摘要:

AbstractThe effects of sulfated polysaccharides on the growth and chemotaxis of endothelial cells promoted by basic fibroblast growth factor (bFGF), a heparin‐binding growth factor, and epidermal growth factor (EGF), a non‐heparin‐growth factor, were examined. The binding abilities of these two growth factors to D‐gluco‐galactan sulfate (DS‐4152) were the same as to heparin. DS‐4152 inhibited the growth and chemotaxis of the cells stimulated by bFGF, and prevented the binding of bFGF to the cells at both its low and high affinity binding sites: the former and the latter are heparin‐like molecules and receptor proteins for bFGF, respectively. However, DS‐4152 affected neither the binding of EGF to endothelial cells nor the proliferation and chemotaxis of the cells stimulated by the factor. Heparin also inhibited the binding of bFGF to low affinity binding sites to the same degree as DS‐4152, but had little effect on the binding of bFGF to high affinity sites and no effects on bFGF‐induced endothelial cell growth. Chondroitin sulfate A prevented neither the binding of bFGF to both sites of the cells nor bFGF‐induced cell proliferation. We thus concluded that the inhibitory effects of DS‐4152 against the growth and chemotaxis of endothelial cells induced by bFGF might be due to the prevention of bFGF binding to its receptor proteins resulting from the binding of DS‐4152 to b

ISSN:0021-9541    

DOI:10.1002/jcp.1041540102

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1993

数据来源: WILEY

摘要:

AbstractThe effects of rapamycin (RAP) on cell cycle progression of human T cells stimulated with PHA were examined. Cell cycle analysis showed that the RNA content of cells stimulated with PHA in the presence of RAP was similar to that of control T cells stimulated with PHA for 12–24 hr in the absence of the drug. This level was substantially higher than that seen in cells stimulated in the presence of cyclosporin A (CsA), an immunosuppressant known to block cell cycle progression at an early point in the cycle. However, the point in the cell cycle at which RAP acted appeared to be well before the G1/S transition, which occurs about 30–36 hr after stimulation with PHA. In an attempt to further localize the point in the cell cycle where arrest occurred, a set of key regulatory events leading to the G1/S boundary were examined, including p110Rbphosphorylation, which occurred at least 6 hr prior to DNA synthesis, p34cdc2synthesis, and cyclin A synthesis. In control cultures, p110Rbphosphorylation was detected within 24 hr of PHA stimulation; p34cdc2and cyclin A synthesis were detected within 30 hr. Addition of RAP to the cultures inhibited each of these events. In contrast, early events, including c‐fos, IL‐2, and IL‐4 mRNAs expression, and IL‐2 receptor (p55) expression, were only marginally affected, if at all, in PHA‐stimulated T cells. Furthermore, the inhibition of cell proliferation by RAP could not be overcome by addition of exogenous IL‐2. These results indicate that RAP blocks cell cycle progression of activated T cells after IL‐2/IL‐2 receptor interaction but prior to p110Rbphosphorylation and other key regulatory events signaling G1/S transition. ©

ISSN:0021-9541    

DOI:10.1002/jcp.1041540103

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1993

数据来源: WILEY

摘要:

AbstractNormal and SV40 virus‐transformed WI‐38 human lung fibroblasts were serum starved and refed, or synchronized by double thymidine block and released from the block. At different time points in the cell cycle, steady state levels of P120 mRNA and P120 protein content of the cells were determined by densitometric scans of Northern and Western blots. At the same time points, [3H]thymidine uptake was measured and flow cytometric analysis performed for DNA content and P120 antigen staining. Levels of P120 protein and P120 mRNA were approximately 4 times greater in non‐synchronous, exponentially growing transformed cells than in similarly growing normal cells. Early G1‐cells, synchronized either with serum deprivation or with metabolic block, contained only a trace amount of P120 protein and mRNA. The P120 gene was transcribed early in G1and P120 protein synthesis initiated in middle G1. A dramatic increase of P120 protein level occurred in S‐phase with a corresponding mRNA peak preceding the P120 protein peak. These results indicate that P120 is overexpressed in transformed WI‐38 cells and that P120 is temporally regulated during the cell cycle of both transformed and normal fibroblasts. The dramatic increase in P120 protein expression at the G1to S boundary suggests that P120 may play a role in the regulation of cell cycle and increased nucleolar activity that is associated with cell proliferation. © 1993 Wil

