bluuebunny/crossref_metadata_2025 huggingface dataset:
Insulin‐like growth factor binding protein (IGFBP‐3), an inhibitor of serum growth factors other than IGF‐I and ‐II
Authors: Liu L. , Delbé J. , Blat C. , Zapf J. , Harel L.
Published: February 2005

Our results show that an insulin‐like growth factor binding protein, IGFBP‐3, purified from rat serum, is an inhibitor of chick embryo fibroblast (CEF) growth. It abolished DNA synthesis in CEF stimulated by IGF‐l as well as by human serum. Rat IGFBP‐3 and IDF45 (an inhibitory diffusible factor secreted by mouse cells) had the same activities, confirming that they have an intrinsic capacity to inhibit serum stimulation and may be considered as growth inhibitors. Our data show that inhibition by IGFBP‐3 of serum stimulation was not simply the result of its inhibition of IGF present in the serum: 1) While anti‐I lgG was able to completely inhibit stimulation induced by added IGF‐I, it did not decrease stimulation induced by 1% human serum. Anti‐IGF‐II IgG inhibited the stimulation induced by added IGF‐II, but only 25% decreased the stimulation induced by 0.7% serum. The percent inhibition was not significantly increased when the concentration of serum was decreased to 0.2%, which induced 140% stimulation of DNA synthesis; 2) stimulation by 0.2% serum was much more inhibited by IGFBP‐3 than by IgG anti IGF‐II; 3) after separation of IGF‐I and IGF‐II from serum by chromatography of acidified serum proteins on BioGel P150, the remaining serum proteins (with a molecular mass greater than 45 kDa) which were depleted in IGF‐I and ‐II (verified by RIA determination) still stimulated DNA synthesis, and this stimulation was 80% inhibited by IGFBP‐3. © 1992 Wiley‐Liss, Inc.
Quantitative fatty acid analyses in cultured porcine pulmonary artery endothelial cells: The combined effects of fatty acid supplementation and oxidant exposure
Authors: Hart C. Michael , Tolson J. Keith , Block Edward R.
Published: February 2005

Supplemental fatty acids can modify the oxidant susceptibility of pulmonary artery endothelial cells (PAEC) in monolayer culture. In addition, in vivo dietary modifications have altered tissue and animal susceptibility to a variety of forms of oxidant stress. These modifications of oxidant injury have been attributed to changes in the numbers of fatty acid double bonds in cell lipids. We tested this hypothesis by incubating porcine PAEC in culture medium supplemented with either 0.1 mM oieic acid (18: 1ω9) or with an equivalent volume of ethanol vehicle alone (ETOH‐0.1%) for 3 h. After supplementation, PAEC were exposed to either oxidant stress, 100 μM hydrogen peroxide (H2O2) in Hanks' balanced salt solution (HBSS), or to control condition, HBSS alone, for 30 min. Supplemented PAEC were exposed to HBSS or H2O2 either immediately or 24, 48, or 72 h after supplementation. Supplementation with 18:1 protected PAEC from H2O2‐induced injury at all time points. The fatty acid composition of PAEC phospholipid (PL), triglyceride (TG), and free fatty acid (FFA) subclasses was determined using thin layer and gas chromatography. The PL fraction contained the majority of PAEC fatty acids, and H2O2 reduced the polyunsaturates in this fraction regardless of supplementation. Supplementation with 18:1 increased the 18:1 content of PAEC PL, TG, and FFA at all time points, modified other fatty acids to a lesser extent, but failed to alter the overall number of fatty acid double bonds at all time points. These results indicate that modification of double bond number does not fully explain the mechanisms by which changes in lipid composition can modulate oxidant injury. © 1992 Wiley‐Liss, Inc © 1992 Wiley‐Liss, Inc.
Use of peanut lectin and rat mammary stem cell lines to identify a cellular differentiation pathway for the alveolar cell in the rat mammary gland
Authors: Rudland Philip S.
Published: February 2005

The presence of the carbohydrate receptor for PNL has been used to identify the previously described morphological types of epithelial cell produced as the stem cell line rat mammary 25 (Rama 25) differentiates to casein secretory alveolar‐like cells in vitro. Thus when cultures of the epithelial stem cell line Rama 25 are treated with neuraminidase, fluorescently‐conjugated PNL fails to stain cuboidal cells, stains weakly grey cells, and stains strongly the surface of dark cells. When superconfluent cultures of Rama 25 are treated with dimethyl sulfoxide or retinoic acid and prolactin, estradiol, hydrocortisone, and insulin to induce differentiation to alveolar cells, PNL stains strongly the untreated surfaces of droplet cells and casein‐secreting vacuolated cells. PNL‐staining of the derivative cell lines with truncated cellular pathways, and quantitative binding of [125]‐labeled PNL to the cultured cells are consistent with this cellular staining pattern. The presence of the carbohydrate receptor for peanut lectin (PNL) has also been used to identify specific epithelial cell types in different mammary structure of the developing rat mammary gland, as they differentiate to casein secretory alveolar cells in vivo. Thus when different structures of the developing rat mammary gland are treated with neuraminidase, peroxidase‐conjugated PNL fails to stain histochemically the majority of epithelial cells in ducts, stains the cytoplasm of the majority of epithelial cells in terminal end‐buds (TEBs), and stains strongly the luminal surfaces of the majority of epithelial cells in alveolar buds (ABs). PNL also stains the untreated luminal surfaces of alveolar cells, whether or not the cells can be stained with a monoclonal antibody to rat β‐casein. Stimulation of mammary differentiation by an analogue of ethyl retinoate or by perphenazine causes cells in end‐buds to bind PNL without the necessity for their desilylation similar to that seen in casein secretory alveoli of lacting rats. In conclusion the different interconvertin cell types of Rama 25 which form a pathway to casein‐secretory cells in vitro are thus equated with recognisable epithelail cells types in vivo. These results suggest that casein‐secretory cells in invo are generated by similar successive interconversions between the major epithelial cell types present in the different mammary structures in the order: ducts, TEBs, ABs, alveoli, and secretory alveoli. © 1992 Wiley‐Liss, Inc.
Hepatocyte growth factor induces calcium mobilization and inositol phosphate production in rat hepatocytes
Authors: Baffy György , Yang Lijun , Michalopoulos George K. , Williamson John R.
Published: February 2005

