bluuebunny/crossref_metadata_2025_split huggingface dataset:
FGF and EGF act synergistically to induce proliferation in BC3H1 myoblasts
Authors: Kelvin David J. , Simard Gilles , Connolly Joe A. 
Published: February 2005

AbstractBC3H1 muscle cells proliferate when grown in high concentrations of FBS (20%). Lowering the FBS concentration to 0.5% causes the cells to stop proliferating and is permissive for the morphological and biochemical differentiation of BC3H1 cells. Exposure of differentiated BC3H1 myocytes to high concentrations of serum or to the purified growth factors FGF or TGF‐b induced a shutdown of this differentiation program but did not induce cell proliferation (Olson et al.,J. Cell Biol.,103: 1799‐1805, 1986; Lathrop et al., Cell Biol.,100:1540–1547, 1985, and Cell Biol., 101:2194–2198,1985). We explored the possibility that BC3H1 cells require factors to act synergistically to induce proliferation. We found that EGF and FGF function in a synergistic fashion to stimulate BC3H1 proliferation. Moreover, the temporal requirement for these growth factors suggest that they are functioning as competence and progression factors for BC3H1 cell proliferation.
Stimulation of cartilage‐matrix proteoglycan synthesis by morphologically transformed chondrocytes grown in the presence of fibroblast growth factor and transforming growth factor‐beta
Authors: Inoue Hiroyuki , Kato Yukio , Iwamoto Masahiro , Hiraki Yuji , Sakuda Mamoru , Suzuki Fujio 
Published: February 2005

AbstractThe effects of transforming growth factor‐beta (TGF‐beta) on the synthesis of cartilage‐matrix proteoglycan by cultured rabbit chondrocytes were examined. Rabbit chondrocytes were seeded at low density and exposed to a 1:1 mixture of Dulbecco's modified Eagle's medium and Ham's F‐12 medium supplemented with 0.5% fetal bovine serum, 1% bovine serum albumin, 50μg/ml ascorbic acid, and 2 × 10−7 M hydrocortisone (Medium A). Various combinations of TGF‐beta, insulin‐like growth factor–l (IGF–I), and fibroblast growth factor (FGF) were also added to Medium A, and the chondrocytes were grown to confluency. Chondrocytes grown with TGF‐beta or FGF alone became flat or fibroblastic, those grown with FGF and TGF‐beta became very elongated and formed distinct foci, and those grown with FGF and IGF‐I showed the spherical configuration characteristic of overtly differentiated chondrocytes. Nevertheless, the incorporation of 3H With glucosamine into the large, chondroitin sulfate proteoglycan synthesized by cultures with FGF and TGF‐beta was similar to that in cells grown with FGF and IGF‐I and five times that in cells cultured with FGF alone. The increases in incorporation of 3H reflected real increases in proteoglycan synthesis, because chemical analyses showed an increase in the accumulation of macromolecules containing uronic acid in cultures with FGF and TGF‐beta or with FGF and IGF‐I. However, FGF in combination with either TGF‐beta or IGF‐I had little effect on the incorporation of 3H into small proteoglycans or hyaluronic acid. These results indicate that chondrocytes morphologically transformed with TGF‐beta and FGF fully express the differentiated proteoglycan phenotype rather than the transformed glycosaminoglycan phenotype.
Differences of Ca2+ regulation in skin fibroblasts from blacks and whites
Authors: Nakamura Akitoshi , Gardner Jeffrey , Hatori Norio , Nakamura Makiko , Fine Burton P. , Aviv Abraham 
Published: February 2005

