bluuebunny/crossref_metadata_2025 huggingface dataset:
Investigation of possible autocrine functions for rat GRO/CINC (cytokine‐induced neutrophil chemoattractant)
Authors: Crippes Barbara A. , Zagorski John , Carr Linda S. , Wittwer Art J. , Dolecki Gregory J. , De Larco Joseph E.
Published: February 2005

Rat cytokine‐induced neutrophil chemoattractant (CINC) is an eight kilodalton polypeptide originally purified from media conditioned by interleukin‐1β stimulated 52E, an epitheloid clone derived from normal rat kidney (NRK) cells. Using a fibroblastic clone of the NRK cells, 49F, we found expression of the CINC gene to be induced by either serum or cytokines in growth‐arrested cultures within 1 hour of stimulation. There was no observable CINC expression in exponentially growing cells in the absence of cytokine stimulation. CINC protein had no significant effect on 3H‐thymidine incorporation or growth rate of NRK49F. We have observed that CINC is constitutively produced by some transformed NRK cells, clone RC20, suggesting an association with the expression of a transformed phenotype. Unlike the parent 49F, RC20 cells are capable of growth in soft agar and serum‐free media and form highly metastatic tumors in nude mice. We have examined the possible autocrine functions of CINC and its possible links to the expression of the transformed phenotype by these cells. The use of a blocking CINC polyclonal antibody demonstrated that CINC did not function as an autocrine growth factor for RC20. Though CINC is a potent chemoattractant for neutrophils, it did not induce migration of either RC20 or 49F cells. CINC only moderately promoted adhesion of RC20 cells when used as a matrix protein. These data do not support the hypothesis that production of CINC by the RC20 cells provides an obvious advantage for the transformed cells constitutively producing it. © 1993 Wiley‐Liss, Inc.
Expression of transforming growth factor α antisense mRNA inhibits the estrogen‐induced production of TGFα and estrogen‐induced proliferation of estrogen‐responsive human breast cancer cells
Authors: Kenney N. J. , Saeki T. , Gottardis M. , Kim N. , Garcia‐Morales P. , Martin M. B. , Normanno N. , Ciardiello F. , Day A. , Cutler M. L. , Salomon D. S.
Published: February 2005

To ascertain if 17β‐estradiol (E2)‐induced proliferation could be attenuated by blocking the expression of endogenous transforming growth factor α (TGFα), estrogen receptor (ER)‐positive, estrogen‐responsive MCF‐7 or ZR‐75‐1 cells and ER‐negative, estrogen‐nonresponsive MDA‐MB‐468 or HS‐578T cells were infected with a recombinant amphotropic, replication‐defective retroviral expression vector containing a 435 base pair (bp) Apa1‐Eco R1 coding fragment of the human TGFα cDNA oriented in the 3′ to 5′ direction and under the transcriptional control of an internal heavy metal‐inducible mouse metallothionein (MT‐1) promoter and containing the neomycin (neo) resistance gene. E2‐stimulated expression of endogenous TGFα mRNA was inhibited by 4–5‐fold, and the production of TGFα protein was inhibited by 50–80% when M‐1 mass‐infected MCF‐7 or MZ‐1 mass‐infected ZR‐75‐1 cells were treated with 0.75‐1 μM CdCl2, whereas in comparably treated parental MCF–7 or ZR‐75‐1 cells there was no significant effect upon these parameters. E2‐stimulated anchorage‐dependent growth (ADG) and anchorage‐independent growth (AIG) of the M‐1 or MZ‐1 cells was inhibited by 60–90% following CdCl2 treatment. In contrast, neither the ADG nor AIG of the parental noninfected MCF‐7 or ZR‐75‐1 cells that were maintained in the absence or presence of E2 was affected by comparable concentrations of CdCl2. The ADG and AIG of TGFα antisense MD‐1 mass‐infected MDA‐MB‐468 cells that express high levels of endogenous TGFα mRNA were also inhibited by 1 μM CdCl2, whereas the ADG and AIG of MH‐1 mass‐infected HS‐578T cells, a TGFα‐negative cell line, were unaffected by CdCl2 treatment. These results suggest that TGFα may be one important autocrine intermediary in regulating estrogen‐induced cell proliferation. © 1993 Wiley‐Liss, Inc.
Amiloride‐dependent transport is the main mechanism implicated in sodium iinflux regulation in rat mast cells
Authors: Cabado Ana G. , Vieytes Mercedes R. , Botana Luis M.
Published: February 2005

Mast cell sodium regulation is a largely unknown field. In our effort to study the mechanisms by which mast cells regulate sodium levels, we have examined the effect of amiloride and ouabain on 22Na entry in rat mast cells in isotonic and hypertonic conditions. Ouabain (0.5 mM) enhances sodium uptake by 32% in isotonic conditions. Hypertonicity increases by 400% the uptake of sodium through an amiloride (1 mM) dependent mechanism. Ouabain has no appreciable effect on the entry of 22Na in hypertonic conditions. © 1993 Wiley‐Liss, Inc.
Apotransferrin stimulation of thyroid hormone dependent rat pituitary tumor cell growth in serum‐free chemically defined medium: Role of FE(III) chelation
Authors: Eby John E. , Sato Hidetaka , Sirbasku David A.
Published: February 2005