ISSN:0021-9541    

DOI:10.1002/jcp.1041540104

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1993

数据来源: WILEY

摘要:

AbstractPrevious studies have shown that atrial natriuretic peptides inhibit mitogenesis in subcultured aortic smooth muscle cells by a mechanism that appears to be mediated via the C‐type or “clearance” receptor. In the current study, we have compared the antimitogenic effect of these peptides in serum‐stimulated primary aortic smooth muscle cell cultures and in subcultured cells. A series of atrial peptides, including rANF99–126, rANF103–126, and rANF103–125, were only poorly antimitogenic in serum‐stimulated primary cultures, whereas des[Cys105, Cys121] rANF104–126which binds selectively to the ANF‐C receptors had no antimitogenic activity. In contrast, in subcultured cells (between subcultures 3 and 25), rANF99–126, rANF103–126, rANF103–126, Cys116rANF102–116, and des[Cys105, Cys121]rANF104–126inhibited serum‐induced [3H]thymidine incorporation (IC50in the range of 10–50 nM), with maximal inhibition of 40–70%. The lack of antimitogenic activity in primary cultures did not appear to be related to the lack of cGMP elevation elicited by atrial peptides or to an inherent insensitivity to the action of antimitogens, because primary cultures were responsive to the cGMP‐elevating effect of atrial peptides and the cells were more rather than less sensitive to the antimitogenic effect of the nitric‐oxide‐vasodilator, SNAP, as compared to subcultured cells. Analysis of the affinity and binding capacity of freshly isolated aortic membranes, and primary or secondary cultures for [125I]rANF99–126, revealed that the number of ANF receptors increased by tenfold, following subculture. Moreover, subcultured cells contained receptors with increased binding affinity for peptide analogues selective for the ANF‐C‐type type receptor. Covalent cross‐linking studies with (125I)rANF99–126confirmed that membranes prepared from fresh aortae predominantly expressed the ANF‐A/guanylate cyclase receptor, whereas in subcultured cells the predominantly cross‐linked protein was the ANF‐C‐type receptor, with receptors in primary cultures occupying an intermediate position. These results suggest that the binding and antimitogenic activity of atrial peptides in aortic smooth muscle cells depends on the phenotypic state of these cells. Moreover, the increased antimitogenic potency of atrial peptides in secondary cultures may reflect increased exp

ISSN:0021-9541    

DOI:10.1002/jcp.1041540105

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1993

数据来源: WILEY

摘要:

AbstractSkeletal 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

ISSN:0021-9541    

DOI:10.1002/jcp.1041540106

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1993

数据来源: WILEY

摘要:

AbstractTransforming growth factor β (TGF‐β) is a cytokine with immunoregulatory properties that acts negatively on T lymphocyte proliferation. However, with the EL 4–6.1 variant of the murine thymoma EL 4 activated with phorbol ester and/or interleukin‐1 (IL‐1), we recently found that it up‐regulates interleukin‐2‐receptor (IL‐2R) expression. Since EL 4–6.1 cells share phenotypic and functional characteristics with the immature thymic subset lacking CD4 and CD8 accessory molecules (DN), we investigated the effect of TGF‐β1 on the IL‐2R 55kD α chain expression and proliferation of activated DN cells and especially in DN cells that do not express CD3. We observed that TGF‐β1 was able to increase both the percentage of CD3−DN cells expressing IL‐2Rα chains and the expression of IL‐2Rα chain in these cells. This stimulatory effect of TGF‐β1 was distal from early transduction events. In addition, TGF‐β1 was found to modulate CD3−DN cell proliferation. During differentiation in the thymus, CD3−DN cells transiently express the IL‐2Rα chain of the IL‐2R and these IL‐2R+CD3−DN cells are preprogrammed to down‐regulate the IL‐2Rα chain and up‐regulate the CD4 and CD8 accessory molecule. We thus also tested the effect of TGF‐β1 on IL‐2Rα chain expression in these in vitro differentiating CD3−DN cells. We found that TGF‐β1 neither significantly affected IL‐2R expression nor changed CD4 or CD8 expression. Hence, in CD3−DN cells, the effect of TGF‐β1 on IL‐2

ISSN:0021-9541    

DOI:10.1002/jcp.1041540107

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1993

数据来源: WILEY

摘要:

AbstractSPARC (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 remode

ISSN:0021-9541    

DOI:10.1002/jcp.1041540108

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1993

数据来源: WILEY

摘要:

Abstract125I‐nerve growth factor (NGF) was found to be internalized and translocated to the nucleus of SKBr5 breast carcinoma cells. The cytoplasm and chromatin isolated from nonmitotic cells accumulated two‐and five‐fold, respectively, more of125I‐NGF than the cells undergoing mitosis. MAb 20.4 developed against the NGF cell surface receptor immunoprecipitated the 80,000 Mrreceptor from plasma membrane and two protein species from the chromatin; 90,000 Mr(major band) and 200,000 Mr(minor band). In SKBr5 cells, binding of NGF to the chromatin did not affect synthesis of rRNA. Proliferation of SKBr5 cells was slightly stimulated by NGF. In control melanoma A875 cells, which express the 230,000 Mrchromatin receptor, NGF inhibited both rRNA synthesis and cell proliferation. We suggest that the 90,000 Mrchromatin receptor expressed by SKBr5 cells represents a “nonactive”, ligand‐binding subunit of the high molecular weight receptor for NGF. The critical role of the chromatin receptor for NGF in rRNA‐dependent cell proliferation is discussed. © 1993

ISSN:0021-9541    

DOI:10.1002/jcp.1041540109

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1993

数据来源: WILEY

摘要:

AbstractTreatment of BALB/c‐3T3 mouse fibroblasts with 3′‐led to a rapid accumulation of 3′‐phosphates and the kinetics of this process has been determined. Concomitant with accumulation of these compounds, the adenine ribonucleotide pool was reduced. The kinetics of the two processes suggested that they were tightly coupled. The inhibitory effect of relatively high concentrations of coformycin indicated that IMP was an intermediate in the catabolic pathway. Similar experiments with Ehrlich ascites tumor cells were performed in Ringer‐Hepes solution at pH 6.5 or 7.5 and with varying concentrations of orthophosphate. The experiments were performed with cells where ATP was [3H]‐. This allowed the determination of the catabolism of adenine ribonucleotides to labeled nucleosides under conditions where added adenosine was phosphorylated. The results showed that at low phosphate concentration (5.8 mM) at pH 6.5 adenosine may be phosphorylated at a rate that was completely balanced to the concomitant catabolism of adenine ribonucleotides; that is, there was apparently a tight kinetic coupling between anabolism of adenosine and catabolism of adenine ribonucleotides. With 3′‐a corresponding effect was obtained although the apparent coupling between phosphorylation of 3′‐and catabolism of adenine ribonucleotides was not complete. When experiments were performed at the same pH but at high concentration of phosphate (45 mM) there was in contrast no coupling between the two processes; that is, ATP was present in constant amounts while 3′‐phosphates accumulated at a high rate. In experiments with adenosine under these conditions there was still some although a relatively limited degree of apparent coupling between phosphorylation of adenosine and catabolism of adenine ribonucleotides. In both lines of cells used and with both adenosine and 3′‐, the main products of the catabolism of adenine ribonucleotides were inosine and hypoxanthine. With 3′‐there was in addition (about 20%) formation of xanthosine, suggesting that IMP dehydrogenase had also been activated. These results lead to the suggestion that adenosine (or 3′‐) may be phosphorylated in two ways. 1) Phosphorylation may depend on an adenosine kinase unrelated to catabolism of adenine ribonucleotides. 2) Phosphorylation may be tightly coupled to catabolism of adenine ribonucleotides. A nucleoside phosphotransferase may catalyze the transfer of a phosphoryl group from IMP to adenosine (or 3′‐) to form AMP (or 3′‐) and inosine, a process that may be tightly coupled to an AMP deaminase reaction. The IMP formed in the latter reaction may not be released but transferred to the phosphotransferase. In contrast, the AMP formed in the phosphotransferase reaction should be in equilibrium with soluble AMP. It is assumed that a physical complex may exist, possibly in a membrane bound form, between AMP deaminase and the nucleosi

ISSN:0021-9541    

DOI:10.1002/jcp.1041540110

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1993

数据来源: WILEY

摘要:

AbstractTo 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 Wi

ISSN:0021-9541    

DOI:10.1002/jcp.1041540111

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1993

数据来源: WILEY