AbstractBlack people have a higher propensity than caucasions toward essential hypertension. To explore the possibility that this racial difference relates to cellular Ca2+ metabolism, we measured 45Ca2+ washout and uptake and cytosolic free concentration of Ca2+ [Ca2+], in serially passed skin fibroblasts from normotensive black and white males. Depending on the experimental conditions, 45Ca2+ washout in these cells was described by either two or three exponential functions, whereas 45Ca2+ uptake was described only by a two‐exponent function. There were no racial differences in 45Ca2+ uptake and washout of unstimulated fibroblasts. However, stimulation by human serum resulted in an increase in the 45Ca2+ washout that was higher in fibroblasts from blacks than from whites. The racial differences were expressed primarily by higher values of the apparent washout rate constant (k1) of 45Ca2+ from the largest and most rapidly exchangeable cellular pool. The effect of human serum was not related to its origin (blacks vs. whites). In 2 mM Ca2+ medium and 10% serum from blacks, the respective k1 (mean ± SEM; × 10−2/min) values for fibroblasts from blacks and whites were 89.68 ± 5.23 and 73.29 ± 4.0; in the presence of 10% serum from whites, the k1 values for cells from blacks and whites were 84.14 ± 2.80 and 76.36 ± 3.23 (overall significance of P .01). In Ca2+‐deficient medium in the presence of 10% human serum, the k1 for fibroblasts from blacks and whites were 115.57 ± 3.76 and 102.15 ± 3.30 (P < .05). Serum substantially increased the 45Ca2+ uptake in fibroblasts from both blacks and whites; however, racial differences were not observed. Basal levels of [Ca2+], were not different in fibroblasts of blacks vs. whites (46.8 ± 6.8 and 43.2 ± 7.1 nM for blacks and whites, respectively). However, the peak response of Ca2+ transients for cell stimulated by 5% human serum was significantly higher in blacks than whites (blacks = 963 ± 213, whites = 481 ± 162 nM; P = .0286). We conclude that Ca2+ regulation is different in serum‐stimulated fibroblasts from blacks and whites and that, at least in part, this difference may relate to a greater agonist‐induced mobilization of Ca2+ in fibroblasts from blacks.
β‐adrenergic control of cell volume and chloride transport in an established rat submandibular cell line
Authors: He Xinjun , Ship Jonathan , Wu Xiaozai , Brown Ashley M. , Wellner Robert B. 
Published: February 2005

AbstractRat submandibular cells treated with methylcholanthrene are able to be propagated in continuous culture while retaining β‐adrenergic responsiveness. A specific clone, RSMT‐A5, has been isolated and studied in detail. RSMT‐A5 cells possess β‐adrnergic receptors (BARS) as judged by [3H]‐dihydroalprenolol ([3H]‐DHA) binding studies. [3H]‐DHA binds to RSMT‐A5 membranes in a specific and saturable manner with respect to time and [3H]‐DHA concentration. Specific binding is saturable within three min of incubation, and a Scatchard analysis reveals a single class of high affinity binding sites with an equilibrium dissociation constant of 0.62 ± 0.03 nM and a receptor density of 101 ± 4 fmole/mg protein. Antagonist competition studies indicate that the BARs are primarily of the β2‐subtype. The BARs are functional since isoproterenol stimulation results in an increased intracellular cAMP content, marked morphological change, and decreased cell volume and chloride content. These same responses can be evoked by treating RSMT‐A5 cells with 8‐bromo‐cAMP. Ion transport inhibitors such as bumetanide (an inhibitor of Na/K/C1 cotransport), SITS and DIDS (inhibitors of chloride‐bicarbonate exchange), amiloride (an inhibitor of Na‐H exchange), ouabain (an inhibitor of Na/K‐ATPase), and dipyridamole and 9‐anthracene carboxylic acid (chloride channel blockers) fail to inhibit the isoproterenol‐stimulated change in chloride content. The effects of either isopro‐terenol or 8‐bromo‐cAMP on both chloride content and cell volume can be inhibited by the chloride channel blocker N‐phenylanthranilic acid, however. Taken together, our results indicate that RSMT‐A5 cells possess a β‐adrenergic receptor system which controls intracellular volume and chloride content by modulating transport processes that are 1) cAMP‐responsive and 2) inhibitable by the putative chloride channel blocker N‐phenylanthranilic acid.
Protein synthesis during induction of DNA replication in thyroid epithelial cells: Evidence for late markers of distinct mitogenic pathways
Authors: Lamy Françoise , Roger Pierre , Lecocq Raymond , Dumont Jacques E. 
Published: February 2005

