine white (W) spotting locus is the site of the c‐kit gene and encodes a tyrosine kinase receptor while the complementary Steel (Sl) iocus encodes its ligand. Mutations at either locus have profound effects on hematopoiesis, particularly erythroid and mast cell proliferation. We added c‐kit antisense oligonucleotides to long‐term suspension cultures of enriched human umbilical cord progenitor cells. This resulted in the suppression of c‐kit gene expression and the preferential suppression of the generation of erythroid burst‐forming cells (BFU‐E) which extended over the life of the culture (3 weeks). The results provide an in vitro model of the “W phenotype” in human hematopoiesis and confirm the importance of c‐kit gene function in early erythropoiesis. Because the generation of BFU‐E was suppressed even after c‐kit gene expression had recovered, this gene product may be critical to the erythroid commitment process. © 1993 Wiley‐Liss, Inc.
Asymmetric origins of the mature glomerular basement membrane
Authors: Lee Larry K. , Pollock Allan S. , Lovett David H.
Published: February 2005

Renal plasma filtration is a critical physiologic function that depends upon the precise composition and arrangement of the constituent extracellular matrix proteins within the glomerular basement membrane (GBM). The GBM develops during renal embryogenesis by the fusion of discrete basement membranes produced independently by endothelial and visceral epithelial cells, and, possibly from matrix secreted by the mesangial cells. In the mature animal, however, the epithelial cell has generally been accepted as the sole source of all GBM constituent proteins. Although the final structures and distributions of the component proteins have been defined by histochemical techniques, the individual contributions of the three resident glomerular cell types to the maintenance and turnover of the mature GBM remain uncertain. We report the application of a new technique, in situ reverse transcription (ISRT), for the localization of RNA transcripts of nine major GBM protein components within the closely apposed cells of the glomerulus. Using this technique, we demonstrate that in normal adult rat glomeruli the RNA transcripts for heparan sulfate proteoglycan and the laminin‐S chain are primarily expressed by visceral epithelial cells, while Type IV α‐1 and α‐2 collagen transcripts were restricted to the endothelial cells in a heterogeneous pattern. RNA transcripts for entactin and the laminin‐A and ‐B2 chains were expressed by all three glomerular cell types, while laminin‐B1 and fibronectin transcripts were limited to the mesangium. These findings demonstrate that GBM synthesis in the mature animal is not restricted to the epithelial cell and that all intrinsic glomerular cells contribute to the production of GBM protein components. The ISRT technique also provided the additional, and unexpected, finding that appreciable synthetic heterogeneity exists within individual glomerular cell types. © 1993 Wiley‐Liss, Inc.
Role of glucose in interleukin‐1β production by lipopolysaccharide‐activated human monocytes
Authors: Orlinska Urszula , Newton Robert C.
Published: February 2005

When monocytes are activated with endotoxin (lipopolysaccharide [LPS]), they make and release several mediators, including interleukin‐1β (IL‐1β). This study was undertaken to investigate the role of glucose in IL‐1β production by these cells. IL‐1β was produced in a dose‐dependent manner to glucose concentration in the culture medium. The uptake of (3H)2‐deoxyglucose in monocytes was stimulated by LPS 1,554% after 10 minutes, 6,095% after 2 hours, then gradually declined after 4 hours of incubation. The inhibition of the uptake of (3H)2‐deoxyglucose by either 10 μM cytochaiasin B or phloretin, added at the time of monocyte activation, was accompanied by significant reduction in ATP/ADP ratio and the inhibition of the production of IL‐1β by activated monocytes. The synthesis of total protein did not change in monocytes activated in the absence of glucose in the culture medium, nor in the presence of either 10 μM cytochalasin B or phloretin. The export of IL‐1β from LPS‐activated monocytes was not inhibited by either 10 μM cytochalasin B or phloretin, nor in the absence of glucose in the culture medium. These data suggest that (1) glucose is required for LPS‐induced IL‐1β production by monocytes; (2) glucose is the major source of ATP for IL‐1β production; (3) glucose transporter (GLUT 1) does not control the export of IL‐1β. © 1993 Wiley‐Liss, Inc.
Recombinant insulin‐like growth factor binding protein‐1 inhibits IGF‐I, serum, and estrogen‐dependent growth of MCF‐7 human breast cancer cells
Authors: Figueroa José A. , Sharma Jivesh , Jackson James G. , McDermott Martin J. , Hilsenbeck Susan G. , Yee Douglas
Published: February 2005

