bluuebunny/crossref_metadata_2025 huggingface dataset:
Regulation of c‐myc and c‐Ha‐ras oncogene expression by cell shape
Authors: Farrell Robert E. , Greene James J.
Published: February 2005

The influence of cell shape on the expression of proto‐oncogenes was examined in normal and malignant human cells that varied in their sensitivities to contact‐inhibition of proliferation. Cells were constrained into varying degrees of roundness by plating onto culture surfaces coated with different concentrations of poly(2‐hydroxyethyl methacrylate) (poly[HEMA]) and assayed for proliferation capacity and levels of c‐myc, c‐ras, c‐fos, and c‐fes mRNAs. Proliferation of contact‐inhibited normal CUA‐1 fibroblasts and the variant HT‐IFNr cells was highly coupled to cell shape. As these cells became more rounded, a critical degree of roundess was reached at which proliferation ceased. In contrast, proliferation of non‐contact‐inhibited maligant HT‐1080 cells was independent of cell shape. Northern analysis revealed that expression of c‐myc and c‐ras was highly sensitive to cell shape in the normal CUA‐1 cells but not in the malignant HT‐1080 or variant HT‐IFNr cells. Levels of c‐myc and c‐ras mRNAs declined to nearly undetectable levels in CUA‐1 cells at degrees of roundness that correlated with loss of proliferative ability. Expression of c‐fos and c‐fes oncogenes were independent of cell shape in all cells tested. Quantification of transcription rates by the nuclear run‐off assay showed that shape modulation of c‐myc and c‐ras oncogene expression occurred at the transcriptional level. These data suggest that changes in cell shape can modulate expression of certain oncogenes and that these changes correlate with the cell's ability to proliferate. Moreover, inability to regulate c‐myc and c‐ras oncogene expression is associated with loss of shape‐dependent growth controls and contact inhibition but that loss of this regulation alone is not sufficient to release cells from contact‐inhibited controls. © 1992 Wiley‐Liss, Inc.
Similar, but not identical, modulation of expression of extracellular matrix components during in vitro and in vivo aging of human skin fibroblasts
Authors: Takeda Koji , Gosiewska Anna , Peterkofsky Beverly
Published: February 2005

Regulation of the synthesis of procollagen and other extracellular matrix components was examined in human skin fibroblasts obtained from donors of various ages, from fetal to 80 years old (in vivo aged), and in fetal fibroblasts at varying passage levels (in vitro aged). Growth rates and saturation densities of fibroblasts decreased with increasing age of the donor and after passage 20 of fetal fibroblasts. The rates of collagen and proteoglycan synthesis also decreased during both types of aging to about 10–25% of the rate in early passage fetal fibroblasts, whereas the synthesis of total noncollagenous proteins was not greatly affected. Decreased collagen synthesis in both types of aging was correlated with lower steady‐state levels of mRNAs for the two subunits of type I procollagen mRNA, although their regulation was not coordinate. Type III collagen mRNA levels also declined in both types of aging. The concentration of fibronectin mRNA also decreased during in vitro aging but more rapidly than the collagen mRNAs, whereas in fibroblasts from 51–80‐year‐old donors, it was similar to or higher than in early passage fetal fibroblasts. This study suggests that the decreased synthesis of procollagen and proteoglycans in in vivo aged fibroblasts represents changes that are responsible for intrinsic degenerative changes that occur in human skin during aging. Furthermore, although in vitro and in vivo aging were similar in many respects, they were not equivalent, as evidenced by the differences in regulation of fibronectin expression. © 1992 Wiley‐Liss, Inc.
Regulation of Cu,Zn‐superoxide dismutase in bovine pulmonary artery endothelial cells
Authors: Kong Xue‐Jun , Fanburg Barry L.
Published: February 2005

To evaluate the regulation of endothelial cell Cu,Zn‐SOD, we have exposed bovine pulmonary artery endothelial cells in culture to hyperoxia and hypoxia, second messengers or related agonists, hormones, free radical generating systems, endotoxin, and cytokines and have measured Cu,Zn‐SOD protein of these cells by an ELISA developed in our laboratory. Control preconfluent and confluent cells in room air contained 196 ± 18 ng Cu,Zn‐SOD/106 cells. A23187 (0.33 μM), for skolin (10 μM), isobutylmethylxanthine (0.1 mM), dexamethasone (1 μM), triiodothyronine (1 μM) and retinoic acid (1 μM) failed to alter this level of Cu,Zn‐SOD. Exposure to anoxia and hyperoxia both elevated the level −1.5–2.0‐fold over 20% oxygen‐exposed controls at 48–72 hr. Similarly, exposures to glucose oxidase (0.0075 units/ml), menadione (12.5 μM), xanthine‐xanthine oxidase (10 μM, 0.03 units/ml) and H2O2 (0.0005%) increased the level up to two–threefold over controls at 24–48 hr. Lipopolysaccharide, TGFβ1, TNFα, and II‐1 also increased levels of cellular Cu,Zn‐SOD, but only in proliferating cells. II‐2, II‐4, interferon‐γ, and GM‐CSF had no effect on Cu,Zn‐SOD. All treatments that elevated SOD resulted in inhibition of cellular growth, but decreased growth of cells at confluence alone was not associated with increased Cu,Zn‐SOD. We propose from these studies that Cu,Zn‐SOD of endothelial cells is not under conventional second messenger or hormonal regulation, but that up‐regulation of the enzyme is associated with (and perhaps stimulated by) free‐radical or oxidant production that also may be influenced by availability of certain cytokines under replicating conditions. © 1992 Wiley‐Liss, Inc.
Inhibition of apoptosis in human tumour cells by okadaic acid
Authors: Song Qizhong , Baxter Glenn D. , Kovacs Eva M. , Findik Duygu , Lavin Martin F.
Published: February 2005