The effects of hepatocyte growth factor (HGF) on intracellular Ca2+ mobilization were studied using fura‐2‐loaded single rat hepatocytes. Hepatocytes microperfused with different amounts of HGF responded with a rapid concentration‐dependent rise in the cytosolic free Ca2+ concentration with a maximum increase of 142% at 80 ng/ml of HGF. The lag period of the Ca2+ response was decreased with increasing HGF concentrations, being 64 ± 12 s, 42 ± 6 s, and 14 ± 2 s, respectively, with 8, 20, and 80 ng/ml of HGF. The detailed pattern of Ca2+ transients, however, was variable. Out of 16 cells tested using 20 ng/ml of HGF, 68% showed sustained oscillatory responses, whereas other cells showed a sustained increase in the cytosolic‐free Ca2+ upon exposure to HGF, which was dependent on the presence of extracellular Ca2+. HGF also induced Ca2+ entry across the plasma membrane. Mobilization of Ca2+ by HGF was accompanied by a rapid accumulation of inositol 1,4,5‐trisphosphate (Ins 1,4,5‐P3). The effects of HGF and epidermal growth factor (EGF) were comparable and partly additive for Ins 1,4,5‐P3 production and for the sustained phase of Ca2+ mobilization. Preincubation of cells with 10 μM of genistein to inhibit protein tyrosine kinases abolished the HGF‐induced Ca2+ response and also inhibited HGF‐induced Ins 1,4,5‐P3 production in rat liver cells. These data indicate that early events in the signal transduction pathways mediated by HGF and EGF have in common the requirements for tyrosine kinase activity, Ins 1,4,5‐P3 production, and Ca2+ mobilization. © 1992 Wiley‐Liss, Inc.
Characterization of interactions between CCK‐33 and CCK receptors in isolated dispersed pancreatic acini
Authors: Hosotani Ryo , Chowdhury Parimal , Doi Ryuichiro , Rayford Phillip L.
Published: February 2005

In isolated dispersed pancreatic acini, we have characterized the interactions between cholecystokinin (CCK) and CCK receptors by simultaneously measuring CCK‐33 immunoreactivity and CCK bioactivity. Incubation of acinar cells with CCK‐33 at cell density of 0.2–0.3 mg acinar protein per ml resulted in stimulation of amylase release concomitant with significant and time‐dependent decrease of the immunoreactive CCK. With L‐364,718 (0.1 μM), a specific CCK receptor antagonist, immunoreactive CCK levels in the media were not significantly altered during incubation; however, CCK‐stimulated amylase release was almost completely abolished (94% inhibition). Vasoactive intestinal peptide (1 nM) significantly potentiated CCK stimulated amylase release without affecting immunoreactive CCK in the media. Insulin (167 nM) did not effect the CCK stimulated amylase release or immunoreactive CCK in the media. Incubation of acinar cells with CCK‐33 at 4°C did not affect the levels of immunoreactive CCK; however, a significant change in levels of immunoreactive CCK were found at 37°C at 90 min. Incubation of cell free medium with CCK‐33 in the presence or absence of secreted enzymes revealed no changes in CCK immunoreactivity in the medium at 90 min. Addition of bacitracin in the incubation media did not affect the CCK immunoreactivity or bioactivity. These findings indicate that in isolated rat pancreatic acini, CCK‐33 stimulates amylase release through a receptor that is specifically blocked by L‐364,718. Specificity of the interactions of CCK‐33 with acinar cells in the media appears to be receptor‐mediated and time‐ and temperature‐dependent. © 1992 Wiley‐Liss, Inc.
In vitro effect of extracellular AMP on MCF‐7 breast cancer cells: Inhibition of glycolysis and cell proliferation
Authors: Hugo Ferdinand , Mazurek Sybille , Zander Ute , Eigenbrodt Erich
Published: February 2005

MCF‐7 human breast cancer cells propagated in vitro were treated with adenosine derivatives added to the culture medium. The effects on cell proliferation, glycolysis, and glutaminolysis were investigated. Of all adenosine derivatives tested, AMP was the most efficient inhibitor of cell proliferation. In AMP‐treated cells, DNA synthesis decreased, whereas RNA and protein syntheses rose normally with time. In terms of carbohydrate metabolism, lactate production from glucose was drastically reduced; therefore, most of lactate produced must have been derived from glutamine. Increase in the enzyme activities involved in glutatmate degradation and in the malate‐aspartate shuttle were observed. In contrast, actual glycolytic flux rates declined, whereas key glycolytic enzyme activities increased. Metabolites such as fructose 1,6‐bisphosphate and pyruvate accumulated in AMP‐arrested cells. Based on the lowered NAD level in the AMP‐treated cells, lactate dehydrogenase, but not malate dehydrogenase, was impaired; thereby the whole of glycolysis was inhibited. In compensation, glutamine catabolism was increased. NAD concentrations fell drastically because of the known inhibition of P‐ribose‐PP synthesis through heightened intracellular AMP levels. A hypothetical metabolic scheme to explain these results and to show how extracellular AMP may influence carbohydrate metabolism and cell proliferation is presented. © 1992 Wiley‐Liss, Inc.
Partial characterization of skeletal myoblast mitogens in mouse crushed muscle extract
Authors: Chen Gang , Quinn Lebris S.
Published: February 2005