The insulin‐like growth factors (IGFs) are potent mitogens for breast cancer cells and their activity is modulated by high affinity binding proteins (IGFBPs). We have recently shown that IGFBP‐1 purified from human amniotic fluid neutralizes IGF‐I‐dependent growth of MCF‐7 cells. In this study we examined the effects of recombinant IGFBP‐1 (rBP‐1) on IGF‐I, estradiol (E2), and serum‐induced monolayer and anchorage independent growth (AIG) of MCF‐7 cells. Under serum‐free conditions, rBP‐1 had no effect on MCF‐7 basal monolayer growth. However, 40 nM rBP‐1 completely blocked the mitogenic action of both IGF‐I and 5% charcoal stripped serum (CSS). This concentration of rBP‐1 partially inhibited E2‐induced growth, while 80 nM rBP‐1 completely abolished E2 mitogenicity. The addition of either excess IGF‐I or 5 nM [Arg3]IGF‐I, a species that does not bind IGFBPs, neutralized rBP‐1 inhibitory effects. In AIG assays, 80 nM rBP‐1 reduced colony number by at least 70% and decreased colony size in all treatment groups compared to control. We examined rBP‐1 effects on both IGF‐I binding to MCF‐7 membranes and activation of type I IGF receptor (IGFR1) and found that 80 nM rBP‐1 reduced IGF‐I receptor binding to levels of nonspecific binding and completely abolished ligand‐dependent IGFR, phosphorylation. However, neither treatment with 5% CSS nor exposure to E2 resulted in IGFR1 phosphorylation suggesting that different mechanism(s) are responsible for rBP‐1 inhibitory action under this condition. Our data suggest rBP‐1 may serve as an antagonist of human breast cancer growth by interfering with growth factor‐mediated cell proliferation. © 1993 Wiley‐Liss, Inc.
Cell substratum modulates responses of preosteoblasts to retinoic acid
Authors: Traianedes K. , Ng K. W. , Martin T. J. , Findlay D. M.
Published: February 2005

The aim of this study was to determine the role of ECM components of bone in regulating the differentiation and function of cells of the osteoblast lineage. Rat UMR 201 cells, phenotypically preosteoblast, were plated onto plastic tissue culture dishes or dishes coated with gelled type I collagen or reconstituted basement membrane (matrigel). Acute cell attachment assays showed that cells adhered to substrates in the following order: collagen > matrigel ≫ plastic. Proliferation rate up to 96 hr were similar on each substrate. However, if cells were treated with 10−6 M retinoic acid (RA), proliferation rates were reduced compared with control for cells grown on collagen and matrigel but not on plastic. Morphological changes were matrix‐specific; in subconfluent cultures, long thin processes were seen with cells grown on collagen and a pattern of interconnecting cell processes formed when cells were plated on matrigel. Striking differences were observed in the constitutive or RA‐induced gene expression of cells grown on the different substrates. When cells plated on collagen were treated with RA, induction of mRNA for alkaline phosphatase (ALP) as well as ALP enzyme activity were much less than with cells grown on plastic. In contrast, RA treatment induced osteopontin (OP) mRNA expression more strongly in cells plated on collagen compared with plastic within 24 hr and this was maintained for 72 hr. RA treatment produced a two fold increase of pro‐α 1(I) collagen mRNA in cells grown on plastic and matrigel but not in cells grown on collagen. Growth on collagen produced changes in the way UMR 201 cells responded to RA from which they did not fully recover in subsequent 48‐hr growth periods on plastic. These results indicate that ECM components regulate the function of and are capable of modulating RA‐induced differentiation of preosteoblasts. © 1993 Wiley‐Liss, Inc.