AbstractThe synthesis of specific proteins has been investigated in primary cultures of dog thyroid epithelial cells, which can be induced to progress into G1 phase, in the presence of insulin, by different types of mitogens: thyrotropin (TSH) acting through cyclic adenosine monophosphate (cAMP), epidermal growth factor (EGF), 12‐O‐tetradecanoyl‐phorbol‐13‐acetate (TPA), or 10% serum. EGF, TPA, or serum specifically induce [35S] methionine labeling of protein 1 (Mr ≈ 80,000). The effect of EGF on protein 1 labeling and DNA replication is dependent on insulin. The level of protein 1 labeling as well as that of DNA synthesis is higher when TSH or TSH + serum are added together with EGF. It peaks in mid‐G1. TSH alone, in the presence of insulin, stimulates DNA replication without inducing protein 1 synthesis, which thus represents a cell‐cycle‐dependent event that is not obligatory in mitogenic activation through cyclic AMP. Among the eight proteins whose synthesis is stimulated by TSH, only the labeling of protein 7, molecular weight ratio (Mr ≈ 38,000), correlates with the DNA synthetic activity of the cells. The present authors identified protein 7 as cyclin/proliferating cell nuclear antigen (PCNA), the auxiliary protein of DNA polymerase‐δ. The effect of TSH on cyclin synthesis is already detectable when most of the cells are in late G1, but its stimulation by EGF or EGF + serum is delayed and detected only after extending the labeling period to the S‐phase. These data support the view that the cAMP‐mediated mitogenic pathway remains partly distinct from the better known pathways induced by growth factors and tumor promoters, even at late stages of the G1‐phase.
An epidermal growth factor‐ricin a chain (EGF‐RTA)‐resistant mutant and an epidermal growth factor‐Pseudomonas endotoxin (EGF‐PE)‐resistant mutant have distinct phenotypes
Authors: Banker Deborah E. , Pastan Ira , Gottesman Michael M. , Herschman Harvey R. 
Published: February 2005

AbstractH20e12 is a mutant HeLa cell line selected for resistance to the toxicity of a chimeric protein conjugate composed of epidermal growth factor (EGF) and the toxic A cham of ricin (RTA). ET‐28 is a mutant KB cell line selected for resistance to the toxicity of a chimeric protein conjugate composed of EGF and Pseudomonas exotoxin (PE). In this report we describe the presence or absence, in these mutants, of cross‐resistance to the two toxic conjugates and the effects of ammonium chloride, leupeptin, and adenovirus cotreatments on toxin efficacies. ET‐28 cells, the EGF‐PE‐resistant cells, are resistant to both EGF‐PE and EGF‐RTA. In contrast, H20e12 cells, the EGF‐RTA‐resistant cells, are as sensitive to EGF‐PE toxicity as are their parent HeLa cells. Ammonium chloride cotreatment substantially reduces the resistance of H20e12 cells to EGF‐RTA but has little or no effect on the resistance of ET‐28 cells to either EGF‐RTA or EGF‐PE. Leupeptin has no effect on the toxicity of either chimeric conjugate on any of the four cell lines, despite its demonstrated ability to inhibit cellular degradation of EGF. In contrast, adenovirus cotreatment enhances the toxicity of EGF‐RTA and EGF‐PE on all cells tested, and completely nullifies the relative resistance of H20e12 and ET‐28 cells to these toxic conjugates. H20e12 and ET‐28 cells appear to be altered in distinct, possibly endosomal, functions.
Multiple intracellular pathways induce expression of a zinc‐finger encoding gene (EGR1): Relationship to activation of the Na/H exchanger
Authors: Jamieson Gordon A. , Mayforth Ruth D. , Villereal Mitchel L. , Sukhatme Vikas P. 
Published: February 2005