Triiodothyronine (T3) dependent growth of GH1 rat pituitary tumor cells in serum‐free defined culture requires apotransferrin (apo Tf) (Sirbasku et al.: Mol. Cell. Endocrinol., 77:C47–C55, 1991). Diferric transferrin (2Fe · Tf) also is necessary as an iron source (Eby et al.: Anal. Biochem., 203:317–325, 1992). Further, T3 dependence is prevented by soluble Fe(III) addition to the medium (Sato et al.: In Vitro Cell. Dev. Biol., 27A:599–602, 1991). While our data suggested that apo Tf caused growth by chelation of Fe(III), direct evidence was required. We used urea polyacrylamide gel electrophoresis along with autoradiography and Western immunoblotting to measure the Fe(III) content of growing GH1 cell cultures and identify the apo Tf, mono‐metal transferrins and 2Fe · Tf present. We found that apo Tf per se did not cause growth but instead chelated inhibitory levels of Fe(III). In fact, apo Tf need not be present at all provided that Fe(III) is reduced to ≤0.6 μM. In addition, other protein and non‐protein Fe(III) chelators were shown to be as effective as apo Tf. Here, we report that pituitary cells are completely inhibited by ≥1.2 μM Fe(III), which are concentrations which might be expected in many culture media and usually are not thought to influence growth. The high sensitivity of pituitary cells to Fe(III) suggests further study to determine what cellular functions are affected and how they interfere with thyroid hormone dependence. © 1993 Wiley‐Liss, Inc.
Density‐dependent inhibitory effect of transforming growth factor‐β on human fibroblasts involves the down‐regulation of platelet‐derived growth factor α‐receptors
Authors: Paulsson Y. , Karlsson C. , Heldin C.‐H. , Westermark B.
Published: February 2005

We have previously found that transforming growth factor‐β1 (TGF‐β1) inhibits the mitogenic activity of platelet‐derived growth factor (PDGF) in cultures of human neonatal fibroblasts in a density‐dependent fashion. In the present investigation we determined the effect of TGF‐β1 on the PDGF α‐receptor, which binds all PDGF isoforms, as well as on the β‐receptor, which binds only PDGF‐BB with high affinity. We found that the inhibitory effect of TGF‐β1 on PDGF‐AA‐induced mitogenesis was density‐dependent; when dense cell cultures were preincubated with TGF‐β1, there was an complete inhibition of 3H‐thymidine incorporation, whereas the effect was less in sparse cultures. A similar density‐dependent effect of TGF‐β1 was seen in PDGF‐BB treated cells, although less pronounced. The binding of 125I‐labeled PDGF‐AA and PDGF‐BB to the α‐receptor was significantly reduced after treatment with TGF‐β1 in dense cultures, whereas the sparse cultures were less affected. A decrease of α‐receptor mRNA was also seen. The levels of β‐receptor protein and mRNA were unaffected. We conclude that the growth inhibitory effect of TGF‐β1 is cell density‐dependent and involves down‐regulation of PDGF α‐receptors. © 1993 Wiley‐Liss, Inc.
Investigation of the ability of several naturally occurring and synthetic polyanions to bind to and potentiate the biological activity of acidic fibroblast growth factor
Authors: Belford David A. , Hendry Ian A. , Parish Christopher R.
Published: February 2005

The ability of several animal, plant, and bacterial derived polyanions (PAs) as well as synthetic PAs to compete with heparin for the binding of acidic fibroblast growth factor (aFGF) was correlated with their ability to potentiate the mitogenic and neurotrophic actions of this factor. Dextran sulphate, K‐carrageenan, pentosan sulphate, polyanethole sulfonate, heparin, and fucoidin competed for the heparin binding site on aFGF at relatively low concentrations (≤50 μg/ml). λ‐carrageenan, ι‐carrageenan, and polyvinyl sulphate exhibited lower affinity for aFGF, whereas hyaluronic acid, dermatan sulphate, chondroitin‐6‐sulphate, chondroitin‐4‐sulphate, and uncharged dextran displayed very low or no demonstrable affinity. Potentiation of the mitogenic action of aFGF for Balb/c 3T3 fibroblasts tended to be in general agreement with the aFGF binding affinity of the PAs. However, polyanethole sulfonate, the carrageenans, polyvinyl sulphate, fucoidin, and pentosan sulphate exerted a mitogenic action on the 3T3 cells that was independent of, and in addition to, the ability of these GAGs to potentiate the action of aFGF. The ability to potentiate the neurotrophic action of aFGF for E8 chick ciliary neurons was a general property of those PA with low or no activity in the mitogen assay. Thus hyaluronic acid, dermatan sulphate, chondroitin‐4‐sulphate, chondroitin‐6‐sulphate, and even uncharged dextran all potentiated aFGF induced neuronal survival. The differential effects of these PA in potentiating the biological activities of aFGF are discussed in relation to their ability to compete for the heparin‐binding site of aFGF. © 1993 Wiley‐Liss, Inc.
Involvement of the conserved acidic amino acid domain of FGF receptor 1 in ligand‐receptor interaction
Authors: Chaudhuri Murari M. , Moscatelli David , Basilico Claudio
Published: February 2005

The fibroblast growth factor receptor 1 (flg) contains eight acidic amino acids between the first and second immunoglobulin domain. This report examines the role of the acidic domain in the interaction of the flg receptor with its ligands. We observed a marked inhibition of binding of bFGF to the receptor when the acidic domain was completely deleted, but mutants with two and four amino acids deleted (flgΔ2 and flgΔ4, respectively) still bound the ligand. After addition of a bifunctional cross‐linking reagent, cross‐linked complexes (between bFGF and receptor) with the expected size were observed in cells expressing mutants lacking two or four acidic residues, but not in cells expressing mutants lacking six or eight acidic residues. Immunoprecipitation with anti‐flg antibody followed by electrophoresis produced a band of 90 Kd in tunicamycin‐treated cells expressing the mutant as well as the wild‐type receptors, indicating that the inhibition of binding was not due to defective expression of the protein. The ability of flgΔ8 to mediate a mitogenic response to FGFs was also greatly reduced when this mutated receptor was expressed in receptor‐negative cells. The effect of replacing the acidic amino acids with lysine residues was also studied. Binding of bFGF to cells transfected with a plasmid encoding a mutated protein with four amino acid substitutions was totally inhibited, but an eight amino acid substitution did not alter ligand binding to the receptor. In this case the mutation with four amino acids substitution caused a drastic impairment of protein expression. Thus the acidic domain of the FGFR‐1 plays an essential role in receptor function, either because it is important for a stable protein configuration or for ligand‐receptor interaction. © 1993 Wiley‐Liss, Inc.
Inhibitors of vacuolar‐type H+‐ATPase suppresses proliferation of cultured cells
Authors: Manabe Tsukasa , Yoshimori Tamotsu , Henomatsu Nobuhiro , Tashiro Yutaka
Published: February 2005