We have utilized a model system to investigate myotrophic factors released by normal adult mouse muscles following a crush injury. We found that saline extracts from gently crushed mouse muscles (CME) contain potent mitogenic activities which act on primary newborn mouse myoblast cultures, as well as on mouse C2 cells, a mouse myoblast cell line. We compared the activity of CME on mouse myoblasts with that of basic fibroblast growth factor (bFGF) and insulin‐like growth factor I (IGF‐I), two growth factors known to be mitogenic for primary myoblasts (Allen, Dodson, and Lutein: Exp. Cell. Res., 152:154–160, 1984; DiMario and Strohman: Differentiation, 39:42–49, 1988; Allen and Boxhorn: J. Cell. Physiol., 138:311–315, 1989; Dodson, Allen, and Hossner: Endocrinology, 117:2357–2363, 1985; Florini and Magri: Am. J. Physiol., 256:C701–C711, 1989). We found that CME could act in an additive fashion to saturating doses of bFGF to increase proliferation in myoblast cultures. Additionally, CME acted additively to the combination of saturating amounts of bFGF and IGF‐I on both C2 and primary myoblast cultures. We also examined additivity of CME with the combination of saturating doses of bFGF, IGF‐I, transferrin (Tf), platelet‐derived growth factor (PDGF), epidermal growth factor (EGF), adrenocorticotrophin (ACTH), and macrophage colony‐stimulating factor (M‐CSF). Our data indicate that CME contains Tf, as well as one or more uncharacterized mitogens for myoblasts which are distinct from Tf, the IGFs, bFGF, EGF, PDGF, M‐CSF, and ACTH. These uncharacterized mitogens may act independently of known growth factors to stimulate myoblast proliferation, or may act through modulation of known growth factor activities. © 1992 Wiley‐Liss, Inc.
Human aortic endothelial cells express the type I but not the type II receptor for interleukin‐1 (IL‐1)
Authors: Akeson Ann L. , Mosher Laura B. , Woods Connie W. , Schroeder Kendra K. , Bowlin Terry L.
Published: February 2005

Endothelial cells (EC) are very responsive to the proinflammatory cytokine inter‐leukin‐1 (IL‐1). EC are induced by IL‐1 to secrete chemotactic factors and to increase expression of cell surface adhesion molecules leading to increased leukocyte adhesion. Activated EC turther contribute to the inflammatory response by secreting additional cytokines. IL‐1 interacts with EC through high‐affinity cell‐surface receptors. However, the low number of receptors present on EC has made characterization difficult. Further, recent evidence has suggested diversity in the responses of EC from different regions of the vascular system. Interested in the effect of IL‐1 on early atherosclerotic lesion formation, we have characterized the IL‐1 receptors on human aortic endothelial cells (HAEC). Using a direct binding assay, we found that HAEC have 1,000–3,000 IL‐1 receptors per cell and bind IL‐1α with a Kd of 3.5 × 10−10 M. We found that a monoclonal antibody specific for the type I receptor completely blocks IL‐1α binding. The blocking antibody also completely inhibits the IL‐1 induced increase in intracellular adhesion molecule 1 (ICAM‐1) expression by HAEC. Using solution hybridization and ribonuclease protection with an antisense probe, a sensitive method for detection of low abundance mRNA species we found that HAEC as well as human umbilical vein EC (HUVEC) have significant levels of mRNA for the type I IL‐1 receptor. To test whether HAEC might also contain transcripts for the type II IL‐1 receptor, we compared levels of mRNAs by polymerase chain reaction (PCR) amplification of cDNAs reverse‐transcribed from total RNA. We found only transcripts for the type I receptor and not the type II receptor in HAEC. Based on this data, we conclude that aortic endothelial cells respond to IL‐1 through the type I receptor. © 1992 Wiley‐Liss, Inc.
Proliferating cell nuclear antigen (PCNA) is expressed in activated rat skeletal muscle satellite cells
Authors: Johnson Sally E. , Allen Ronald E.
Published: February 2005

Skeletal muscle satellite cells from uninjured muscle of adult animals are generally found to be in a quiescent state, and when cultured, they remain quiescent in vitro for a period of time which is directly related to the age of the donor animal. A technique for studying the activation of satellite cells in primary cultures has been developed and employs proliferating cell nuclear antigen (PCNA) as a marker for entrance into the S phase of the cell cycle. PCNA is a protein involved in DNA replication and is maximally expressed in S phase of the cell cycle. We monitored PCNA expression in satellite cells isolated from young (3 week) and adult (9 month) rats, and our results indicate that satellite cells begin to accumulate PCNA prior to changes in cell number in both age groups. Using ELISA techniques, we demonstrated that addition of an extract of crushed muscle (CME) activated satellite cells and significantly reduced the length of the lag phase in cells from both age groups. Addition of bFGF shortened the lag phase of PCNA synthesis in satellite cells from 3‐week‐old rats but had no effect on the kinetics of PCNA expression in cells from 9‐month‐old rats. Based on our experiments, PCNA expression can be used as a marker to follow the entry of satellite cells into the cell cycle in primary mass cultures. © 1993 Wiley‐Liss, Inc.
Suramin interrupts androgen‐inducible autocrine loop involving heparin binding growth factor in mouse mammary cancer (Shionogi carcinoma 115) cells
Authors: Kasayama Soji , Saito Hiroshi , Kouhara Haruhiko , Sumitani Satoru , Sato Bunzo
Published: February 2005

