bluuebunny/crossref_metadata_2025_split huggingface dataset:
Granulocyte‐Macrophage colony‐stimulating factor and steel factor induce phosphorylation of both unique and overlapping signal transduction intermediates in a human factor‐dependent hematopoietic cell line
Authors: Hallek Michael , Druker Brian , Lepisto Eva M. , Wood Kenneth W. , Ernst Timothy J. , Griffin James D. 
Published: February 2005

AbstractSteel factor (SF), the ligand for the proto‐oncogene c‐kit, acts synergistically with GM‐CSF or IL‐3 to support the growth of normal human hematopoietic progenitor cell. We examined the effect of SF on GM‐CSF or IL‐3 induced proliferation of a human factor‐dependent cell line, MO7. SF supported MO7 cell proliferation as well as IL‐3 or GM‐CSF alone, and its addition dramatically enhanced (three‐to sixfold) maximal GM‐CSF or IL‐3 stimulated proliferation. SF did not increase the number of affinity of cell surface GM‐CSF receptors. We examined several early events of signal tranduction in an effort to elucidate the biochemical mechanisms of synergy of these factors. Since each of these three cytokines is believed to function in part through activation of a tyrosine kinse, we examined their effects on cellular phosphotyrosine containing proteins. Each cytokine induced rapid, transient, and concentration dependent tyrosine phosphorylation of a number of substrates. For GM‐CSF and IL‐3, these phosphoproteins were indistinguishable (150, 125, 106, 93, 80, 79, 73, 44, 42, and 36 kDa), while SF induced major or minor tyrosine phosphorylation of 205, 140–150, 116, 106, 90, 90, 80, 79, 73, 44, 42, 39, 36, 32 kDa phosphoproteins. Two other signal tranduction intermediates known to be phosphorylated and activated by GM‐CSF and IL‐3, the 70–75 kDa Raf‐1 kinase, and p42 mitogen‐activated protein kinase‐2 (MAPK) were also phosphorylated by SF. Combinations of GM‐CSF or IL‐3 with SF did not further increase the phosphorylation of Raf‐1 or p42 MAPK when compared to any of the factors alone. In contrast SF, but not GM‐CSF or IL‐3, induced tyrosine phosphorylation of phospholipase C‐γ (PLC‐γ). These results indicate that SF and GM‐CSF/IL‐3 have partially overlapping effects on early signal tranducing events, as well as striking differences, such as tyrosine phophorylation of PLC‐γ. This cell line should provide a useful model system to investigate the complicated process of hematopoietic growth factor synergy. © 1992 Wiley‐Liss, Inc.
Leukemia inhibitory factor and interleukin‐11 promote maturation of murine and human megakaryocytes in vitro
Authors: Burstein Samuel A. , Mei Rui‐Lian , Henthorn James , Friese Paul , Turner Katherine 
Published: February 2005

AbstractThe growth‐promoting activities of optimally stimulating concentrations of leukemia inhibitory factor (LIF) and interleukin‐11 (IL‐11), a stromal cell‐derived cytokine, on megakaryocytes in liquid marrow cultures were compared to interleukin‐6 (IL‐6), a known megakaryocytic maturation factor. Maximally stimulating concentrations of LIF (25 ng/ml), IL‐11 (10 ng/ml), or IL‐6 alone (10 ng/ml) promoted an 81, 157, and 153% increase, respectively, in acetylcholinesterase (AchE) activity in murine serum‐free cultures compared with controls (n = 5). In combination with 25 U/ml murine interleukin‐3 (IL‐3), LIF, IL‐6, and IL‐11 showed increases, respectively, of 35%, 49%, and 174% in AchE activity compared with IL‐3 alone (n = 4). Flow cytometric analysis of 4‐day‐old cultures showed that LIF alone had minimal effect on megakaryocytic ploidy, whereas IL‐11 and IL‐6 alone markedly augmented high ploidy cells. Enumeration of cells stained for AchE showed that IL‐11 increased the numbers of Mks in comparison to LIF, IL‐6 or controls by up to 59%. Moreover, a twofold increment in Mk number was noted when IL‐11 was used in combination with IL‐3 (compared with either IL‐3 alone of IL‐3 + IL‐6). Flow cytometric ploidy analysis of 8‐day‐old human liquid marrow cultures showed that either LIF, IL‐11, or IL‐6 alone markedly augmented the percentage of 32N cells compared with cultures containing only human IL‐3. The data suggest that LIF and IL‐11 promote murine and human Mk maturation in vitro, although the effect of IL‐11 exceeds that of LIF in mice. Despite the comparable influence of IL‐11 and IL‐6 on Mk ploidy, IL‐11 has the additional characteristic of enhancing the number of Mks, particularly in combination with IL‐3. © 1992 Wiley‐Liss, Inc.
Atypical receptor‐mediated signal transduction events in the EGF‐dependent growth‐inhibited cell line, MDA‐468
Authors: Church Jon G. , Richardson Vernon J. , Lockwood Ann G. 
Published: February 2005