AbstractIntracellular pathways that rapidly stimulate the expression of a mitogeninducible, zinc‐finger encoding gene, EGR1 (Sukhatme et al., Cell 53:37–43), have been characterized in two human fibroblasts strains (WI‐38 and HSWP). Serum and epidermal growth factor (EGF) were each found to strongly stimulate EGR1 expression in both cell types. Comparably high levels of expression could also be induced by treatment with the phorbol ester TPA. In cells rendered deficient in PK‐C, serum and EGF were each still capable of inducing high levels of EGR1 mRNA. demonstrating that additional non‐protein kinase C pathways are capable of stimulating EGR1 expression. In both fibroblasts strains, stimulation of EGR1 expression by all these agents exhibited rapid, transient kinetics and could be superinduced if protein synthesis was inhibited through the addition of cycloheximide. Finally, various agents, known to stimulate/inhibit the activation of another early mitogenic response, the activation of Na/H exchange, were analyzed for their effect on EGR1 expression. Interestingly bradykinin, vasopressin, and Ca ionophores, which dramatically stimulate Na/H exchange, were only weak stimulants of EGR1 expression. Conversely, EGF, which stimulates Na/H exchange poorly, strongly activated EGR1 expression. Hence while EGR1 expression could be triggered by multiple intracellular pathways, its expression does not appear to require the prior activation of Na/H exchange.
Proliferation of fetal rat hepatocytes in response to growth factors and hormones in primary culture
Authors: Hoffmann Birgit , Piasecki Angelika , Paul Dieter 
Published: February 2005

AbstractFetal rat hepatocytes (day 19 of gestation) multiply in primary culture in arginine‐free, hydrocortisone‐containing chemically defined medium MX‐82 supplemented either with epidermal growth factor (EGF) or insulin or both. In contrast, hepatocytes did not multiply under similar culture conditions using Dulbecco's minimum essential medium (DMEM). Cells underwent two divisions within 10 days in cultures maintained in MX‐82 medium without a medium change, and cells grew to increased final cell densities when the medium was renewed every third day. When the medium MX‐82 was enriched by the addition of lipids, intermediary metabolites, and trace metals (medium MX‐83), cells grew to higher densities. In the absence of the growth factors, cells became quiescent and subsequently could be induced to synthesize DNA in response to EGF. With the increasing numbers of cells per dish, the growth response of the hepatocytes diminished. Levels of hepatocyte‐specific albumin and α‐fetoprotein mRNAs at day 0 were similar to those observed at day 10 in primary fetal rat hepatocyte cultures and were maintained at higher levels in medium MX‐83 than in medium MX‐82.
Dexamethasone effects on creatine kinase activity and insulin‐like growth factor receptors in cultured muscle cells
Authors: Whitson Peggy A. , Stuart Charles A. , Huls M. Helen , Sams Clarence F. , Cintron Nitza M. 
Published: February 2005