The androgen‐dependent clonal cell line SC‐3, derived from Shionogi carcinoma 115, secretes a fibroblast growth factor (FGF)‐autocrine growth factor in response to androgen, which is able to bind to FGF receptors. In SC‐3 cells, FGF receptor expression is upregulated by the SC‐3‐derived growth factor, providing a means of amplifying an autocrine loop of cell growth. In the present investigations, the effect of the polysulfonated naphthylurea suramin on this autocrine loop and its amplification in SC‐3 cells were studied. Suramin inhibited androgen‐dependent growth of SC‐3 cells in a concentration‐dependent fashion: ∼50% inhibition was observed at 25 μM. [3H]Thymidine incorporation into the cells stimulated with partially purified SC‐3‐derived growth factor was inhibited by suramin in a similar way. Additionally, suramin inhibited acidic (a) or basic (b) FGF‐induced cell proliferation, though relatively high concentrations were necessary to achieve the maximal inhibition. Pretreatment of SC‐3 cells with suramin decreased cell surface 125I‐bFGF binding without altering dissociation constant (Kd) of the binding sites. When the cells were incubated with 250 μM suramin for 24 h, the maximum binding (Bmax) decreased to almost 50% of the control. Treatment with suramin also decreased the levels of FGF receptor‐1 mRNA to a similar extent, whereas it appeared not to affect the levels of β‐actin mRNA. Moreover, suramin completely blocked androgen‐ or bFGF‐induced accumulation of FGF receptor‐1 mRNA. The inhibitory effects of suramin on FGF receptor expression were reversed by simultaneous addition of high concentrations of bFGF. These results indicate that suramin exerts its potent antiproliferative action on SC‐3 cells through inhibition of an androgen‐inducible autocrine loop involving SC‐3‐derived growth factor and FGF receptor. © 1993 Wiley‐Liss, Inc.
Effect of low extracellular Ca2+ on growth spreading area, cytoplasmic Ca2+ concentration, and intracellular pH in normal and transformed human fibroblasts
Authors: Yoshida Toshimichi , Takahashi Yasuo , Takashima Shiro
Published: February 2005

The transformation of certain cells reduces the requirement of extracellular Ca2+ for growth. The SV‐40 transformed human lung fibroblasts, WI‐38 VA13, require less Ca2+ than normal WI‐38 cells. Spreading area of normal cells decreases when cultured in 10 μM Ca2+ medium. Intracellular calcium concentration ([Ca2+]i), of the normal and transformed cells cultured in 10μM and 2 mM Ca2+ media was measured by the fluorescence microscope technique using fura‐2 as a probe. The [Ca2+], is measured in the resting state and during mobilization by serum or bradykinin stimulation. The lowering of extracellular calcium concentration results in a decrease in the resting state [Ca2+],i of both normal and transformed cells. Although the total decrease in [Ca2+]i is the same for both cell, the rate of decrease is much faster in normal cells than in transformed cells. Low extracellular Ca2+ reduces the number of cells responsive to the serum or bradykinin stimulation and decreases the peak [Ca2+]i value in both cells. In addition, we investigated, using BCECF as a fluorecent probe, the intracellular pH (pHi) of normal and transformed cells maintained at low and normal Ca2+. The low Ca2+ condition makes pHi acidic in normal cells but not in transformed cells. The acidification of the normal cell is accompanied by a decrease in the spreading area of the cells. The decrease of the cell attacment, followed by the reduced spreading area, induced the acidic pHi. These results suggest that the reduced Ca2+ requirement of transformed cells for growth is related to the mechanism of pHi regulation rather than Ca2+ homeostasis and, possibly, to the anchorage‐independent growth, which is a unique feature of transformed cells. © 1993 Wiley‐Liss, Inc.
Establishment of a hepatocytic epithelial cell line from the murine fetal liver capable of promoting hemopoietic cell proliferation
Authors: Hata Masahiro , Nanno Masanobu , Doi Hideyuki , Satomi Susumu , Sakata Tsuneaki , Suzuki Ryuji , Itoh Tsunetoshi
Published: February 2005

Although the fetal liver has been thought to be the main hemopoietic organ in the embryonal period, whether or not hepatocytes play a major role in hemopoiesis remains obscure. We have established an epithelial cell line from the murine fetal liver, which can support hemopoiesis in vitro. The proliferation of the epithelial cells was promoted synergistically by both epidermal growth factor (EGF) and insulin. The cells were identified as epithelial cells by the presence of desmosomes and tight junctions. Cytoplasmic organelles including small mitochondria and dilated Golgi apparati as well as intercellular canalicular structures similar to bile canaliculus also helped in confirming the hepatic origin of the cell line (designated as FHC). The cells in the primary culture were positive for both α ‐fetoprotein and albumin, indicating the hepatocytic nature of the cell line. Cloned FHC cells were demonstrated to have the ability to maintain hemopoietic progenitors in fetal liver and adult bone marrow in the coculture, and among them, FHC‐4D2 clone displayed the greatest activity. Hemopoiesis‐supporting function could also be seen even when bone marrow cells were separated from FHC‐4D2 cells by nitrocellulose membrane. Column chromatography revealed three distinct peaks of hemopoietic activities with different molecular sizes in the supernatant of FHC‐4D2. Neutralization test with antibodies and proliferative response to interleukin‐3 (IL‐3)/granulocyte‐macrophage colony stimulating factor (GM‐CSF)‐IC2 cells demonstrated that the hemopoietic activities were attributed to GM‐CSF and macrophage colony stimulating factor (M‐CSF). Transcripts of GM‐CSF and M‐CSF were readily detectable in Northern blot analysis, whereas no messages for IL‐3, IL‐6, CSF for granulocytes (G‐CSF) or erythropoietin (EPO) were identified. Therefore, this is the first report on the fetal hepatocyte cell line capable of supporting hemopoiesis. © 1993 Wiley‐Liss, Inc.
Cytokine‐induced expression of mRNAs for chemotactic factors in human synovial cells and fibroblasts
Authors: Bédard Pierre‐André , Golds Elaine E.
Published: February 2005