AbstractIt is now generally considered that early signalling from tyrosine kinases that induce mitogenesis is initiated through the formation of heteromeric complexes consisting of the autophosphorylated tyrosine kinase and a number of tyrosylphosphorylated proteins, including phospholipase C‐γ (PLC‐γ) and GTPase activating protein (GAP). However, since much of this work has been performed on proliferative, chimeric cell lines expressing heterologous receptor molecules, we examined the nature of the epidermal growth factor receptor (EGFR) signalling complex formation in the human breast cancer cell line, MDA‐468. This cell line has an amplified, native EGFR gene, correspondingly overexpresses the EGFR, and its growth in culture is inversely related to the EGF concentration. Our results indicate that in MDA‐468 cells, both the EGFR and PLC‐γ are phosphorylated on tyrosine residues and can be co‐immunoprecipitated. This occurs at both high and low EGF concentrations regardless of the proliferative endpoint. The molecular association is correlated with a significant increase in total inositol phosphates formed in response to the growth factor treatment. In contrast, however, there is no evidence that GAP is either phosphorylated on tyrosine residues or forms a complex with the activated EGFR in EGF‐treated MDA‐468 cells. These observations suggest that as a model for growth factor action, the formation of heteromeric protein signalling complexes may demonstrate considerable diversity depending upon both cell type and physiology. © 1992 Wiley‐Liss, Inc.
Heat‐induced transcription from RNA polymerases II and III and HSF binding activity are co‐ordinately regulated by the products of the heat shock genes
Authors: Price Brendan D. , Calderwood Stuart K. 
Published: February 2005

AbstractHeat shock leads to co‐ordinate increases in transcription of a family of heat shock genes, including the mouse hsp70.1 and B2 genes. Activation of the heat shock transcription factor (HSF) by heat shock stimulates transcription of the murine hsp70.1 gene (by RNA polymerase II). B2 genes are short, repetitive sequences whose transcription (by RNA polymerase III) are also increased after heat shock. We have studied whether heat‐induced transcription is auto‐regulated by the products of the heat shock genes. The results indicate: (1) after an initial heat shock, transcription of the heat shock genes by RNA polymerases II and III becomes desensitized to further heat shock, and the heat‐induced DNA binding activity of the HSF is lost, (2) if accumulation of heat shock gene products is inhibited, the desensitizing effect of a prior heat shock is removed, and (3) transcription of the hsp70. 1 and the B2 gene apparently involves different mechanisms, with hsp70.1 employing the HSF and the B2 gene using a separate, heat‐activated transcriptional mechanism. However, the level of transcription from the hsp70.1 and B2 genes and the stability of their respective RNAs are co‐ordinately regulated by the level of heat shock protein in the cell. The data indicate that auto‐regulation of the level of mouse heat shock gene products is mediated by RNA polymerase II transcripts but that the regulatory mechanism can control transcription from RNA polymerase III genes as well. © 1992 Wiley‐Liss, Inc.
Lactoferrin binding sites and nuclear localization in K562(S) cells
Authors: Garré Cecilia , Bianchi‐Scarrà Giovanna , Sirito Mario , Musso Marco , Ravazzolo Roberto 
Published: February 2005

AbstractLactoferrin, a single chain cationic glycoprotein, present in the secondary granules of neutrophils, acts as a negative feedback regulator of myelopoiesis. Specific receptors for lactoferrin were detected on the surface of different hematopoietic cell types. The influence of lactoferrin on cell growth in culture has been reported. Interactions of lactoferrin with DNA were also demonstrated.In the present paper we confirm the presence of lactoferrin specific binding sites on K562 cells and we estimate the number of binding sites and the dissociation constant. By Western blotting analysis performed on K562 lysates we find a band of about 120 kDa responsible for specific binding of lactoferrin. We also show that lactoferrin, after binding at the cell surface, is internalized in a temperature dependent way and is immunologically detectable as a DNA‐linked protein in nuclear extracts. © 1992 Wiley‐Liss, Inc.
Effect of membrane phosphatidylethanolamine‐deficiency/phosphatidylcholine‐excess on the metabolism of phosphatidylcholine and phosphatidylethanolamine
Authors: Fisk Harold A. , Kano‐Sueoka Tamiko 
Published: February 2005