We investigated effects of bafilomycin A1, a specific inhibitor of vacuolar‐type H+‐ATPase (V‐ATPase), and its analogues on proliferation of various cultured cells. The proliferation of the various cell lines was suppressed by adding bafilomycin A1 to the culture medium. This inhibitory effect appeared at a concentration of nanomolar order and was dose dependent. Although the suppression was reversible, the drug exerted not only suppression of the proliferation but also death to some cell lines. Drug concentration required for 50% inhibition of the cell proliferation during 48 h differed markedly depending on cell species and the sensitivity appears to increase by the transformation of the cells. Two derivatives of concanamycin A, an analogue of bafilomycin A1, also inhibited strongly V‐AT‐Pase in vitro and in vivo, and simultaneously cell proliferation. Two concanamycin A derivatives which have lost inhibitory effect on V‐ATPase lost inhibitory effect on cell proliferation as well. These results suggest that V‐ATPase is involved in the machinery maintaining the cell proliferation. © 1993 Wiley‐Liss, Inc.
Polyamine transport regulation by calcium and calmodulin: Role of Ca2+‐ATPase
Authors: Khanp Naim A. , Sezan Alphonse , Quemener Véronique , Moulinoux Jacques‐Ph
Published: February 2005

The study was conducted on human leukemia (K 562) cells to characterize the mechanisms implicated in the regulation of the polyamine spermicine (Spd) transport process. The antagonists of calmodulin, trifluoperazine (TFP), W‐7 (N‐[6‐aminohexyl]‐5‐chloro‐1‐naphthelenesulfonamide), or mellitin inhiblted significantly polyamine Spd uptake in these cells. The translocation of calmodulin towards plasma membrane and a concomitant decrease in its contents in cytosol were directly correlated with the time course increases similar to that of Spd uptake, indicating that calmodulin is recruited towards plasma membrane during the Spd transport process. Diminution of free intracellular calcium, (Ca2+)i, by preincubating the cells in BAPTA (bis[2‐amino‐5‐methylphenoxyl]‐ethane‐N,N′,N′,‐tetraacetate) buffer inhibited Spd transport significantly. Addition of lanthanum (LAN), a molecule known to inhibit Ca2 efflux via Ca2+‐ATPase, curtailed Spd uptake by these cells. LAN inhibited Vmax, but not the Km, of Spd uptake, indicating that the former does not directly interact with the polyamine transporter; rather it regulates the transport process, probably via its action on Ca2+‐ATPase. Calmodulin‐stimulated uptake of 45Ca2+ by inside‐out vesicles of K 562 cells, a measure of Ca2+‐ATPase activity. Furthermore, addition of LAN inhibited both basal and calmodulin‐stimulated activity of Ca2+‐ATPase. Thapsigargin (THAP), a molecule known to elevate (Ca2+) i due to its action on the endoplasmic reticulum, increased Spd transport whereas addition of LAN inhibited THAP‐stimulated Spd transport activity. THAP increased free (Ca2+)i in these cells, and a pre‐addition of LAN to these cells curtailed the THAP‐stimulated increases of (Ca2+)i concentrations. Addition of Spd brought about elevations in (Ca2+)i contents. Caffeine also increased (Ca2+)i in these cells; however, it failed to stimulate significantly the Spd uptake process, indicating that (Ca2+)i which is involved in the regulation of polyamine transport pathways does not belong to the calcium‐induced calcium‐release (CICR) pool. Replacement of Ca2+ from the incubation medium (i.e., 0% Ca2+) resulted in higher uptake activity as compared to that in 100% Ca2+ medium, demonstrating that in 100% Ca2+ medium the calcium efflux process is quickly compensated by calcium refilling/influx from the extracellular medium, while in 0% Ca2+ medium there is perpetual efflux of (Ca2+)i which contributes to higher Spd uptake process. The results of this study suggest that an increase in free (Ca2+)i and its release from the cells via Ca2+ ATPase, and concomitant activation of calmodulin, which controls Ca2+‐pump activity, are involved in the regulation of the Spd uptake process in human leukemia cells. © 1993 Wiley‐Liss, Inc.
Endothelial cell hypoxia associated proteins are cell and stress specific
Authors: Graven Krista K. , Zimmerman Leslie H. , Dickson Eric W. , Weinhouse Gerard L. , Farber Harrison W.
Published: February 2005