In response to interleukin 1 or tumor necrosis factor, human synovial cells and fibroblasts expressed several genes encoding known chemotactic factors or related proteins. Transcripts for interleukin 8 (IL‐8), gro/MGSA, pAT 464, IP‐10, pAT 744 and Monocyte Chemotactic and Activating Factor (MCAF) accumulated rapidly in IL‐1 and TNF‐treated cells. The inhibition of protein synthesis led to the superinduction of IL‐8 and gro/MGSA mRNAs in IL‐1, but not in TNF‐treated cells. Thus, IL‐1 and TNF are likely to regulate the expression of these mRNAs by different mechanisms. Important cell‐specific differences in mRNA accumulation characterized the expression of chemotactic factor genes. Moreover, only a subset of the same genes was activated in quiescent cells stimulated by serum. Therefore, genes encoding closely related proteins each had a distinct pattern of expression. Continuous stimulation of fibroblasts and synovial cells with IL‐1 resulted in high and prolonged expression of IL‐8 and gro/MGSA mRNAs. These results extend the list of chemotactic factor genes expressed by mesenchymal cells in vitro and suggest a pivotal role for these cells in processes such as chronic inflammation. © 1993 Wiley‐Liss, Inc.
Extracellular matrix produced by cultured corneal and aortic endothelial cells contains active tissue‐type and urokinase‐type plasminogen activators
Authors: Korner Gil , Bjornsson Thorir D. , Vlodavsky Israel
Published: February 2005

Incubation of plasminogen with the subendothelial extracellular matrix (ECM) synthesized by cultured bovine corneal and aortic endothelial cells resulted in generation of fibrinolytic activity, indicated by proteolysis of 125I‐fibrin in a time‐and dose‐dependent manner. Both tissue‐type plasminogen activator (t‐PA) and urokinase‐type plasminogen activator (u‐PA) were identified in the ECM by fibrin zymography, immunoblotting, and inhibition of plasminogen activation by anti‐u‐and anti‐t‐antibodies. Most of the ECM‐resident plasminogen activator (PA) activity did not originate from intracellular PA release occurring when the endothelial cells were lyzed and the ECM exposed, since a comparable amount of PA was associated with the ECM when the cells were lyzed with Triton X‐100 or removed intact by treatment with 2 M urea. Active u‐PA and t‐PA were released from ECM by treatment with heparanase (endo‐β‐D‐), indicating that some of the ECM‐resident PA activity is sequestered by heparan sulfate side chains. These results indicate that both u‐PA and t‐PA produced by endothelial cells are firmly sequestered in an active form by the subendothelial ECM. It is suggested that ECM‐resident plasminogen activators participate in sequential matrix degradation during cell invasion and tumor metastasis. PA activity may also function in release of ECM‐bound growth factors (i.e., basic fibroblast growth factor) and activation of proenzymes (i.e., prothrombin), resulting in modulation of the ECM growth‐promoting and thrombogenic properties. © 1993 Wiley‐Liss, Inc.
Elevated glutathione S‐transferase gene expression is an early event during steroid‐induced lymphocyte apoptosis
Authors: Flomerfelt Francis A. , Briehl Margaret M. , Dowd Diane R. , Dieken Ellen S. , Miesfeld Roger L.
Published: February 2005

Based on the finding that glutathione S‐transferase Yb1 (GST) gene expression is elevated in the regressing prostate of androgen‐ablated rats, we analyzed GST transcript levels during steroid‐induced lymphocyte cell death. It was found that GST gene expression was induced in steroid‐sensitive cells within 4 hr of dexamethasone treatment, required functional glucocorticoid receptor, and was dose‐dependent with regard to hormone. GST expression was not induced in an apoptosis‐defective variant that contained normal levels of functional receptor, indicating that GST up‐regulation was the result of secondary events that occur during steroid‐mediated apoptosis. Using the calcium ionophore A23817 to induce lymphocyte cell death, GST RNA levels were increased in both steroid‐sensitive and steroid‐resistant cell lines, supporting the conclusion that elevated GST expression was the result of cellular processes associated with apoptosis, rather than a direct consequence of steroid‐mediated transcriptional control. The cells were also treated with dibutyryl cAMP to cause cell death; however, this mode of killing did not result in GST up‐regulation. Taken together, these results suggest that GST induction in dexamethasone‐treated T‐lymphocytes occurs early in the steroid‐regulated apoptotic pathway and that this may be a marker of calcium‐stimulated cell death. Based on the known function of GST as an antioxidant defense enzyme and its transcriptional regulation by reactive oxygen intermediates, we propose that the gene product of a primary GR target gene(s) directly or indirectly effects the redox state of the cell. Thus activation of GST gene expression in apoptotic lymphocytes is likely a indicator of oxidative stress, rather than a required step in the pathway. © 1993 Wiley‐Liss, Inc.
Fibroblast growth factor receptor deficiency in dystrophic retinal pigmented epithelium
Authors: Malecaze F. , Mascarelli F. , Bugra K. , Fuhrmann G. , Courtois Y. , Hicks D.
Published: February 2005

The retinal pigmented epithelium (RPE) is known to be site of the primary lesion in inherited retinal dystrophy in the Royal College of Surgeons (RCS) rat, a model for retinitis pigmentosa. Although the only functional defect so far detected in these cells is their failure to efficiently phagocytose shed photoreceptor outer segment debris, the actual cause of photoreceptor cell death is still unknown. Recently the possibility of “trophic factors” important in photoreceptor survival produced by normal RPE but not by dystrophic RPE has been suggested. Hence we decided to investigate the presence and abundance of two candidate diffusible factors, the acidic and basic fibroblast growth factors (aFGF and bFGF, respectively), as well as their high affinity cell surface receptors (FGF‐R). mRNA was isolated from primary cultures of purified normal and dystrophic RPE and analyzed by PCR amplification using specific oligonucleotide primers for aFGF and bFGF: the size and abundance of amplified fragments was similar for both cell types. Also, aFGF protein, detected by immunocytochemistry using specific antisera, appeared to be present in approximately equal amounts and distributed in a similar pattern. However, scatchard analysis of radio‐labelled bFGF binding to primary cultures of normal and dystrophic rat RPE revealed that dystrophic RPE possess only 29% the number of surface receptors compared to congenic normal cells. Furthermore, the level of expression of FGF‐R2 mRNA, but not that of FGF‐R1, was significantly different. Other parameters measured (receptor affinity, profile of ligand internalization and degradation, receptor molecular weight and mitogenic activity) did not show any significant differences between normal and dystrophic RPE. The precise role of FGF‐R deficiency in the etiology of the disease hence remains to be determined, but it indicates the importance of trophic factors in the normal functioning of the retina. © 1993 Wiley‐Liss, Inc.
Role of Ca2+ in stimulation of DNA synthesis by epidermal growth factor and tumor promoters in cultured rat hepatocytes
Authors: Petronijevic Tadija , Edwards Anthony M.
Published: February 2005