AbstractCells of epithelial origin generally require ethanolamine (Etn) to grow in defined culture medium. When such cells are grown without Etn, the membrane phospholipid composition changes drastically, becoming phosphatidylethanolamine (PE)‐deficient due to a reduced de novo rate of PE synthesis, and growth stops. We have hypothesized that the cessation of growth occurs because this membrane phospholipid environment is no longer suitable for membrane‐associated functions. Phospholipid has long been known to play a role in the transduction of some signals across membranes. In addition to the well‐known phosphatidylinositol cycles, hydrolysis of phosphatidylcholine (PC) and PE has recently been shown to play a central role in signal transduction. Using an Etn‐requiring rat mammary cell line 64‐24, we have studied the metabolism of PC and PE in response to the phorbol ester phorbol 12, 13‐dibutyrate (PDBu) under conditions where cells have either normal or PE‐deficient membrane phospholipid. In cells having normal membrane phospholipid, the synthesis of PC was stimulated by PDBu (∼fourfold), as was the degradation of PC and PE (by twofold and fourfold, respectively). Product analysis suggested that PDBu stimulated hydrolysis of PC by both phospholipases C and D (PLC and PLD), and of PE by PLD. However, in PE‐deficient cells, neither lipid synthesis or degradation were significantly stimulated by PDBu. Analysis of the CDP‐choline pathway of PC sythesis indicated that the regulatory enzyme, CTP: phosphorylcholine cytidylyltransferase, was stimulated about twofold by PDBu in cells having normal membrane, but not in PE‐deficient cells. These results indicate that the membrane phospholipid environment profoundly affects phospholipid metabolism, which no doubt influences cell growth and regulation. © 1992 Wiley‐Liss, Inc.
Rapamycin blocks cell cycle progression of activated T cells prior to events characteristic of the middle to late G1 phase of the cycle
Authors: Terada Naohiro , Lucas Joseph J. , Szepesi Agota , Franklin Richard A. , Domenico Joanne , Gelfand Erwin W. 
Published: February 2005

AbstractThe effects of rapamycin (RAP) on cell cycle progression of human T cells stimulated with PHA were examined. Cell cycle analysis showed that the RNA content of cells stimulated with PHA in the presence of RAP was similar to that of control T cells stimulated with PHA for 12–24 hr in the absence of the drug. This level was substantially higher than that seen in cells stimulated in the presence of cyclosporin A (CsA), an immunosuppressant known to block cell cycle progression at an early point in the cycle. However, the point in the cell cycle at which RAP acted appeared to be well before the G1/S transition, which occurs about 30–36 hr after stimulation with PHA. In an attempt to further localize the point in the cell cycle where arrest occurred, a set of key regulatory events leading to the G1/S boundary were examined, including p110Rb phosphorylation, which occurred at least 6 hr prior to DNA synthesis, p34cdc2 synthesis, and cyclin A synthesis. In control cultures, p110Rb phosphorylation was detected within 24 hr of PHA stimulation; p34cdc2 and cyclin A synthesis were detected within 30 hr. Addition of RAP to the cultures inhibited each of these events. In contrast, early events, including c‐fos, IL‐2, and IL‐4 mRNAs expression, and IL‐2 receptor (p55) expression, were only marginally affected, if at all, in PHA‐stimulated T cells. Furthermore, the inhibition of cell proliferation by RAP could not be overcome by addition of exogenous IL‐2. These results indicate that RAP blocks cell cycle progression of activated T cells after IL‐2/IL‐2 receptor interaction but prior to p110Rb phosphorylation and other key regulatory events signaling G1/S transition. © 1993 Wiley‐Liss, Inc.
Localization and quantitation of calcium pools and calcium binding sites in cultured human keratinocytes
Authors: Pillai Sreekumar , Menon Gopinathan K. , Bikle Daniel D. , Elias Peter M. 
Published: February 2005