Vascular endothelial cells (EC) are one of the initial cells exposed to decreases in blood oxygen tension. Bovine EC respond not only by altering secretion of vasoactive, mitogenic, and thrombogenic substances, but also by developing adaptive mechanisms in order to survive acute and chronic hypoxic exposures. EC exposed to hypoxia in vitro upregulate a unique set of stress proteins of Mr 34, 36, 39, 47, and 56 kD. Previous studies have shown that these proteins are cell associated, upregulated in a time and oxygen‐concentration dependent manner, and are distinct from heat shock (HSPs) and glucose‐regulated proteins (GRPs). To further characterize these hypoxia‐associated proteins (HAPs), we investigated their upregulation in human EC from various vascular beds and compared this to possible HAP upregulation in other cell types. Human aortic, pulmonary artery, and microvascular EC upregulated the same set of proteins in response to hypoxia. In comparison, neither lung fibroblasts, pulmonary artery smooth muscle cells, pulmonary alveolar type II cells, nor renal tubular epithelial cells upregulated proteins of these Mr. Instead, most of these cell types induced synthesis of proteins of Mrs corresponding to either HSPs, GRPs, or both. Further studies demonstrated that exposure of EC to related stresses such as cyanide, 2‐deoxyglucose, hydrogen peroxide, dithiothreitol, and glucose deprivation did not cause upregulation of HAPs. Evaluation of cellular damage during hypoxia using phase‐contrast microscopy, trypan blue exclusion, chromium release, and adherent cell counts showed that EC survived longer with less damage than any of the above cell types. The induction of HAPs, and the lack of induction of HSPs or GRPs, by EC in response to hypoxia may be related to their unique ability to tolerate hypoxia for prolonged periods. © 1993 Wiley‐Liss, Inc.
Phorbol 12‐myristate 13‐acetate stimulates proliferation and ductal morphogenesis and inhibits functional differentiation of normal rat mammary epithelial cells in primary culture
Authors: Wada Tsutomu , Darcy Kathleen M. , Guan Xiaoping , Ip Margot M.
Published: February 2005

The effect of the tumor promoter phorbol 12‐myristate 13‐acetate (PMA) on proliferation and differentiation of normal mammary epithelial cells from 50‐day‐old virgin rats was investigated using a model system that allows for full morphological and functional development of the cells. In this model, mammary epithelial cells are grown within a reconstituted basement membrane in a defined serum‐free medium. PMA at a concentration of 10−6 M effected translocation of protein kinase C from cytosol to membrane. At the same concentration, it stimulated cell proliferation both in the presence and absence of EGF, and this stimulation was observed even when PMA exposure was limited to 15 min at the time of each media change. In contrast to the effect on proliferation, PMA at concentrations of 10−7 and 10−6 M inhibited functional differentiation as assessed by casein accumulation. Phorbol 12,13‐dibutyrate at 10−6 M also stimulated proliferation and inhibited casein accumulation and was more effective than PMA in both cases. In contrast, the nonactive tumor promoter 4‐α PMA had no effect on either proliferation or differentiation. One of the most striking effects of PMA was its ability to stimulate an atypical ductal morphogenesis, as manifested by the formation of intricate web‐like colonies, and to inhibit the development of the well‐differentiated alveolar‐like multilobular colonies. PMA was also shown to completely suppress the growth of the squamous‐like colonies that develop when EGF is absent or deficient. These effects of phorbol esters in mammary epithelial cells to stimulate proliferation, inhibit functional differentiation, and stimulate the development of ductal colonies are consistent with the suggestion that the signal transduction pathways evoked by PMA could act to stimulate the growth of initiated cells or render normal cells more sensitive to carcinogen. © 1994 Wiley‐Liss, Inc.
Calphostin‐C stimulates epidermal growth factor receptor phosphorylation and internalization via light‐dependent mechanism
Authors: Gamou Shinobu , Shimizu Nobuyoshi
Published: February 2005

Calphostin‐C with perylenequinone structure is known to bind the regulatory domain of protein kinase C (PKC) and to inhibit kinase activity in vitro in a light‐dependent fashion. We have found that calphostin‐C induces substantial serine and threonine phosphorylation of the epidermal growth factor (EGF) receptor in a light‐dependent fashion in the EGF receptor‐hyperproducing squamous carcinoma cell line NA. Tryptic phospho‐peptide mapping and phospho‐amino acid analysis revealed that calphostin‐C–‐enhanced phosphorylation was on threonine 669, serine 671, serine 1046/1047, and serine 1166. However, caiphostin‐C did not inhibit phosphorylation of the 80 K protein, a cytosolic major substrate of PKC (MARCKS). Staurosporine, a potent PKC inhibitor with affinity for the catalytic domain of PKC, inhibited phosphorylation of the 80 K protein and 12‐O‐tetradecanoyl‐13‐phorbol acetate induction of EGF receptor phosphorylation but did not inhibit the calphostin‐C induction of the EGF receptor phosphorylation. These results suggest that the target of calphostin‐C in vivo is different from that of staurosporine and thus calphostin‐C in vivo does not inhibit PKC. Furthermore, calphostin‐C enhanced the internalization of phosphorylated EGF receptor. Thus, calphostin‐C apparently activates a novel signal transduction pathway which involves phosphorylation and internalization of the EGF receptor via light‐dependent mechanism. © 1994 Wiley‐Liss, Inc.
Stimulation of hepatocyte DNA synthesis by neurotensin
Authors: Hasegawa Kaoru , Kar Siddhartha , Carr Brian I.
Published: February 2005

Epidermal growth factor and transforming growth factor alpha stimulated DNA synthesis in primary cultures of adult rat hepatocytes. Neurotensin amplified epidermal growth factor‐stimulated or transforming growth factor alpha‐stimulated DNA synthesis by three‐ to eightfold. Neurotensin by itself did not stimulate DNA synthesis. Amplification of DNA synthesis by neurotensin was observed as low as 10−10 M, and it was increased in a dose‐dependent manner with maximal effects at 10–8 M. These results were obtained when hepatocytes were cultured in Williams' medium E, but not in Leibovitz L‐15 medium, suggesting that a minor component(s) in the medium is required for hepatocytes to fully respond to neurotensin. Neurotensin effect on DNA synthesis was observed not only in normal rat hepatocytes but also in partially hepatectomized rat hepatocytes, although its effect was stronger in normal hepatocytes. Amplified DNA synthesis was inhibited by transforming growth factor β. Secondary mitogens (co‐mitogens) such as insulin, vasopressin, or angiotensin II interacted additively with low concentrations of epidermal growth factor as well as with neurotensin. Neurotensin‐related peptides such as kinetensin or neuromedin‐N, which was released from blood plasma by pepsin digestion, did not have this amplifying effect on DNA synthesis at any concentrations tested. Neurotensin mRNA was found in several organs including brain and intestine, but not liver. These results suggest that neurotensin can be regarded as a new secondary mitogen and that it may be involved in cell proliferation, including regenerating liver as a gastrointestinal hormone and/or a neurotransmitter. © 1994 Wiley‐Liss, Inc.
Consequences of aberrant ornithine decarboxylase regulation in rat hepatoma cells
Authors: Tome Margaret E. , Fiser Steven M. , Gerner Eugene W.
Published: February 2005