Gamma‐radiation, tetrandrine, bistratene A, and cisplatin were all found to induce pronounced morphological changes characteristic of apoptosis and extensive DNA fragmentation in the human BM13674 cell line 8 h after treatment. Apoptosis induced in BM13674 cells by these diverse agents was markedly inhibited by 1 μM okadaic acid, a tumour promoter that inhibits protein phosphatases 1 and 2A. This compound also inhibited the appearance of apoptosis in fresh human leukaemia cells that had been exposed to gamma‐radiation. The inhibition of apoptosis was confirmed using fluorescence microscopy and DNA gel electrophoresis. Dephosphorylation of a limited number of proteins was shown to be associated with apoptosis and okadaic acid prevented these dephosphorylations. Previous studies on the BM13674 cell line showed that an inhibitor of protein synthesis failed to prevent apoptosis in these cells. The present data provides further support that posttranslational modification of proteins, in particular, phosphorylation/dephosphorylation status, plays an important role in inhibition/activation of programmed cell death in different human cells after exposure to several cytotoxic agents. © 1992 Wiley‐Liss, Inc.
Transforming growth factor‐β1 (TGF‐β1) up‐regulation of collagen type II in primary cultures of rabbit articular chondrocytes (RAC) involves increased mRNA levels without affecting mRNA stability and procollagen processing
Authors: Galéra Philippe , Vivien Denis , Pronost Stéphane , Bonaventure Jacky , Rédini Françoise , Loyau Gérard , Pujol Jean‐Pierre
Published: February 2005

The effect of transforming growth factor‐β1 (TGF‐β1) on collagen biosynthesis was investigated in confluent primary monolayer cultures of rabbit articular chondrocytes (RAC). Exposure to TGF‐β (0.1, 1, and 10 ng/ml) in serum‐free medium caused a dose‐ and time‐dependent stimulation of collagen biosynthesis associated with an increase of steady‐state levels of procollagen type II mRNA. Elevation of the mRNA steady‐state did not result from a stabilization of the transcript, as shown by measure of the mRNA half‐life. Electrophoresis (SDS‐PAGE) showed that TGF‐β stimulates the synthesis of most collagen isotypes, including type II, without qualitative change in their distribution. Moreover, pulse‐chase experiments revealed that TGF‐β did not affect the processing rate of type II procollagen.

TGF‐β slightly stimulated the production of prostaglandin E2 (PGE2), which could in turn exert an inhibition on collagen synthesis. However, addition of indomethacin to block prostaglandin synthesis did not further enhance the TGF‐β‐induced stimulation of collagen production, suggesting that this mediator was not implicated in the effect. Moreover, TGF‐β increased steady‐state levels of procollagen type II, I, and III mRNAs even in the presence of indomethacin. Despite these increased mRNA levels, only the production of type II collagen was significantly augmented, suggesting that type I procollagen mRNA was not fully translated. In addition, the TGF‐β‐induced stimulation of collagen synthesis was observed whenever ascorbic acid is added or not in the culture medium. In conclusion, TGF‐β, which is present in great amount in bone and cartilage, can increase the collagen production of cultured RAC and might therefore play a role in the early events of cartilage repair, such as those observed in osteoarthritis. © 1992 Wiley‐Liss, Inc.
Differential effects of SPARC and cationic SPARC peptides on DNA synthesis by endothelial cells and fibroblasts
Authors: Funk Sarah E. , Sage E. Helene
Published: February 2005

SPARC (secreted protein, acidic and rich in cysteine), also known as osteonectin, is an extracellular Ca+2‐glycoprotein that inhibits the incorporation of [3H]‐and delays the onset of S‐phase in synchronized cultures of bovine aortic endothelial (BAE) cells. This effect appears not to be dependent on the functional properties of SPARC associated with changes in cell shape or inhibition of cell spreading. In this study we investigate the conditions under which cell cycle modulation occurs in different types of cells. Human umbilical vein endothelial cells, a transformed fetal BAE cell line, and bovine capillary endothelial cells exhibited a sensitivity to SPARC and a cationic peptide from a non‐Ca+2‐region of SPARC (peptide 2.1, 0.2—0.8 mM) similar to that observed in BAE cells. In contrast, human foreskin fibroblasts and fetal bovine ligament fibroblasts exhibited an increase in the incorporation of [3H]‐in the presence of 25 μM—0.2 mM peptide 2.1; inhibition was observed at concentrations in excess of 0.4 mM. This biphasic modulation could be further localized to a sequence of 10 amino acids comprising the N‐terminal half of peptide 2.1. A synthetic peptide from another cationic region of SPARC (peptide 2.3) increased [3H]‐incorporation by BAE cells and fibroblasts in a dose‐dependent manner. In endothelial cells, a stimulation of 50% was observed at a concentration of 0.01 mM; fibroblasts required ∼ 100‐fold more peptide 2.3 for levels of stimulation comparable to those obtained in endothelial cells. The observation that SPARC and unique SPARC peptides can differentially influence the growth of fibroblasts and endothelial cells in a concentration‐dependent manner suggests that SPARC might regulate proliferation of specific cells during wound repair and remodeling. © 1993 Wiley‐Liss, Inc.
Role of the plasma membrane in signal transduction in human polymorphonuclear leukocytes
Authors: DelBuono Brian J. , Simons Elizabeth R.
Published: February 2005