AbstractCalcium plays a crucial role in regulating the growth and differentiation of cultured keratinocytes. However, the mechanism(s) of this regulation is not clear. Prior studies have shown that intracellular free calcium (Cai) increases with keratinocyte differentiation. In this study, in order to evaluate the role of cytosolic free calcium and organelle‐bound calcium in keratinocyte differentiation, we quantitated and localized calcium pools in keratinocytes, utilizing the fluorescence probe indo‐1 and ion‐capture cytochemistry, respectively. Cai of undifferentiated keratinocytes was 80–120 nM, whereas Cai of differentiated keratinocytes was 200–300 nM depending on the extent of differentiation. The Cai of individual cells in an undifferentiated colony was heterogeneous (60–160 nM) with larger cells displaying higher Cai. Heterogeneity also was observed in the intracellular calcium‐containing precipitates in the different layers of stratifying keratinocyte cultures using the cytochemical technique. Calcium precipitates were abundant in the lower cell layers, progressively decreasing apically, with the uppermost layer devoid of precipitates. Calcium‐containing precipitates appeared as fine‐tocoarse electron‐dense granules on the plasma membrane, within the cytosol, mitochondria, nucleus, and vacuolar organelles. Whereas ionomycin in the presence of extracellular calcium increased the amount of intracellular calcium precipitates, EGTA removed calcium precipitates from organelles. Unlike intact epidermis, keratinocytes displayed no extracellular calcium reservoirs. Putative calcium binding sites, visualized by trivalent lanthanum (La) binding, were abundant on cell membranes and desmosomes of basaloid cells, but decreased in the upper cell layers. These studies revealed differences in the distribution of free ionic calcium (as determined by the fluorescence technique) and organelle‐bound calcium (as determined by the cytochemical technique). Striking differences were also observed in calcium localization between intact epidermis and cultured epidermal cells. The localization pattern of calcium in cultured keratinocytes may reflect the hyperproliferative state of these cells, as in psoriatic epidermis, and/or the absence of a normal permeability barrier in these submerged cultures. © 1993 Wiley‐Liss, Inc.
Major influence of cell differentiation status on characteristics of proteoglycans synthesized by cultured rabbit renal proximal tubule cells: Role of insulin and dexamethasone
Authors: Lelongt B. , Vandewalle A. , Brenchley P. E. C. , Baudouin B. , Géniteau‐Legendre M. , Verroust P. J. , Ronco P. M. 
Published: February 2005

AbstractTo analyze the influence of epithelial cell differentiation and the effects of hormones on the characteristics of cell‐associated and secreted proteoglycans (PGs), we studied their distribution, synthesis, and biochemical features in a model of renal proximal tubule cells in primary culture in which cell differentiation could be controlled by medium composition. In cells cultured in serum‐free, hormonally defined medium supplemented with insulin and dexamethasone that exhibited a high degree of morphological and functional proximal differentiation (Ronco et al., 1990), cell‐associated PGs were similar to those extracted in vivo by their size estimated by Sepharose CL‐6B chromatography (Kav = 0.27, vs. 0.26), composition (heparan‐sulfate), and localization in a continuous basal layer of extra‐cellular matrix (ECM). In contrast, major quantitative and qualitative anomalies of cell‐associated PGs were observed in poorly differentiated cells grown in 1% fetal calf serum‐supplemented medium (FCS). PGs alterations included: (1) reduced and irregular expression of PGs at the cell basal pole, (2) a 2.8‐fold decrease in [35S]‐incorporation into cell‐associated PGs, (3) a 3.1‐fold increase in trypsin‐releasable PGs, and (4) the emergence of a high MW PG composed exclusively of chondroitin‐sulfate (CS) (Kav = 0.09 on Sepharose CL‐6B) as well as of putative free CS‐glycosaminoglycan (GAG) chains (Kav = 0.49 on Sepharose CL‐6B). The same alterations were identified in the basal defined medium devoid of hormones but were partially or totally abolished by addition of insulin and dexamethasone, respectively. At variance with cell‐associated PGs, production and biochemical features of secreted PGs were not influenced by cell differentiation status and medium composition. © 1993 Wiley‐Liss, Inc.
Sequestration and secretion of insulin‐like growth factor‐I by bovine aortic endothelial cells
Authors: Gajdusek Corinne M. , Luo Zhengyu , Mayberg Marc R. 
Published: February 2005

AbstractEndothelial cells elaborate growth promoting activities in culture medium that support limited smooth muscle cell and fibroblast growth in vitro in the absence of serum. We investigated whether insulin‐like growth factor‐I (IGF‐I) was synthesized and secreted by bovine aortic endothelial cells in vitro. Subconfluent endothelial cell cultures in serum‐free medium secreted severalfold higher IGF‐I levels than confluent cultures by acid‐sizing chromatography and IGF‐I radioimmunoassay. The IGF‐I secretory level was not sustained during a second serum‐free incubation. In contrast, secretion of IGF binding proteins persisted and was maintained at constant levels throughout the same observation periods. Analysis of poly (A+)RNA by northern blots revealed hybridization of an IGF‐I cDNA to a 7.5‐ to 7.0‐kb transcript and superinduction of the 7.5–7.0‐kb mRNA by the translational inhibitor, cyclohexamide. However, no endogenously labeled IGF‐I was detected in conditioned media after incubation of cultures with [35S]cysteine or [3H]leucine. When cultures were incubated in the presence of serum supplemented with IGF‐I, subconfluent cultures sequestered and released more IGF‐I than confluent cultures. We concluded that the majority of IGF‐I secreted in vitro was sequestered from serum. © 1993 Wiley‐Liss, Inc.
Transduced endothelial cells expressing high levels of tissue plasminogen activator have an unaltered phenotype in vitro
Authors: Jaklitsch Michael T. , Biro Sadatoshi , Casscells Ward , Dichek David A. 
Published: February 2005