DH23A cells, an α‐difluoromethylornithine (DFMO)–resistant variant of rat hepatoma tissue culture cells (HTC), contain high levels of very stable ornithine decarboxylase (ODC). In the absence of DFMO, the high ODC activity results in a large accumulation of endogenous putrescine. Concomitant with the putrescine increase is a period of cytostasis and a subsequent loss of viable cells. In contrast, HTC cells with a moderate polyamine content can be maintained in exponential growth. This suggests that a moderate polyamine concentration is necessary for both optimal cell growth and survival. The cytoxicity observed in the DH23A cells is apparently not due to byproducts of polyamine oxidation or alterations in steady state intracellular pH or free [Ca2+]. It is possible to mimic the effects of high levels of stable ODC by treatment of cells with exogenous putrescine in the presence of DFMO. This suggests that overaccumulation of putrescine is the causative agent in the observed cytotoxicity, although the mechanism is unclear. These data support the hypothesis that downregulation of ODC may be necessary to prevent accumulation of cytotoxic concentrations of the polyamines. © 1994 Wiley‐Liss, Inc.
Loss of cytotoxic effect of epidermal growth factor (EGF) on EGF receptor overexpressing cells is associated with attenuation of EGF receptor tyrosine kinase activity
Authors: Gardner David P. , Shimizu Nobuyoshi
Published: February 2005

The biological activity of epidermal growth factor (EGF) is mediated through the intrinsic tyrosine kinase activity of the EGF receptor (EGFR). In numerous cell types, binding of EGF to the EGFR stimulates the tyrosine kinase activity of the receptor eventually leading to cell proliferation. In tumor‐derived cell lines, which overexpress the EGFR, however, growth inhibition is often seen in response to EGF. The mechanism for growth inhibition is unclear. To study the relationship between growth inhibition and EGFR kinase activity, we have used a cell line (PC‐10) derived from a human squamous cell carcinoma that overexpresses EGFR. When exposed to 25 ng/ml EGF at low cell densities (1,300 cells/cm2), PC‐10 cells exhibit cell death. In contrast, if EGF is added to high density cultures, no EGF mediated cell death is seen. When PC‐10 cells were maintained at confluency in the presence of 25 ng/ml EGF for a period of 1 month, they were subsequently found competent to proliferate at low density in the presence of EGF. We designate these cells APC‐10. The APC‐10 cells exhibited a unique response to EGF, and no concentration of EGF tested could produce cell death. By 125I‐EGF binding analysis and [35S]methionine labeling of EGFR, it was found that the total number of EGFR on the cell surface of APC‐10 was not decreased relative to PC‐10. No difference between PC‐10 and APC‐10 was seen in EGF binding affinity to the EGFR. Significantly, EGF stimulated autophosphorylation of the EGFR of APC‐10 was 8–10‐fold lower than that of PC‐10. This reduced kinase activity was also seen in vitro in membrane preparations for EGFR autophosphorylation as well as phosphorylation of an exogenously added substrate. No difference between PC‐10 and APC‐10 in the overall pattern of EGFR phosphorylation in the presence or absence of EGF was detectable. However, the serine and threonine phosphorylation of the EGFR of APC‐10 cells was consistently 2–3‐fold lower than that seen in PC‐10 cells. These results suggest a novel mechanism for EGFR overexpressing cells to survive EGF exposure, one that involves an attenuation of the tyrosine kinase activity of the EGFR in the absence of a change in receptor levels or receptor affinity. © 1994 Wiley‐Liss, Inc.
Age related induction of platelet‐derived growth factor A‐chain mRNA in normal human fibroblasts
Authors: Karlsson Christina , Paulsson Ylva
Published: February 2005

We have previously found that stimulation of normal neonatal fibroblasts with PDGF or EGF leads to a transient induction of PDGF A‐chain mRNA and the synthesis of PDGF‐AA proteins. This finding may imply the existence of an autocrine feedback mechanism to amplify the mitogenic signal under certain conditions. We have now studied the PDGF‐BB mediated PDGF A‐chain induction in a set of fibroblasts from young and old donors to clarify if the levels of induction are correlated to the donor age and the replicative capacity of the cells. The PDGF A‐chain induction was found to be reduced in cells from old donors compared with cells from embryonic and neonatal donors. The different cell strains were also characterized further with respect to PDGF receptor expression and PDGF binding properties. PDGF β‐receptors were found to be enhanced in old donor cells strains, whereas the PDGF α‐receptors showed more variability in expression between the strains. The PDGF A‐chain mRNA induction was also decreased or absent in late passage human fibroblasts (senescent cells) when compared with early passage cells. These data suggest that the PDGF A‐chain mRNA induction is regulated by an age related mechanism in human fibroblasts. © 1994 Wiley‐Liss, Inc.
Effects of fibronectin on actin organization and respiratory burst activity in neutrophils, monocytes, and macrophages
Authors: Yang Kuender D. , Augustine Nancy H. , Shaio Men‐Fang , Bohnsack John F. , Hill Harry R.
Published: February 2005