To more closely examine the role of the cell surface in transmembrane signal transduction in human neutrophils, sealed right‐side‐membrane vesicles free of organellar membrane components were used as models of the plasma membrane. These vesicles, incubated with a fluorescent analogue of the chemotactic peptide fMLP, bound this ligand similarly in extent and kinetics to intact neutrophils. Vesicles responded to this stimulation with a slow increase in internal [Ca++] which was inhibited by EGTA but not by verapamil; the cytosolic Ca++ transient seen in intact cells within 10 sec of stimulation was absent in vesicles. The vesicles also maintained a transmembrane potential (ψ) and were depolarized by the K+ ionophore valinomycin. However, unlike intact cells which hyperpolarized and then depolarized in response to fMLP, the vesicles demonstrated only a sustained hyperpolarization. Vesicles also differed from intact cells by not producing superoxide (O2−) in response to fMLP. Finally, fMLP caused dramatic alterations in membrane vesicle lipid metabolism: at early time points (within 5–10 sec), there was a transient production of diacylglycerol (DAG) concomitant with inositol lipid breakdown, with no apparent hydrolysis of non‐inositol phospholipids. For up to 5 min after stimulation, there was no increase in the levels of phosphatidic acid or of inositol lipids. Thus, a significant portion of the signalling pathway in neutrophils is located at the cell surface or in the plasma membrane and functions independently of intracellular components. Furthermore, the plasma membrane is intimately involved in events occurring during both the early (DAG generation) and late (slow, prolonged rise in [Ca++]) phases of cellular response. In contrast, several of the responses to fMLP (the Ca++ transient, depolarization, generation of O2−, recycling of lipid metabolites) involve signalling machinery not constitutively resident on the neutrophil surface. © 1993 Wiley‐Liss, Inc.
Internalization of basic fibroblast growth factor (bFGF) in cultured endothelial cells: Role of the low affinity heparin‐like bFGF receptors
Authors: Rusnati Marco , Urbinati Chiara , Presta Marco
Published: February 2005

We have shown (Presta et al., Cell Regul., 2:719–726, 1991) that a long‐lasting interaction of basic fibroblast growth factor (bFGF) with endothelial GM 7373 cells is required to induce cell proliferation. In the present work, we have investigated the interaction of 125I‐bFGF with GM 7373 cells, its pathway of internalization, and its intracellular fate under the same experimental conditions previously utilized to assess the mitogenic activity of the growth factor. Cell cultures were incubated with 10 ng/ml 125I‐bFGF for 2 h at 4°C. Then, cells were shifted to 37°C without changing the medium. A rapid down‐regulation of high affinity sites, paralleled by a rapid internalization of 125I‐bFGF, was observed during the first 1–2 h at 37°C. After 6–8 h, also low affinity sites down‐regulate. This was paralleled by a continuous internalization of 125I‐bFGF and by a slow disappearance of the growth factor from the culture medium. This suggests that GM 7373 cells activate, when exposed to bFGF for a long period of time, a late internalization pathway mediated by low affinity sites. This hypothesis was supported by the following experimental evidence: 1) soluble heparin inhibited the prolonged internalization of 125I‐bFGF and its binding to low affinity sites with the same potency; 2) treatment of GM 7373 cells with heparinase, which removes most of the low affinity sites, also inhibited the prolonged internalization of 125I‐bFGF internalized via low affinity sites was partially protected from lysosomal degradation. This was the case also when 125I‐bFGF was internalized in the presence of soluble heparin, suggesting that the complexes bFGF—surface glycosaminoglycan and bFGF—soluble heparin are maintained during the internalization of the growth factor. Moreover, the capacity of soluble heparin to inhibit the mitogenic activity of bFGF also when added to cell cultures several hours after the growth factor indicates that the requirement for a prolonged interaction of bFGF with GM 7373 cells in order to induce cell proliferation might be related to the late internalization of the growth factor via low affinity sites. © 1993 Wiley‐Liss, Inc.
Induction of glucose regulated proteins during growth of a murine tumor
Authors: Cai Jie‐Wei , Henderson Barbara W. , Shen Jun‐Wen , Subjeck John R.
Published: February 2005

Chronic anoxia, glucose starvation, low pH, and numerous other conditions induce the glucose‐regulated system of stress proteins (GRPs), whose principal members are observed at 78, 94, and 170 kDa. These stresses may be expected to occur during growth in untreated tumors. To examine the possibility that GRPs are correspondingly induced, we have examined the protein profiles of small (<0.1 g), intermediate (0.2–0.8 g), and large (>1.8 g) radiation‐induced fibrosarcoma (RIF) tumors grown on C3H mice. One and two‐dimensional gel electrophoresis indicate that the principal GRPs at 78 and 94 are coordinately and substantially increased in large tumor masses, relative to the small, and may be partially increased in the intermediate tumors. Necrotic material removed from large tumors exhibited an identical pattern of GRP induction with no visible indication of protein degradation and also contained a significant fraction of viable cells. Western blot analysis using rabbit antisera raised against the 78 and 170 kDa GRPs also demonstrated the enhanced accumulation of these proteins in the large tumors. The antibody against the 170 kDa GRP was also capable of detecting the induction of this stress protein in large tumors by indirect immunofluorescence analysis. Northern blot studies using a probe for the GRP 78 gene also showed an increase in GRP 78 message in large tumors as well as in RIF cells exposed to anoxic stress in vitro. Two‐dimensional gel electrophoresis indicated that the major heat shock proteins at 70 and 90 kDa were not increased in the larger tumors, and the amount of the 90 kDa species was reduced. Finally, the quantity of vimentin and its degradation products is significantly diminished in large tumors and in anoxic cells. This study demonstrates that RIF tumor cells undergo a glucose regulated stress response in situ during tumor growth. © 1993 Wiley‐Liss, Inc.
Kinetics of 125I‐PDGF binding and down‐regulation of PDGF receptor in arterial smooth muscle cells derived from patients with moyamoya disease
Authors: Aoyagi Masaru , Fukai Naomi , Matsushima Yoshiharu , Yamamoto Mari , Yamamoto Kiyotaka
Published: February 2005