AbstractWe examined the effects of dexamethasone on creatine kinase (CK) activity and insulin‐like growth factor I (IGF‐I) binding in two skeletal muscle‐derived cell lines (mouse, C2C12; rat, L6) and in one cardiac muscle‐derived cell line (rat, H9c2). Dexamethasone treatment during differentiation of cultured cells caused a dose‐dependent increase in CK activity as well as an increase in the degree of myotube formation in C2C12 and L6, whereas H9c2 cells did not exhibit significant CK activities during culture or dexamethasone treatment. Dexamethasone treatment of C2C12 did not stimulate proliferation in differentiating cultures, but a dose‐dependent increase in the number of nuclei was observed for L6 concomitant with increased CK activity. In L6 the increased CK activity may therefore reflect a dose‐dependent increase in proliferation. Short‐term (48 hr) treatment of C2C12 with dexamethasone (20 nM) did not appear to alter myoblast fusion but reversibly increased CK activity. In C2C12 the observed increase in CK, alanine aminotransferase (ALT), and aspartate aminotransferase (AST) activities with dexamethasone treatment suggest modulation of protein expression and/or turnover. Although the data for dexamethasone effects on CK activities varied in each of the cell lines, consistent behavior was observed in all three cell lines when IGF‐I binding was examined. IGF‐I binding to dexamethasone‐treated cells (50 nM for 24 hr the day prior to confluence) resulted in an increased number of available binding sites, with no effect on the binding affinities. Affinity cross linking and autoradiography indicated that the increase in IGF‐I binding was the result of dexamethasone up‐regulation of type I IGF receptors. Our data for all three muscle cell lines suggest that similar heterologous hormone receptor modulation of type I IGF receptor sites occurs with dexamethasone treatment.
Endogenous regulation of 2‐deoxyglucose uptake in c6 glioma cells correlates with cytoskeleton‐mediated changes of surface morphology
Authors: Lange Klaus , Brandt Ursulac , Keller Konrad , Zimmermann Bernd 
Published: February 2005

AbstractThe cellular basis of the membrane‐limited state of glucose utilization and the mechanism of the endogenous regulation of hexose uptake in dense monolayers of C6 glioma cells were investigated. In an earlier study, it was shown that at high rates of glucose transport and phosphorylation combined with the inhibition of glycolytic adenosine triphosphate (ATP) production by iodoacetate, an endogenous regulatory response occurred that resulted in rapid, periodic variations of the glucose uptake rates (Lange et al., 1982). Similar time‐dependent periodic changes of uptake rates also occurred during incubation of C6 glioma cells with 2 mM 2‐deoxyglucose (2‐DG) without pretreatment of the cells with iodoacetate. These changes were accompanied by variations of the intracellular ATP content, by distinct alterations of the shape and arrangement of microvilli and lamellae (lamellipodia) on the cell surface, and by changes of the cytoskeletal F‐actin content. Because the changes of 2‐DG uptake rates occurred independent of the intracellular 2‐DG concentration, the bulk of this 2‐DG pool was assumed to be localized apart from the membranal transport sites. Downregulation of 2‐DG uptake appeared to be triggered by a rapid decrease of a small pool of the cellular ATP involved in the phosphorylation of transported hexose. Scanning and transmission electron microscopic observations of cells fixed in different states of the endogenous uptake regulation supported the assumption that the interior of lamellae and microvilli may represent a small entrance compartment for transported hexoses in which occurred the observed close coupling between hexose transport and phosphorylation as well as the rapid variations of ATP content. Hexose uptake is supposed to be regulated by cytoskeleton‐mediated changes of volume and diffusional accessibility of this compartment, modulating the degree of its metabolic coupling with the cytoplasmic main compartment.
Differential effects of hyperoxia and hydrogen peroxide on DNA damage, polyadenosine diphosphate‐ribose polymerase activity, and nicotinamide adenine dinucleotide and adenosine triphosphate contents in cultured endothelial cells and fibroblasts
Authors: Junod Alain F. , Jornot Lan , Petersen Hilke 
Published: February 2005