Previous studies have shown that fibronectin (Fn) enhances phagocytosis and killing of antibody‐coated bacteria by neutrophils and macrophages. In an attempt to understand the mechanism of this enhancement, we have investigated the effects of Fn on phagocytosis‐related actin organization as well as respiratory burst activity in neutrophils, monocytes and culture‐derived macrophages. Employing an NBD‐phallacidin flow cytometric analysis of filamentous actin formation, we found that Fn promotes rapid actin polymerization within 30 seconds in neutrophils, monocytes, and macrophages, but not lymphocytes. Enhancement of actin polymerization by Fn was concentration‐dependent and mediated by a pertussis toxin‐ but not cholera toxin‐ sensitive G protein. Inhibition of protein kinase C by sphingosine (20 μM), calcium influx by verapamil (0.1 mM), or intracellular calcium mobilization by 8‐(N, N‐diethyl‐amino) octyl‐3,4,5‐trimethoxybenzoate HCI (TMB‐8; 0.1 mM) did not block Fn‐enhanced actin polymerization in phagocytes. Incubation of neutrophils and macrophages on microtiter plates precoated with Fn suppressed superoxide (O2−) production induced by IgG‐ and IgA‐ opsonized group B streptococci. In contrast, Fn significantly enhanced IgA‐ and IgG‐mediated O2− production by freshly isolated monocytes. These data suggest that Fn enhances phagocytosis, presumably through G protein‐coupled cytoskeleton reorganization and augments O2− production by circulating monocytes. In contrast, it appears to suppress O2− production by the active phagocytic cells, neutrophils and macrophages. This may result in enhanced phagocytosis and intracellular killing of microorganisms without damaging interstitial tissues. © 1994 Wiley‐Liss, Inc.
Up‐regulation of aFGF expression in quiescent cells is related to cell survival
Authors: Renaud F. , Oliver L. , Desset S. , Tassin J. , Romquin N. , Courtois Y. , Laurent M.
Published: February 2005

Exogenously administrated acidic FGF modulates the proliferation of several cell types, controls cell differentiation, and promotes cell survival. Most cells that are sensitive to exogenous aFGF are also capable of expressing it at very low levels. Thus in order to establish the role of endogenous aFGF as a mitogenic, differentiation, or survival factor, we studied the regulation of aFGF expression by evaluating the level of mRNA by PCR amplification and the concentration of protein by Enzyme Immuno Assay (EIA). In the lens, the amount of aFGF transcripts in nondividing cells of the central epithelium and in the differentiated fiber cells located at the periphery of the lens is similar, suggesting that endogenous aFGF is not involved with lens differentiation. In cultures, depending on the growth conditions, the endogenous aFGF expressed by Bovine Epithelial Lens (BEL) cells is sub ect to modulation. Cells arrested either by contact inhibition or by serum deprivation express more aFGF transcripts and protein than in exponentially growing cells, implying that endogenous aFGF has no mitogenic role under these conditions. In serum‐deprived cells, the addition of specific aFGF antisense primers inhibits endogenous aFGF express on and leads to the death of these cells. These results associated with the higher expression of aFGF in nondividing BEL cells, suggesting that, contrary to exogenous aFGF, endogenous aFGF is not a mitogenic factor but a survival factor.
Mitosis and cytokinesis in subconfluent endothelial cells exposed to increasing levels of shear stress
Authors: Wechezak Arlene R. , Viggers Robert F. , Coan Daniel E. , Sauvage Lester R.
Published: February 2005

An in vitro flow apparatus in combination with cultured endothelium was used to determine the effects of fluid‐generated shear stress on cells undergoing mitosis and cytokinesis. Cell responses were recorded by time‐lapse video microscopy under phase contrast or Hoffman modulation contrast optics. Completion of cell division in mitotic cells was dependent upon both the initial presence of intercellular attachments and the magnitude of fluid wall shear stress. In nonisolated populations, 95.3%, 69.5%, and 57.1% of the cells completed cell division as opposed to 66.6%, 20.4%, and 11.9% in the isolated cell groups at 2.8, 14.1, and 33 dynes/cm2, respectively. Prestressing cells for 14 h prior to monitoring failed to increase retention of isolated mitotic cells. The presence of neighboring cells facilitated replication by providing an anchoring attachment or a luminal surface for completion of division. Cell detachment most commonly occurred at the onset of cytokinesis when substrate contact areas were minimal and focal contacts were absent. A comparison between no flow controls and shear stress specimens indicated no significant differences in transit times for mitosis and cytokinesis. Thus, subconfluent endothelial cells may be more susceptible to detachment during cell division due to increases in shear stress, the absence of intercellular attachments, and reduced cell‐substrate contacts. © 1994 wiley‐Liss, Inc.
Growth regulation of human glioblastoma t98g cells by insulin‐like growth factor‐1 and its receptor
Authors: Ambrose Diane , Resnicoff Mariana , Coppola Domenico , Sell Christian , Miura Masahiko , Jameson Steve , Baserga Renato , Rubin Raphael
Published: February 2005