Progressive stenosis or occlusion of bilateral internal carotid arteries by fibrocellular intimal thickening results in cerebral ischemia in moyamoya disease. We recently found that cultured smooth muscle cells (SMC) derived from arteries of patients with moyamoya disease responded poorly to serum mitogens, especially to platelet‐derived growth factor (PDGF). In the present study, we investigated further the binding and processing of 125I‐PDGF, as well as down‐regulation of the PDGF receptors in arterial SMC derived from patients with moyamoya disease. The specific binding sites of 125I‐PDGF were reduced significantly at both 4°C and 22°C on SMC from moyamoya disease compared with those from controls (4.78 vs. 11.92 × 104/cell at 4°C), though the apparen dissociation constant (Kd) were the same. Kinetics of 125I‐PDGF binding at 37°C in cells from moyamoya disease showed fewer binding sites (less than 1/3 of controls) and lower degradation per cell than in those from controls, though no difference was observed in either internalization or degradation of each receptor. When SMC were exposed to lower concentrations of nonlabeled PDGF at 37°C, the percentage of remaining binding sites on cells from moyamoya disease was significantly less than that from controls. This excess down‐regulation of PDGF receptor in SMC from moyamoya disease may be interpreted as insufficent recycling or a decreased intracellular pool of the PDGF receptor. These results are closely correlated with the diminished proliferation responses to PDGF in SMC from moyamoya disease and provide evidence that functional alterations in vascular cells are involved in the mechanism of development of intimal thickening in moyamoya disease. © 1993 Wiley‐Liss, Inc.
Timing of protooncogene expression varies in toxin‐induced liver regeneration
Authors: Schmiedeberg Phyllis , Biempica Luis , Czaja Mark J.
Published: February 2005

Hepatic expression of the protooncogenes c‐fos and c‐myc occurs within 2 h after partial hepatectomy, and these immediate early genes are thought to prime the hepatocytes for subsequent proliferation. To examine whether such gene activation occured in the setting of hepatocyte proliferation after toxic liver injury, protooncogene expression was examined during the regenerative response following liver injury from carbon tetrachloride (CCI4) or galactosamine (GaIN). The pattern of protooncogene expression after CCI4 mirrored that seen after partial hepatectomy, with rises in c‐fos and c‐myc mRNA content within 2 h, and then a rapid return to baseline levels. In contrast, early c‐fos and c‐myc expression did not occur after GaIN injury. Instead GaIN‐induced regeneration led to a delayed and prolonged c‐fos an c‐myc activation which peaked 24–48 h after injury. Increase in c‐jun, jun‐B, and jun‐D mRNA levels also occured in both models at times similar to the rises of c‐fos and c‐myc expression. Although the timing of DNA synthesis was identical after GaIN or CCI4 treatment the proliferative response after GaIN injury was significantly less than that of CCI4, and marked by the histologic appearance of oval cells. The coadministration of 2‐acetylaminofluorene, an inhibitor of differentiated hepatocyte proliferation, together with CCI4 altered the usual pattern of post‐CCI4 protooncogene expression to one resembling that seen after GaIN injury. Thus, the timing of protooncogene expression during liver regeneration may vary considerably. These variations may influence the nature of the proliferative response in terms of which cell types(s) proliferates, and the amount of regeneration that ensures. © 1993 Wiley‐Liss, Inc.
Increase in portal flow induces c‐myc expression in isolated perfused rat liver
Authors: Isomura Hiroshi , Sawada Norimasa , Nakajima Yasuo , Sakamoto Hirofumi , Ikeda Tatsuru , Kojima Takashi , Enomoto Katsuhiko , Mori Michio
Published: February 2005

We examined expression of the c‐myc oncogene in isolated perfused livers to elucidate the mechanisms involved in triggering the proliferation of hepatocytes after partial hepatectomy (PH). During perfusion with a 1:1 mixture of Dulbecco's modified Eagle's medium and the oxygen transport fluid FC‐43, rat livers were two‐thirds resected (PH), and further perfused for 1 1/2 hours at the physiological portal flow throughout the perfusion. Expression of c‐myc in the perfused livers with PH(+) was ten times higher than in those with PH(−). Furthermore, expression of c‐myc in the PH(−) livers perfused with a threefold volume of the physiological portal flow was 5–10 times higher than that in the livers perfused with the physiological portal flow. The perfusates that passed through the livers did not induce DNA synthesis of primary cultured hepatocytes. These results suggest that an increase in the portal flow volume may act as a trigger for hepatocyte proliferation after PH. © 1993 Wiley‐Liss, Inc.
Sex‐dependent effects of 17‐beta‐estradiol on chondrocyte differentiation in culture
Authors: Nasatzky E. , Schwartz Z. , Boyan B. D. , Soskolne W. A. , Ornoy A.
Published: February 2005