AbstractElevated cellular plasminogen activator activity has been associated with significant alterations in the in vitro phenotype of both malignant cell lines and nonmalignant endothelial cells. To examine the role of elevated cellular plasminogen activator activity in the production of altered endothelial cell behavior, bovine coronary artery endothelial cells were transduced with a retroviral vector expressing large amounts of tissue plasminogen activator. Cells transduced with the tissue plasminogen activator vector were compared with both untransduced cells and cells transduced with a control vector in a series of in vitro assays of cellular attachment, proliferation, migration, and invasion. The morphology of the 2 transduced populations was unchanged. There was a small decrease (5–15%) in the horizontal migration rate of both transduced cell populations, as compared with that of untransduced cells. No significant differences were detected among the three cell populations in any of the other assays. We conclude that expression of high levels of tissue plasminogen activator does not specifically affect endothelial cell phenotype in vitro. These data lend support to the hypothesis that elevated plasminogen activator activity is necessary but not sufficient to produce alterations in endothelial cell behavior. © 1993 Wiley‐Liss, Inc.
Altered calcium homeostasis and cell injury in silica‐exposed alveolar macrophages
Authors: Rojanasakul Yongyut , Wang Liying , Malanga Carl J. , Ma Jane Y. C. , Banks Daniel E. , Ma Joseph K. H. 
Published: February 2005

AbstractThere is evidence to suggest that cell injury induced in alveolar macrophages (AM) following phagocytic activation by silica particles may be mediated through changes in intracellular free calcium [Ca2+]i. However, the mechanism of silica‐ induced cytotoxicity relative to [Ca2+]i overloading is not yet clear. To provide a better insight into this mechanism, isolated rat AMs were exposed to varying concentrations of crystalline silica (particle size < 5 μm in diameter) and the fluctuation in their [Ca2+]i and cell integrity were quantitatively monitored with the fluorescent calcium probe, Fura‐2 AM, and the membrane integrity indicator, propidium iodide (PI). Results from this study indicate that silica can rapidly increase [Ca2+]i in a dose‐dependent manner with a characteristic transient calcium rise at low doses (<0.1 mg/ml) and an elevated and sustained rise at high doses (>0.1 mg/ml). Depletion of extracellular calcium [Ca2+]o markedly inhibited the [Ca2+]i rise (≈90%), suggesting that Ca2+ influx from extracellular source is a major mechanism for silica‐induced [Ca2+]i rise. When used at low doses but sufficient to cause a transient [Ca2+]i rise, silica did not cause significant increase in cellular PI uptake during the time of study, suggesting the presevation of membrane integrity of AMs under these conditions. At high doses of silica, however, a marked increase in PI nuclear fluorescence was observed. Depletion of [Ca2+]o greatly inhibited cellular PI uptake, induced by 0.1 mg/ml or higher doses of silica. This suggests that Ca2+ influx, as a result of silica activation, is associated with cell injury. Indeed, our results further demonstrated that the low dose effect of silica on Ca2+ influx is inhibited by the Ca2+ channel blocker nifedipine. At high doses of silica (>0.1 mg/ml), cell injury was not prevented by nifedipine or extracellular Ca2+ depletion, suggesting that other cytotoxic mechanisms, i.e., nonspecific membrane damage due to lipid peroxidation, are also responsible for the silica‐induced cell injury. Silica had no significant effect on cellular ATP content during the time course of the study, indicating that the observed silica‐induced [Ca2+]i rise was not due to the impairment of Ca2+‐pumps, which restricts Ca2+ efflux. Pretreatment of the cells with cytochalasin B to block phagocytosis failed to prevent the effect of silica on [Ca2+]i rise. Taken together, these results suggest that the elevation of [Ca2+]i caused by silica is due mainly to Ca2+ influx through plasma membrane Ca2+ channels and nonspecific membrane damage (at high doses). Neither ATP depletion nor Ca2+ leakage during phagocytosis was attributed to the silica‐induced [Ca2+]i rise. © 1993 Wiley‐Liss, Inc.