AbstractThe effects of oxidative stress on DNA damage and associated reactions, increased polyadenosine diphosphate‐ribose polymerase (PARP) activity and decreased nicotinamide adenine dinucleotide (NAD) and adenosine triphosphate (ATP) contents, have been tested in primary cultures of porcine aortic endothelial cells. The cells were treated with 50‐500 μM H2O2 for 20 min or 100 μM paraquat for 3 days or were exposed to 95% O2 for 2 and 5 days. The administration of 250‐500 μM H2O2 resulted in a marked increase in PARP activity and a profound depletion of ATP and NAD. Although hyperoxia had no effect on PARP activity and reduced only slightly the ATP and NAD stores, it markedly reduced the ability of endothelial cells to increase PARP activity upon exposure to DNase. Paraquat had a similar effect. Human dermal fibroblasts were also exposed to 50‐500 μM H2O2 for 20 min or 95% O2 for 5 days. Their response to H2O2 differed from that of endothelial cells by their ability to maintain the ATP content at a normal level. Fibroblasts were also insensitive to the effect of hyperoxia. These results suggest that the oxidant‐related DNA damage is a function of the type of oxidative stress used and may be cell‐specific.
Diacylglycerol, but not inositol 1,4,5‐trisphosphate, accounts for platelet‐derived growth factor‐stimulated proliferation of BALB 3T3 cells
Authors: Suzuki‐Sekimori Reiko , Matuoka Koozi , Nagai Yoshitaka , Takenawa Tadaomi 
Published: February 2005

AbstractRecently we found that an intracellular event related to phosphatidylinositol 4,5‐bisphosphate (PIP2) is crucial for platelet‐derived growth factor (PDGF)‐induced mitogenesis in fibroblastic cells (Matuoka, K., et al.: Science 239:640‐643, 1988). In the present study we examined the mitogenic effects of PIP2 and its hydrolysis products introduced into the cytoplasm of BALB 3T3 cells by micro‐injection to confirm the role of PIP2 hydrolysis in PDGF stimulation of cell proliferation. Injection of 1,2‐dioleylglycerol (diolein) into serum‐deprived quiescent cells induced DNA synthesis with the same time course as that induced by exposure of the cells to PDGF and, in the presence of PDGF, caused no additional increase in the cell population entering S phase. The injection of PIP2, inositol 1,4,5‐trisphosphate, or 1,2‐dioleylphosphatidic acid into the cells did not induce mitogenesis. Consistent results were obtained in experiments in which the cells were exposed to 1‐oleyl‐2‐acetylglycerol (OAG) and ionomycin; namely, OAG stimulated proliferation of BALB 3T3 cells, but ionomycin did not induce any mitogenesis. Desensitization of the protein kinase C pathway by prolonged exposure of the cells to phorbol ester abolished the induction of cell proliferation by subsequent injection of diolein or exposure to phorbol ester or OAG as well as by PDGF challenge. These findings strongly suggest that activation of the protein kinase C system following formation of diacylglycerol by PIP2 hydrolysis is mainly responsible for the mitogenic action of PDGF on BALB 3T3 cells.
Involvement and relative importance of at least two distinct mechanisms in the effects of 2‐mercaptoethanol on murine lymphocytes in culture
Authors: Pruett Stephen B. , Obiri Nicholas , Kiel Johnathan L. 
Published: February 2005

Abstract2‐Mercaptoethanol (2‐ME) exerts several effects on murine lymphocytes in culture that might explain its ability to enhance survival and growth of these cells. The uptake of the essential amino acid cystine and consequently the maintenance of intracellular glutathione levels are enhanced by 2‐ME. Furthermore, 2‐ME (even in the disulfide form) causes lymphocytes to release thiols into the culture medium. These effects might protect the cells from oxidative damage. The additional cystine provided by treatment of lymphocyte cultures with 2‐ME might also allow adequate protein synthesis to support survival and/or growth. This study was conducted to assess the relative importance of the antioxidant and protein synthesis effects of 2‐ME. As expected, 2‐ME increased cystine uptake at all concentrations that enhanced growth and survival, but four nonthiol antioxidants that enhanced growth and/or survival either did not substantially affect cystine uptake or decreased it and did not affect the release of cystine or its products. The results presented here demonstrate that antioxidant protection is necessary and sufficient for lymphocyte survival and that cystine uptake in untreated lymphocytes is sufficient to support the protein synthesis needed for survival and limited growth. However, we also noted that concentrations of 2‐ME that stimulated maximal growth more than doubled protein synthesis as measured at 8 hr. Thus the portion of the effects of 2‐ME not accounted for by antioxidant action could be accounted for by enhanced protein synthesis.