This study examines the effects of extracellular Ca2+ concentrations, [Ca2+]o, and of treatments known to modulate intracellular Ca2+ levels on the extent and timing of DNA synthesis in primary cultures of adult rat hepatocytes. In cultures exposed to insulin and EGF, the extent of DNA synthesis between 40 h and 70 h in culture was independent of [Ca2+]o in the range 25–1,800 μM, although the peak of DNA synthesis occurred 5–10 h earlier with 1.2 mM Ca2+ than with 25 μM Ca2+. Complete removal of extracellular Ca2+ using EGTA blocked DNA synthesis if Ca2+ was removed on the second day after EGF addition but not if Ca2+ was absent only on day 1. Treatment of cultures in 1.2 mM Ca2+‐containing media with Ca2+‐ionophore A23187 or with thapsigargin, agents expected to raise cytosolic [Ca2+], failed to augment the stimulation of DNA synthesis by EGF. These observations suggest that hepatocytes may have a permissive requirement for [Ca2+]o > 0 at least late in the sequence of events leading from growth factor stimulation to DNA synthesis. However, sustained elevation of cytosolic [Ca2+] does not appear to be important as an early signalling event either in mediating or augmenting EGF action in hepatocytes. The ability of liver tumor promoters α‐hexachlorocyclohexane or DDT to stimulate DNA synthesis in combination with EGF was independent of [Ca2+]o. By contrast, the skin tumor‐promoting phorbol ester, TPA, or liver tumor promoter, phenobarbital, were without effect or inhibitory at low [Ca2+]o but in combination with EGF, stimulated DNA synthesis at [Ca2+]o > 0.4 mM, suggesting that Ca2+ may have some role in mediating or modulating the stimulatory effects of these agents. © 1993 Wiley‐Liss, Inc.
Mechanical stimulation of skeletal muscle generates lipid‐related second messengers by phospholipase activation
Authors: Vandenburgh Herman H. , Shansky Janet , Karlisch Patricia , Solerssi Rosa Lopez
Published: February 2005

Repetitive mechanical stimulation of cultured avian skeletal muscle increases the synthesis of prostaglandins (PG) E2 and F2α which regulate protein turnover rates and muscle cell growth. These stretch‐induced PG increases are reduced in low extracellular calcium medium and by specific phospholipase inhibitors. Mechanical stimulation increases the breakdown rate of 3H‐arachidonic acid labelled phospholipids, releasing free 3H‐arachidonic acid, the rate‐limiting precursor of PG synthesis. Mechanical stimulation also increases 3H‐arachidonic acid labelled diacylglycerol formation and intracellular levels of inositol phosphates from myo‐[2‐3H]inositol labelled phospholipids. Phospholipase A2 (PLA2), phosphatidylinositol‐specific phospholipase C (PLC), and phospholipase D (PLD) are all activated by stretch. The stretch‐induced increases in PG production, 3H‐arachidonic acid labelled phospholipid breakdown, and 3H‐arachidonic acid labelled diacylglycerol formation occur independently of cellular electrical activity (tetrodotoxin insensitve) whereas the formation of inositol phosphates from myo‐[2‐3H]inositol labelled phospholipids is dependent on cellular electrical activity. These results indicate that mechanical stimulation increases the lipid‐related second messengers arachidonic acid, diacylglycerol, and PG through activation of specific phospholipases such as PLA2 and PLD, but not by activation of phosphatidylinositol‐specific PLC. © 1993 Wiley‐Liss, Inc.
Proliferative and migratory responses of murine microvascular endothelial cells to granulocyte‐colony‐stimulating factor
Authors: Bocchietto Elena , Guglielmetti Antonella , Silvagno Francesca , Taraboletti Giulia , Pescarmona Gian Piero , Mantovani Alberto , Bussolino Federico
Published: February 2005

Microvascular murine endothelial cells lines transformed by middle T oncogene of polyoma virus maintain the biological characteristics of nontransformed microvascular endothelial cells (EC). By using cell lines originated from different anatomical districts (thymus, brain, heart, and skin), we demonstrated that murine granulocyte‐colony‐stimulating factor (G‐CSF) induces proliferation of murine microvascular endothelial cells at nanomolar concentrations without any cooperation with fetal calf serum. The proliferative effect on murine cells is less than that elicited by epidermal growth factor (EGF), used as standard for this function. G‐CSF also promotes the migration of tEnd.1 endothelial cell line assayed by Boyden chamber technique. The analysis of transcript for G‐CSF receptor (G‐CSFR) by Northern blot hybridization and by reverse‐transcriptase polymerase chain reaction (RT‐PCR) shows that these cell lines have specific mRNA, with the size of that present in myeloid cells. These results indicate that G‐CSF operates in the microvascular endothelial cells by a mechanism related to the presence of a specific receptor. © 1993 Wiley‐Liss, Inc.
Proteases are implicated in the changes in the Sertoli cell cytoskeleton elicited by follicle‐stimulating hormone or by dibutyryl cyclic AMP
Authors: Tung Pierre S. , Burdzy Krystyna , Fritz Irving B.
Published: February 2005