This study examined the effects of 17‐beta‐estradiol (E2) on chondrocyte differentiation in vitro. Cells derived from male or female rat costochondral growth zone and resting zone cartilage were used to determine whether the effects of E2 were dependent on the stage of chondrocyte maturation and whether they were sex‐specific. [3H]‐incorporation, cell number, alkaline phosphatase specific activity, and percent collagen production were used as indicators of differentiation. Alakaline phosphatase specific activity in matrix vesicles and plasma membranes isolated from female chondrocyte cultures was measured to determine which membrane fraction was targeted by the hormone. Specificity of the E2 effects was assessed using 17‐alpha‐estradiol. The role of fetal bovine serum and phenol red in the culture medium was also addressed. The results demonstrated that E2 decreases cell number and [3H]‐incorporation in female chondrocytes, indicating that it promotes differentiation of these cells. Alkaline phosphatase specific activity is stimulated in both growth zone and resting zone cells, but the effect is greater in the less mature resting zone chondrocytes. The increase in enzyme activity is targeted to the matrix vesicles in both cell types, but the fold increase is greater in the growth zone cells. In male chondrocytes, there was a decrease in [3H]‐incorporation at high E2 concentrations in resting zone cells at the earliest time point examined (12 hours) and a slight stimulation in alkaline phosphatase activity in growth zone cells at 24 hours. Cells cultured in serum‐free medium exhibited a dose‐dependent inhibition in alkaline phosphatase activity when cultured with E2, even in the presence of phenol red. E2‐stimulation of enzyme activity is seen only in the presence of serum, suggesting that serum factors are also necessary. E2 increased percent collagen production in female cells only; the magnitude of the effect was greatest in the resting zone chondrocyte cultures. The results of this study indicate that the effects of E2 are dependent on time of exposure, presence of serum, and the sex and state of maturation of the chondrocytes. E2‐stimulation of alkaline phosphatase specific activity is targeted to matrix vesicles. © 1993 Wiley‐Liss, Inc.
Effect of amino acid analogs on the development of thermotolerance and on thermotolerant cells
Authors: Laszlo Andrei , Li Gloria C.
Published: February 2005

Exposure of HA‐1 Chinese hamster fibroblasts to amino acid analogs has been shown to have a heat‐sensitizing effect as well as inducing the heat shock response (Li and Laszlo, 1985a). In this study, we have examined the effect of amino acid analogs on the development of thermotolerance after a brief heat shock or exposure to sodium arsenite and the effect of amino acid analogs on cells that are already thermotolerant. Exposure of HA‐1 cells to amino acid analogs inhibited the development of thermotolerance following a mild heat shock or treatment with sodium arsenite. However, cells that were already thermotolerant were resistant to the sensitizing action of amino acid analogs. The refractoriness of thermotolerant cells to amino acid analog treatment developed in parallel with thermotolerance. The uptake of the arginine analog, canavanine, and its incorporation into proteins was not altered in the thermotolerant cells. Furthermore, another biological consequence of exposure to amino acid analogs, sensitization to ionizing radiation, also was not altered in the thermotolerant cells. The inhibition of the development of thermotolerance by amino acid analogs and the refractoriness of thermotolerant cells to the heat‐sensitizing action of amino acid analogs lend further support the role of heat‐shock proteins in the phenomenon of thermotolerance. © 1993 Wiley‐Liss, Inc.
Stimulation of bone resorption and osteoclast clear zone formation by low pH: A time‐course study
Authors: Murrills Richard J. , Stein Linda S. , Dempster David W.
Published: February 2005

The significance of low pH‐induced stimulation of osteoclastic bone resorption has recently been questioned following the finding that embryonic chick osteoclasts were only weakly stimulated by extremely low pH (6.5) and that the effect was transient, apparently due to cytotoxicity. Although low pH in the range 6.8–7.2 is known to stimulate rat osteoclasts over 24 h, the long‐term effects of low pH on mammalian osteoclasts are not known. We have therefore conducted time‐course studies over 72 h on the effect of pH in the range 6.3–7.3 on bone resorption and cytotoxicity in both rat and chick osteoclasts. In neonatal rat osteoclasts, lowering extracellular pH produced a powerful and significant stimulation of resorption over 24 h. Detailed analysis of the resorption focus revealed that this was due mainly to a higher proportion of active osteoclasts at lower pH. In addition, osteoclasts excavated slightly larger pits at low pH. Stimulation was no longer significant at 72 h, however, due to a pH‐dependent slowing of resorption at acid pH associated 1) with cytotoxicity primarily of nonosteoclastic cells and 2) with an acceleration of bone resorption after 24 h at more alkaline pH. Resorption stimulated by low pH was associated with the formation of actin‐rich “clear zones” within the osteoclast. Chick osteoclasts were less sensitive to low pH than rat osteoclasts but nonetheless showed a consistently higher level of resorption at low pH over 24–72 h. These results suggest that protons play an important regulatory role in neonatal rat osteoclasts, and stimulate the formation of clear zones. The lower sensitivity of the chick osteoclast to acid pH may be due to a species difference or the chick osteoclast's higher basal level of resorption. © 1993 Wiley‐Liss, Inc.
Ruffling and locomotion: Role in cell resistance to growth factor‐induced proliferation
Authors: Heckman C. A. , Oravecz K. I. , Schwab D. , Pontén J.
Published: February 2005