The interaction of insulin‐like growth factors (IGFs) with the IGF‐1 receptor is an important step in the control of cell proliferation and development. In particular, IGF‐1 and IGF‐2 are key regulators of central nervous system development, and may modulate the growth of glial tumors. We have investigated the growth factor regulation of the human glioblastoma cell line T98G. These cells growth arrested in serum‐free medium at 34°C, despite their secretion of substantial amounts of bioactive IGF‐1. To be stimulated to divide, growth‐arrested cells required the addition of platelet‐derived growth factor (PDGF) or its equivalent, 1% serum. Cell proliferation in serum‐free medium could also be obtained by shifting the cells to a temperature of 39.6°C. Treatment of growth‐arrested cells with PDGF or temperature shift was accompanied by a transient increase in the expression of the mRNA for the IGF‐1 receptor. Transfection with a plasmid constitutively expressing the full cDNA for the human IGF‐1 receptor allowed autonomous growth in serum‐free medium at 34°C. By contrast, growth induction by growth factors or temperature shift was abrogated by transfection of the cells with a plasmid expressing a 300 bp segment of mRNA antisense to the IGF‐1 receptor mRNA. Cloning in soft agar was also inhibited by expression of antisense IGF‐1 receptor mRNA. These results demonstrate that the IGF‐1 receptor is strictly required for the growth of T98G glioblastoma cells. Moreover, the autocrine interaction of IGF‐1 with its receptor regulates both autonomous and anchorage‐independent growth of these cells. © 1994 wiley‐Liss, Inc.
Synthesis of and response to 1,25 dihydroxycholecalciferol by subpopulations of murine epidermal keratinocytes: Existence of a paracrine system for 1,25 dihydroxycholecalciferol
Authors: Rizk‐Rabin Marthe , Zineb Rougui , Zhor Bouizar , Michele Garabedian , Jana Pavlovitch
Published: February 2005

The epidermis is both a target tissue for and a source of 1,25 dihydroxycholecal‐ciferol. The present study determines which of the epidermal cell populations synthesizes 1,25 dihydroxycholecalciferol and which responds to this hormone. Epidermal keratinocytes from new born rat epidermis were separated by unit gravity sedimentation into poorly differentiated cells, slow‐cycling more differentiated cells, actively proliferating cells, and terminally differentiating subpopulations. The keratinocyte populations were characterized by cell size analysis, cell morphology, and DNA and RNA contents (acridine orange flow cytometry). 1,25(OH)2D3 synthesis was studied by measuring the conversion of [3H] 25(OH)D3 to [3H] 1,25(OH)2D3. The purified product was tested for its ability to compete with synthetic [3H] 1,25(OH)2D3 for binding to chick intestinal cytosol. The responses of the keratinocyte subpopulations to exogeneous 1,25(OH)2D3 were evaluated by the increase in 25(OH)D3‐24 hydroxylase activity. Furthermore the expression of 1,25(OH)2D3 receptors (VDR) was examined in these cell populations. The results show that only the least differentiated cells produced 1,25(OH)2D3. In contrast, immunocytochemical detection of VDR, the VDR mRNA, and a 25(OH)D3‐24 hydroxylase response to 1,25(OH)2D3 were mainly found in the more differentiated cells. Thus, the ability of epidermis to synthesize 1,25(OH)2D3 and be simultaneously sensitive to it depends on the state of cell differentiation. This suggests that the mammalian epidermis contains a paracrine system in which the more differentiated keratinocytes are sensitive to the 1,25(OH)2D3 produced locally by neighboring immature ones. © 1994 wiley‐Liss, Inc.
Release of potassium, lipids, and proteins from nonionic detergent treated chicken red blood cells
Authors: Kellermayer Miklós , Ludáany Andrea , Miseta Attila , Kǒszegi Tamás , Berta Gábor , Bogner Péter , Hazlewood Carlton F. , Cameron Ivan L. , Wheatley Denys N.
Published: February 2005

The plasma membrane of erythrocytes, as of other cells, is thought to act as the barrier responsible for maintaining intracellular gradients of most ions and small molecular species between the cell and its environment. Controlled application of the nonionic detergent Brij 58 effectively opened the erythrocyte plasma membrane, as judged by electron microscopy and lipid mobilization, but the cytoplasm maintained much of its integrity for about 30 min. Release of K+ correlated well with release of protein into the surrounding medium. The results demonstrate that permeabilization of the erythrocyte plasma membrane does not result in an instantaneous equilibration of small ions, such as K+, between the cell and its environment. A comparison was made between erythrocytes treated with Brij 58 and Triton X‐100. The lipid and protein solubilizing actions of Triton X‐100 were not as easily separable in time as those of Brij 58. The results of treatment of the erythrocytes with different types of nonionic detergents suggest that the membranolytic and cytoplasmic protein destabilizing actions of nonionic detergents correspond with their hydrophilic‐lipophilic balance numbers (HLB values). © 1994 wiley‐Liss, Inc.
Modulation of the Na–;H antiport by insulin: Interplay between protein kinase C, tyrosine kinase, and protein phosphatases
Authors: Incerpi S. , Baldini P. , Bellucci V. , Zannetti A. , Luly P.
Published: February 2005

The insulin modulation of Na‐H antiport in rat hepatocytes was studied using the fluorescent, pH‐sensitive intracellular probe, 2′,7′ bis (carboxyethyl)‐5(6)‐carboxyfluorescein (BCECF). Our data show that insulin stimulates the NaH antiport. The dose‐response of insulin effect shows a behavior typical of other insulin responses: a maximum in the physiological range (1 nM) and smaller effects at higher and lower hormone concentrations. The time‐course of activation is very fast at high hormone concentrations and slow, but reaching a higher value, for the physiological concentrations (0.26± 0.05 and 0.18 ± 0.022 pH units for 1 nM and 1 μM insulin respectively). The use of phorbol, 12‐myristate, 13‐acetate (PMA), a potent activator of protein kinase C and its inhibitor staurosporine, and the inhibitor of tyrosine kinase erbstatin analog, suggests that both protein kinase C and tyrosine kinase could be involved in the mechanism leading to NaH antiport activation by insulin. We suggest that the activation of the antiport involves the two pathways depending on the hormone concentration. In particular, protein kinase C would mediate the effects of high hormone concentrations, acting as a growth factor, since staurosporine fully inhibited insulin 1 μM, but only partially 1 nM effects, and tyrosine kinase would mediate the effect of insulin 1 nM and only partially 1 μM. Okadaic acid 1 μM, a potent inhibitor of protein phosphatases, mimicked the hormone effects on the antiport and abolished the different time‐course due to hormone concentration, suggesting a role of kinases and phosphatases in the signal transduction. The effect of all activators was abolished by amiloride analog, 5‐(N‐ethyl‐N‐isopropyl) amiloride (EIPA), confirming the specificity of these effects. © 1994 wiley‐Liss, Inc.
Nitric oxide decreases in vitro phagocytosis of photoreceptor outer segments by bovine retinal pigmented epithelial cells
Authors: Becquet F. , Courtois Y. , Goureau O.
Published: February 2005