Follicle‐stimulating hormone (FSH) or dibutyryl cyclic AMP (dbcAMP) elicits striking morphological changes in Sertoli cells in culture in serum‐free medium, resulting in a transition from an epithelial type of cell association pattern to that of an astrocytic or fibroblast‐like cell, with attenuated cytoplasmic extensions between cells, and with diminished F‐actin stained stress fibers. These responses of Sertoli cells do not occur in the presence of normal untreated serum, but they do take place in the presence of acid‐treated serum which is depleted of antiproteases. The addition of α2‐macroglobulin to serum‐free medium or to antiprotease‐depleted serum resulted in the blockage of morphological responses of Sertoli cells to FSH or to dbcAMP. Changes in pattern of arrangements of F‐actin in Sertoli cells in culture, which occur in response to FSH or to dbcAMP, were also prevented by the presence of α2‐macroglobulin. Thus, the diminution in bundles of F‐actin containing stress fibers, which otherwise takes place in Sertoli cells stimulated by FSH or by dbcAMP, did not occur in cells in culture in the presence of α2‐macroglobulin, in the presence or absence of acid‐treated serum. The inhibiting effects of dbcAMP on the migration of Sertoli cells in serum‐free medium became nondetectable in medium containing normal untreated serum, but remained evident in Sertoli cells in culture in medium containing acid‐treated serum depleted of antiproteases. Addition of α2‐macroglobulin blocked the inhibitory effects of dbcAMP on Sertoli cell migration. Similarly, the presence of α2‐macroglobulin prevented the inhibitory effects of dbcAMP on the contractility of TM4 cells which had been embedded in collagen type‐I and incubated in serum‐free medium. We discuss the possibility that cellular proteases may be implicated in the disintegration of microfilament bundles, either by favoring depolymerization of actin filaments; by facilitating breakage of the link of the transmembrane molecular assembly between cytoskeletal extracellular matrix components; or by catalyzing a disruption of the modular organization of one or more of the actin cross‐linking proteins. By inference, we postulate that morphological responses of Sertoli cells to FSH require the activation of cellular proteases for one or more of these reactions, and that α2‐macroglobulin blocks the Sertoli cell morphological responses to FSH by inhibiting the proteases involved. © 1993 Wiley‐Liss, Inc.
Expression and function of gingival fibroblast C1q receptors are upregulated by interleukin‐1β and transforming growth factor‐β
Authors: Luddington Stephen , Qwarnström E. E. , Page R. C. , Bordin S.
Published: February 2005

In injury and inflammation, complement (C) component C1q, in addition to its central role in initiation of classical pathway of complement activation, modulates diverse cellular functions by binding to specific cell surface receptors. Interaction of substrate‐bound C1q with receptors for the collagen‐like domain of C1q (C1qRC) of human gingival fibroblasts (HGF) promotes cell attachment. We investigated modulation of the adhesive function and expression of C1qRC by interleukin‐1β (IL‐1β) and transforming growth factor‐β (TGF‐β). Confluent fibroblast monolayers were incubated under standard culture conditions with or without cytokines. C1qRC function was measured by attachment assays. IL‐1β and TGF‐β increased fibroblast adhesion to C1q to 146% and 131% of controls, respectively. Cytokine enhancement of HGF adhesion was concentration‐dependent, saturable (20 ng/ml IL‐1β; 1 ng/ml TGF‐β) and time‐dependent (IL‐1β 12‐hr peak; TGF‐β 24‐hr peak). Effect of IL‐1β and TGF‐β on C1qRC expression was assessed by flow cytometry measurements of fluorescence intensity of cells stained with C1q and FITC anti‐C1q antibody, and by binding studies with 125l‐C1q. Cells treated with cytokines displayed a two‐ to four‐fold increased fluorescence of cell‐bound C1q compared to controls. Binding studies indicated the increased fluorescence correlated with increase in number of C1qRC in both IL‐1β (4.7 × 106/cell) and TGF‐β (3.9 × 106/cell)‐treated cells, compared to control (3.0 × 106/cell), but had no effect on binding affinity. Rates of internalization of receptor‐bound C1q were similar in cytokine‐treated cells and controls. We propose from these data that IL‐1β and TGF‐β have the ability to upregulate C1qRC expression, and this effect contributes to increased adhesion of HGF to substrate‐bound C1q. © 1993 Wiley‐Liss, Inc.
Cell cycle dependent regulation of cdc2 mRNA in mouse fibroblasts: Requirement of protein synthesis and of continued mitogenic stimulation
Authors: Buquet‐Fagot Christine , Fagot Dominique , Mester Jan
Published: February 2005

In the chemically transformed mouse fibroblasts (BP‐A31) placed in a serum‐free medium, the cdc2 mRNA content decreases in parallel with the cessation of [3H]thymidine incorporation. Extinction of the cdc2 gene expression is also observed in BP‐A31 cells overexpressing the human c‐myc oncogene. At quies‐cence, the cdc2 gene expression can be reinduced with serum or with other mitogens such as insulin or 12‐O‐tetradecanoyl phorbol 13‐acetate (TPA). The kinetics of induction is characterized by a lag period which differs according to the mitogen used and reflects the length of the G1 phase (4–6 h with insulin or serum, 9–12 h with TPA). The cdc2 mRNA accumulation is prevented when protein synthesis is blocked with cycloheximide, also if the drug is added at a time when the synthesis of cdc2 mRNA is already under way. Similarly, removal of the mitogen leads to a cessation of the cdc2 mRNA accumulation. These results suggest that the increased expression of the cdc2 gene is mediated by (a) short‐lived, growth factor‐regulated protein(s). © 1993 Wiley‐Liss, Inc.
Stretch and anesthetic dependency of atrial natriuretic peptide release demonstrated by an ultrastructural assay
Authors: Newman T. M. , Severs N. J.
Published: February 2005