It has long been known that the growth rate of cells in vitro can be retarded by providing substrates of restricted area. Such experiments were performed with adhesive islets, made by depositing metals onto agarose layers through templates of various sizes. Since normal cells are unable to adhere to agarose, they become confined to the metallic surface. Using such haptotactic islets, we have studied the role of membrane ruffling and cell locomotion in the resistance of AG1523 human fibroblasts to growth factor‐induced mitogenesis. Cells plated on small substrates, i.e., 2,150 μm2 in area, initially showed vigorous ruffling, which was suppressed by 8 h after plating but had resumed again by 12 h. In contrast, cells on larger‐size islets showed a rapid decline and stabilization of ruffling activity. When the growth rate was measured for single cells cultured on haptotactic islets, it was found to increase linearly from areas of 4,280 μm2 up to 425,000 μm2. Since the area needed to saturate the growth response was ∼50‐fold larger than the area occupied by a single cell, the growth inhibition was attributed in part to an interference with locomotion. The implication that locomotion provided positive input into growth control mechanisms was subjected to a direct test by evaluating the effect of nine polypeptide growth factors on the motility of serum‐starved cells. All except TGF‐β1 stimulated movement. Finally, the mitogenic effect of growth factors was measured by [3H]thymidine incorporation and found to be proportional to motile activities, as quantitatively assayed. We conclude that locomotion suppression is a factor in AG1523 cell resistance to growth factor‐induced mitogenesis. © 1993 Wiley‐Liss, Inc.
Retinoic acid induces adult muscle cell differentiation mediated by the retinoic acid receptor‐α
Authors: Halevy Orna , Lerman Orna
Published: February 2005

Retinoic acid (RA) is known to induce differentiation in many cell systems. This induction is mediated by nuclear RA receptors (RARs), which act as transactivating factors belonging to the superfamily of steroid/thyroid nuclear receptors. In this study, effects of RA were studied in a mouse myogenic C2 cell line and in primary chicken satellite cells, the myogenic precursor cells in adult muscle. Addition of RA decreased the rate of DNA synthesis in a dose‐dependent manner in both cultures. Differentiation was more rapid in cells treated with RA, and these cells exhibited large myotubes after 48 hr of incubation as compared to untreated cells, which were still proliferating. Addition of 10−8 M RA to C2 cells elevated mRNA levels of myogenin, a skeletal muscle‐specific transcription factor. In parallel, activity levels of the muscle‐specific creatine kinase were enhanced in the RA‐treated cells nearly twofold as compared to the untreated cells. RA treatment of both mouse C2 and chicken satellite cells caused rapid induction of the RAR‐α mRNA levels. Maximal mRNA levels were observed after 2 to 5 hr followed by a sharp reduction to nearly zero levels at 9 hr. The RAR‐α mRNA levels augmented in a dose‐dependent manner between concentrations of 10−10 M and 10−8 M RA, whereas higher concentrations caused mRNA levels to decrease. These results indicate that RA induces differentiation in both adult skeletal muscle primary satellite cells and a myogenic cell line. The rapid and specific induction of RAR‐α mRNA in these cells upon exposure to RA may suggest that this receptor is the primary target and a mediator of RA. © 1993 Wiley‐Liss, Inc.
Regulation of fibronectin and laminin binding activity in cultured human lymphoblastic cell lines
Authors: Stoolman L. M. , Wang Tai‐Ling , Situ Rui , Varani J.
Published: February 2005

The current study shows that a clonal derivative of the Jurkat cell line up‐regulates both the avidity and density of the α 6/β1 receptor in response to phorbol 12‐myristate 13‐acetate (PMA). This derivative attaches to fibronectin and, to a lesser degree, laminin constitutively. Adhesion and spreading are dramatically up‐regulated following treatment with PMA. The response on fibronectin peaks within 4 hours, is insensitive to cyclohexaminde, can be blocked by monoclonal antibodies (Mabs) to the β1 and α 5 subunits of the β1 family of integrins, and is not associated with increased expression of the α 5 or β1 epitopes at the cell surface. In contrast, the response on laminin is biphasic. The early phase parallels the response on fibronectin. The second phase peaks after 48–72 hours of treatment with PMA, is sensitive to cycloheximide, can be blocked by Mabs to the β1 and α 6 subunits, and is associated with increased expression of the α 6 epitope. Both the density independent and dependent responses to PMA in Jurkat cells are blocked by the protein kinase inhibitor staurosporine. The HSB‐2, CEM, Molt‐4, and HPB‐ALL T‐lymphoblastic cell lines also up‐regulate attachment to fibronectin and laminin following treatment with PMA. All four lines constitutively attach to fibronectin and show rapid up‐regulation of attachment following treatment with PMA. None of the lines attach to laminin prior to PMA treatment; however, specific adhesion developed after 4–120 hours of treatment. The most mature lines (Jurkat and HPB‐ALL) up‐regulated adhesion on laminin more rapidly than the less phenotypically mature lines (CEM, Molt‐4, and HSB‐2). In summary, clonal derivatives of the Jurkat cell line up‐regulated attachment to laminin through protein kinase dependent increases in α /β1 receptor avidity and density. In addition, the expression of functional receptors for laminin is linked to developmental maturity in a series of T‐lymphoblastic cell lines. © 1993 Wiley‐Liss, Inc.
Intracellular signalling by nucleotide receptors in PC12 pheochromocytoma cells
Authors: Raha Sandeep , De Souza Lynne R. , Reed Juta K.
Published: February 2005