The presence of nitric oxide synthase (NOS) in the retina, the constitutive isoform in photoreceptor outer segments and the inducible form in retinal pigmented epithelial (RPE) cells, has been demonstrated, but the role of the free radical NO produced, remains unknown. We have investigated the effect of NO on the process of rod outer segment (ROS) phagocytosis. Using an in vitro assay for phagocytosis in primary cultures of bovine RPE cells, we demonstrate that NO released by SIN‐1 (3‐morpholinosydnonimine) in the culture medium inhibits the phagocytosis of ROS. Furthermore, endogenous NO, produced by RPE cells cotreated with lipopolysaccharide (LPS) and interferon‐γ (IFN‐γ), is also able to decrease RPE cell phagocytic activity. This effect depends upon the continuous presence of NO during the assay and is abolished by the scavenging of NO by hemoglobin or by the inhibition of NO synthase activity by L‐arginine analog, NG‐monomethyl‐L‐arginine. Pretreatment of ROS with SIN‐1 failed to impair subsequent phagocytosis, demonstrating that NO directly affects the RPE cells ability to phagocytose ROS. The inhibitory effect of NO is cGMP independent, since 8‐bromo‐cGMP does not modify this process. This decrease of ROS phagocytosis by RPE cells caused by NO may occur as a result of retinal inflammation, and could lead to photoreceptor degeneration. © 1994 wiley‐Liss, Inc.
Enhancement of differentiation induction of mouse myelomonocytic leukemic cells by extracellular ATP
Authors: Yamaguchi Masahiro , Hirayoshi Kazunori , Okuma Minoru , Nagata Kazuhiro
Published: February 2005

We examined the effect of ATP on the terminal differentiation of mouse myelomonocytic leukemic M1 cells to macrophages. Although ATP alone did not induce M1 cell differentiation, addition of ATP with the differentiation inducer, interleukin 6 (IL‐6), enhanced the induction of differentiation by IL‐6 about twofold. Comparison among several adenine nucleotides revealed that the order of effectiveness on differentiation enhancement was ATP > ADP > AMP ≥ adenosine. Using reactive blue 2, a P2 receptor antagonist, we confirmed that the effect of ATP on the stimulation of differentiation was mediated through the P2 purinergic receptor. Examination of cytosolic [Ca2+] elevation by ATP and comparison of potency of differentiation enhancement among several ATP analogs demonstrated that the effect of differentiation enhancement was mediated through P2y purinergic receptors expressed on M1 cell surface. Within 3 h of exposure, ATP alone slightly increased the expression of differentiation‐related immediate early response genes, c‐myc and JunB, and ATP also enhanced the IL‐6–induced expression of these genes. Induction of JunB expression by ATP analogs correlated with their potency of differentiation enhancement, which suggested that induction of JunB by ATP is one of signaling pathways involved in the exertion of its differentiation‐enhancing effect. © 1994 wiley‐Liss, Inc.
Calmodulin concentrated at the osteoclast ruffled border modulates acid secretion
Authors: Radding Wilson , Williams John P. , Hardy Robert W. , McDonald Jay M. , Whitaker Charles H. , Turbat‐Herrera Elba A. , Blair Harry C.
Published: February 2005

Osteoclasts mediate acid dissolution of bone for maintenance of serum [Ca2+] and for replacement of old bone in terrestrial vertebrates. Recent findings point to the importance of intracellular signals, particularly Ca2+, in osteoclast regulation. However, acid degradation of bone mineral subjects the osteoclast to uniquely high extracellular [Ca2+]. We hypothesized that this high calcium environment would affect calcium signalling mechanisms, and studied the calcium binding regulatory protein, calmodulin, in the osteoclast. Avian osteoclast bone resorption was inhibited 30% at 1 μM and 90% at 7 μM by the calmodulin antagonist trifluoperazine. Osteoclast bone attachment was not affected by 10 μM trifluoperazine. Quantitative immunofluorescence using fluorescein‐labelled calmodulin monoclonal antibody showed a severalfold increase of calmodulin concentration in bone attached relative to plastic attached osteoclasts. Western blots confirmed this, showing two to threefold increased osteoclast calmodulin per milligram of cell protein in 3‐day bone‐attached vs. nonattached cells. Scanning confocal microscopy showed calmodulin polarization to areas of bone attachment. Electron micrographs with 9nm colloidal gold labelling showed calmodulin in the acid secreting ruffled membrane. ATP‐dependent acid transport in osteoclast membrane vesicles was inhibited by the calmodulin antagonist calmidazolium. This effect was reversed by addition of excess calmodulin, showing that the inhibition is specific. Vesicle acid transport inhibition reflects an approximately fourfold shift in the apparent Km for ATP of vesicular acid transport in the presence of the calmodulin antagonist. We conclude that calmodulin concentration and distribution is modified by bone attachment, and that osteoclastic acid secretion is calmodulin regulated. © 1994 Wiley‐Liss, Inc.