Using an ultrastructural assay developed to quantify the secretion of atrial natriuretic peptide‐containing granules, release of the hormone, in response to different degrees of atrial distension, is directly demonstrated at the cellular level. The ultrastructural assay developed uses an in situ tannic acid perfusion technique to arrest the exocytosis of atrial granules in the anesthetized rat. Secretory granules, which retain the capacity to undergo exocytosis throughout a 30‐minute tannic acid perfusion, accumulate at the cell surface in a state of fusion with the plasma membrane, with the core contents retained. Quantification of arrested granules thus provides a measure of the rate of granule release and allows the responses to different stimuli to be assessed. By altering the height of the perfusate, perfusion pressure and hence the degree of distension of the right atrium can be increased, and this causes a proportional rise in the release of secretory granules from individual myocytes. An anesthetic regime incorporating fentanyl citrate was found to increase significantly the rate of granule release, and this was further augmented by atrial distension. Quantification of the numbers of cytoplasmic granules under the same conditions did not reveal a reduction in granules. This is thought to be because only a small pool of granules is recruited for exocytosis, and granule production may continue during the perfusion period. Our assay of atrial secretory granule release allows the effect of a variety of stimulatory and inhibitory agents to be assessed directly at the cellular level and provides an independent comparison with previous biochemical data from whole animal and isolated organ studies. © 1993 Wiley‐Liss, Inc.
Characterization of prostaglandin F2α receptor of mouse 3T3 fibroblasts and its functional expression in Xenopus laevis oocytes
Authors: Nakao Akihide , Watanabe Tsuyoshi , Taniguchi Shigeo , Nakamura Motonao , Honda Zen‐Ichiro , Shimizu Takao , Kurokawa Kiyoshi
Published: February 2005

Prostaglandin (PG) F2α increased [3H]thymidine incorporation into quiescent NIH 3T3 cells, stimulated phosphoinositide breakdown, and raised intracellular Ca2+ concentration ([Ca2+]i) in a dose‐dependent manner with ED50 values of 2.0 × 10−8 M, 4.6 × 10−8 M, and 7.5 × 10−8 M, respectively. The increase in [3H]thymidine incorporation with PGF2α was additive with that seen with epidermal growth factor (EGF) or insulin. The peak [Ca2+]i increase with PGF2α was still obvious in the absence of extracellular Ca2+ and was insensitive to islet activating protein (IAP) pretreatment. Membranes prepared from NIH 3T3 cells exhibited a specific binding for PGF2α, which was sensitive to GTPγS but not sensitive to IAP pretreatment. Xenopus laevis oocytes injected with NIH 3T3 cell mRNA between 18S and 28S rRNA fractionated by sucrose gradient, expressed a PGF2α‐specific Cl− current when examined by voltage clamp. This Cl− current was also insensitive to IAP pretreatment and not affected by extracellular Ca2+ concentration ([Ca2+]o). These results indicate 1) that the NIH 3T3 cells expressed a specific PGF2α receptor which is linked to phosphoinositide‐specific phospholipase C (PLC) activation and to mobilization of Ca2+ via an IAP‐insensitive G‐proteins(s), 2) that this PGF2α receptor may play an active role in the proliferation of NIH 3T3 cells, and 3) that this PGF2α receptor can be expressed in the oocyte system. © 1993 Wiley‐Liss, Inc.
Protein kinase inhibitor, staurosporine, induces a mature neuronal phenotype in SH‐SY5Y human neuroblastoma cells through an α‐, β‐, and ζ‐protein kinase C‐independent pathway
Authors: Jalava Annika , Åkerman Karl , Heikkilä Jari
Published: February 2005

Previous studies have shown that the tumour‐promoting phorbol ester 12‐O‐tetradecanoyl phorbol‐13 acetate (TPA) induces both morphological and functional differentiation in SH‐SY5Y human neuroblastoma cells (Påhlman et al., 1981). In order to investigate the role of protein kinase C (PKC) in TPA‐induced maturation of SH‐SY5Y cells, we have used staurosporine, which is a potent inhibitor of protein kinases including PKC. Treatment of SH‐SY5Y cells with 25 nM staurosporine for 72 hours caused an appearance of long, neuritelike processes with varicosities, terminated by growth cones. The morphological differentiation was accompanied by a cessation of DNA synthesis, induction of growth associated protein 43 (GAP‐43), and neuropeptide Y (NPY) mRNA. These effects of staurosporine were comparable to those elicited by TPA. Staurosporine further induced a time‐dependent increase in the expression of tyrosine hydroxylase protein and a 30‐fold increase in the concentration of noradrenaline. TPA only induced a margginal increase in tyrosine hydroxylase expression. Both TPA and staurosporine induced an appearance of voltage‐gated Ca2+ channels in SH‐SY5Y cells detected with single‐cell fluorescent measurements using fura‐2. The Ca2+ channels were found almost exclusively in growth cones and varicosities. Staurosporine inhibited both basal and a TPA‐induced phosphorylation of an endogenous 80kDa PKC substrate (p80), and also blocked c‐fos proto‐oncogene mRNA expression induced by the phorbol ester. Bryostatin 1, a potent activator of PKC, has failed to induce morphological or functional differentiation in SH‐SY5Y cells (Jalava et al., 1990). Incubation of SH‐SY5Y cells in the presence of 100 nM bryostatin 1 for 24 hours caused a complete disappearance of all immunoreactive α‐, β‐, and ζ‐PKC. The level of ϵ‐PKC decreased by 70%. Staurosporine induced a partial translocation of the ϵ‐isoenzyme but it failed to cause down‐regulation of ϵ‐PKC. Bryostatin 1‐treatment did not interfere in the ability of staurosporine to induce morphological differentiation, cessation of DNA synthesis, and GAP‐43 and NPY mRNA expression. The ability of staurosporine to stimulate tyrosine hydroxylase expression and to increase cellular content of noradrenaline was also unaffected. Taken together the results of this study show that staurosporine induces a mature neuronal noradrenergic phenotype in SH‐SY5Y cells through an α‐, β‐, and ζ‐PKC‐independent pathway. © 1993 Wiley‐Liss, Inc.