The effect of extracellular ATP was studied in PC12 cells, a neurosecretory line that releases ATP. The addition of micromolar concentrations of ATP to PC12 cells evoked a transient increase in the cytosolic free Ca2+ concentration ([Ca2+]i), as measured with the Ca2+‐dye fura 2. AMP and adenosine were without effect, ruling out the involvement of P1 receptors in mediating this response. The increase in [Ca2+]i was reduced in calcium‐free media and virtually eliminated by the addition of EGTA, suggesting that calcium influx was the primary response initiated by extracellular ATP. Nucleotide triphosphates such as UTP and, to a lesser degree, ITP also evoked an increase in [Ca2+]i while GTP and CTP had little effect. In order to identify the receptor subtype mediating this response, the efficacy of ATP and ATP cogeners was assessed. The rank order potency was ATP > adenosine 5′‐[γ‐thio]triphosphate > ADP > 2‐methylthioadenosine triphosphate (2‐MeSATP) ∼ adenosine 5′‐[β‐thio]diphosphate ≫ adenosine 5′‐[αβ‐methylene] triphosphate, adenosine 5′‐[βγ‐imido]triphosphate. This profile is not characteristic of either the P2X or the conventional P2Y receptors. The Ca2+ response exhibited desensitization to ATP that was dependent on the extracellular metabolism of ATP. UTP was equally effective in desensitizing the response. ATP, UTP, ITP, and to a much lesser extent 2MeSATP increased inositol phosphate production in a dose‐dependent manner, suggesting receptor coupling to phosphatidylinositol‐specific phospholipase C. These data are consistent with the view that PC12 cells express a class of non‐P2Y nucleotide receptors (P2N) that mediate calcium influx and the accumulation of inositol phosphates. © 1993 Wiley‐Liss, Inc.
Nitric oxide and endothelin secretion by brain microvessel endothelial cells: Regulation by cyclic nucleotides
Authors: Durieu‐Trautmann O. , Fédérici C. , Créminon C. , Foignant‐Chaverot N. , Roux F. , Claire M. , Strosberg A. D. , Couraud P. O.
Published: February 2005

Endothelin (ET)‐1 was originally characterized as a potent vasoconstrictor peptide secreted by vascular endothelial cells. It possesses a wide range of biological activities within the cardiovascular system and in other organs, including the brain. Also secreted by endothelial cells, nitric oxide (NO), has recently been identified as a relaxing factor, as well as a pleiotropic mediator, second messenger, immune defence molecule, and neurotransmitter. Most of the data concerning the secretion of these two agents in vitro has been collected from studies on macrovascular endothelial cells. Given the remarkable heterogeneity of endothelia in terms of morphology and function, we have analyzed the ability of brain microvessel endothelial cells in vitro to release ET‐1 and NO, which, at the level of the blood‐brain barrier, have perivascular astrocytes as potential targets. The present study was performed with immortalized rat brain microvessel endothelial cells, which display in culture a non transformed phenotype. Our data demonstrate that: (1) these cells release NO when induced by IFNγ and TNFα, (2) they constitutively secrete ET‐1, and (3) cAMP potentiates the cytokine‐induced NO release and exerts a biphasic regulation on ET‐1 secretion: micromolar concentrations of 8‐Br‐cAMP inhibit and higher doses stimulate ET‐1 secretion. This stimulation is blocked by EGTA and the calmodulin antagonist W7, but not by protein kinase C inhibitors, suggesting the involvement of the calmodulin branch of the calcium messenger system. These results suggest that cerebral microvessel endothelial cells may participate in vivo to the regulation of glial activity in the brain through the release of NO and ET‐1. © 1993 Wiley‐Liss, Inc.
Modulation of transforming growth factor‐beta 1 stimulated elastin and collagen production and proliferation in porcine vascular smooth muscle cells and skin fibroblasts by basic fibroblast growth factor, transforming growth factor‐α, and insulin‐like growth factor‐I
Authors: Davidson Jeffrey M. , Zoia Ornella , Liu Ji‐Min
Published: February 2005

During tissue repair and development, matrix accumulation is modulated as multiple signals impinge on target cells. We have investigated the effects of combinations of the mitogenic cytokines, basic fibroblast growth factor (bFGF), transforming growth factor alpha (TGF‐α), and insulin‐like growth factor‐1 (IGF‐1) with transforming growth factor‐beta 1 (TGF‐β1) with respect to the production of two matrix components, elastin and type I collagen. Using specific enzyme‐linked immunoassays for detection of secreted precursors in both vascular smooth muscle cells and skin fibroblasts from the domestic pig, production of these two fibrous proteins was shown to be strongly stimulated by TGF‐β1. In the smooth muscle cell, both bFGF and TGF‐α were potent antagonists of TGF‐β1‐mediated matrix production, whereas IGF‐1 was only weakly additive with respect to elastin production. Antagonism was also evident to a lesser extent in skin fibroblasts. Reduced responsiveness to TGF‐β1 did not appear to be due to a switch to a proliferative state, since TGF‐β1 itself acted as a mitogen in confluent SMC, and TGF‐α was only a weak mitogen in confluent fibroblast cultures. Although a predominant effect of TGF‐β is matrix accumulation, these findings suggest that this property will be significantly modified by the cytokine context. © 1993 Wiley‐Liss, Inc.
Heparin‐like glycosaminoglycans participate in binding of a human trophoblastic cell line (JAR) to a human uterine epithelial cell line (RL95)
Authors: Rohde Larry H. , Carson Daniel D.
Published: February 2005

In vitro studies in our laboratory have indicated that heparan sulfate proteoglycans (HSPGs) play an important role in murine embryo implantation. In order to investigate the potential function of HSPGs in human implantation, two human cell lines (RL95 and JAR) were used to model uterine epithelium and embryonal trophectoderm, respectively. A heterologous cell‐cell adhesion assay was developed to determine if binding of JAR cells to RL95 cells was heparan sulfate–dependent. Labeled, single cell suspensions of JAR cells attached to confluent monolayers of RL95 cells in a dose‐ and time‐dependent manner. Heparin‐like glycosaminoglycans and JAR cell proteoglycans competitively inhibited JAR cell adhesion to RL95 cells by 50% or more. A panel of chemically modified heparins were used to demonstrate that O‐sulfation and amino group substitution were critical for inhibition of cell‐cell adhesion. Treatment with chlorate, an inhibitor of A ATP‐sulfurylase, resulted in a 56% reduction in cell‐cell binding compared to untreated controls. Heparinase and chondroitinase ABC markedly inhibited JARRL95 binding, while chondroitinase AC had no significant effect. These observations indicated that HSPGs as well as dermatan sulfate–containing proteoglycans participated in cell‐cell binding. Collectively, these results indicate that initial binding interactions between JAR and RL95 cells is mediated by cell surface glycosaminoglycans (GAGs) with heparin‐like properties (i.e., heparan sulfate and dermatan sulfate). These observations are consistent with an important role for HS and heparin‐like GAGs as well as their corresponding binding sites in early stages of human trophoblast‐uterine epithelial cell binding.
Characterization of an unusual mutant of human melanoma cells resistant to anticancer drugs that inhibit topoisomerase II
Authors: Campain Julie A. , Padmanabhan Raji , Hwang Jaulang , Gottesman Michael M. , Pastan Ira
Published: February 2005

The topoisomerase II inhibitor, VP‐16 (etoposide), is an important component in many chemotherapeutic regimens. To cahracterize resistance to this drug, the human melanoma cell line, FEM‐X, was selected in multiple steps with VP‐16. To prevent the development of typical multidrug resistance, an inhibitor of P‐glycoprotein, the tiapamil analog, RO‐11–2933, was added to the selections. The resultant clone FVP3 is 56‐fold resistant to VP‐16 and cross‐resistant to doxorubicin (Adriamycin) (9‐fold) and VM‐26 (27‐fold). These cells are also two‐ to fourfold resistant to m‐AMSA, daunorubicin, and mitoxantrone. FVP3 is not resistant to the P‐glycoprotein substrate vinblastine, does not express the MDR1 gene at detectable levels, and does not show reduced 3H‐VP‐16 accumulation. Unlike other cell lines that exhibit resistance to inhibitors of topoisomerase II, FVP3 has the same level of topoisomerase II expression and activity as FEM‐X. Using live cells treated with VP‐16, band depeletion assays and KCI/SDS precipitation assays show that topoisomerase II from FVP3 is much less susceptible to drug‐induced cleavable complex formation than is that from FEM‐X. This difference in sensitivity to VP‐16 is also detected using lysates from disrupted cells, but not with isolated nuclei devoid of cytoplasmic and membrane components. In addijtion, the topoisomerase li present in nuclear edtracts from FVP3 is not resistant to the effects of VP‐16 as measured by: (1)inhibition of strand passing activity during decatenation of kinetoplast DNA, (2) drug‐induced linearization of plasmid DNA, and (3) immunodepletion by VP‐16. These results suggest that some component of the cytoplasm or cellular membranes, or a factor depleted from nuclei during their isolation, is responsible for the resistance to VP‐16 in FVP3. © 1993 Wiley‐Liss, Inc.
Two zinc‐dependent steps during G1 to S phase transition
Authors: Chesters John K. , Petrie Linda , Lipson Kenneth E.
Published: February 2005

The influence of zinc (Zn) availability on thymidine kinase mRNA concentration has been investigated in cells in which production of the mRNA was regulated by either truncated thymidine kinase promoters or by the SV40 early promoter. Thymidine kinase mRNA concentrations were decreased by low Zn availability even when the promoter was truncated to 80 bp but not when it was replaced by the SV40 promoter. However, thymidine incorporation by the SV40 cells was still sensitive to lack of Zn, suggesting a second Zn‐sensitive process involved in commitment to S phase. The increase in histone H3 mRNA production prior to S phase was not inhibited by lack of Zn leading to a preferential increase in this mRNA in exponentially growing cells deprived of Zn. © 1993 Wiley‐Liss, Inc.
Synergistic action of calcium‐lonophores and hyperthermia is best interpreted as thermal enhancement of calcium toxicity
Authors: Stege G. J. J. , Wierenga P. K. , Konings A. W. T. , Kampinga H. H.
Published: February 2005

It has been shown that no relation exists between [Ca2+]i and hyperthermic cell killing, although heat‐induced increase of [Ca2+]i can be observed in some cell lines. When ionophores are used, dose‐dependent rises in [Ca2+]i may be found. Beyond a certain threshold of ionophore‐induced increases in [Ca2+]i, cells may be killed. Different threshold levels of [Ca2+]i exist in different cell lines. Hyperthermia can act synergistically with calcium ionophores to potentiate cell killing. Since there is no causal relation between [Ca2+]i and heat toxicity, this synergism can be explained as heat enhanced Ca2+ toxicity. In the current report, it is shown that both ionophore‐induced Ca2+ toxicity (37°C) and its potentiation by heat are dependent on extracellular calcium and related to sustained increases in [Ca2+]i. With ionomycin concentrations up to 15 μM, no increase in [Ca2+]i was seen in cells maintained in medium without Ca2+. Ionomycin effects on intracellular compartments were absent, and the drug seemed to act solely on the level of the plasmamembrane. Also, the synergism of heat and ionomycin appeared to act at the plasmamembrane, because depletion of extracellular calcium completely abolished this synergistic effect. The data presented are also discussed in the light of controversies existing in the literature for the role of calcium in hyperthermic cell killing. © 1993 Wiley‐Liss, Inc.