摘要:

AbstractTransforming growth factor‐beta (TGF‐β) purified from platelets is a potent growth inhibitor of several normal epithelial cell types in culture. In contrast, some carcinoma cell lines derived from tumors of these same tissues are resistant to this factor. Using recombinant human TGF‐β, the authors have confirmed these results with six normal human epidermal keratinocyte strains and four human epidermal squamous carcinoma cell lines. However, the sensitivity of normal cells to TGF‐β was found to depend on the culture conditions. When grown in a specialized nutrient medium supplemented with pituitary extract, keratinocytes were completely inhibited by the addition of 0.3 ng/ml TGF‐β. In contrast, when their growth was supported by cocultivation with 3T3 fibroblast feeder cells, 30‐ to 100‐fold higher concentrations of TGF‐β were required to achieve comparable growth inbibition. This differential sensitivity occurred despite the fact that in both culture systems TGF‐β in the culture medium had a half‐life of about 50 minutes, becoming tightly bound to the surface of the culture dish. Bound TGF‐β proved to be biologically active and stable for about a week in the absence of 3T3 feeder cells. Incubating 3T3 cells on TGF‐β‐coated dishes, however, resulted in nearly quantitative removal and degradation of the TGF‐β within 2 days, permitting normal rates of keratinocyte growth. The binding of TGF‐β to surfaces and the ability of fibroblasts to attenuate its inhibitory activity for epithelial cells must be considered when evaluating in vitro models and in planning strate

ISSN:0021-9541    

DOI:10.1002/jcp.1041390302

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

年代:1989

数据来源: WILEY

摘要:

AbstractInterleukin‐4 (IL‐4), which was originally identified as a B‐cell growth factor, has been shown to produce diverse effects on hemopoietic progenitors. The present study investigated the effects of purified recombinant murine IL‐4 on early hemopoetic progenitors in methylcellulose culture. IL‐4 supported the formation of blast cell colonies and small granulocyte/macrophage (GM) colonies in cultures of marrow and spleen cells of normal mice as well as spleen cells of mice treated with 150 mg/kg 5‐fluorouracil (5‐FU) 4 days earlier. When the blast cell colonies were individually picked and replated in cultures containing WEHI‐3 conditioned medium and erythropoietin (Ep), a variety of colonies were seen, including mixed erythroid colonies, indicating the multipotent nature of the blast cell colonies supported by IL‐4. To test whether or not IL‐4 affects multipotent progenitors directly, we replated pooled blast cells in cultures under varying conditions. In the presence of Ep, both IL‐3 and IL‐4 supported a similar number of granulocyte/erythrocyte/macrophage/megakaryocyte (GEMM) colonies. However, the number of GM colonies supported by IL‐4 was significantly smaller than that supported by IL‐3. When colony‐supporting abilities of IL‐4 and IL‐3 were compared using day‐4 post‐5‐FU spleen and day‐2 post‐5‐FU marrow cells, IL‐4 supported the formation of fewer blast cell colonies than did IL‐3. IL‐4 and IL‐6 revealed synergy in support of colony formation from day 2 post‐5‐FU marrow cells. These results indicate that murine IL‐4 is another direct‐acting multilineage colonystimulating factor (multi‐CSF),

ISSN:0021-9541    

DOI:10.1002/jcp.1041390303

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

年代:1989

数据来源: WILEY

摘要:

AbstractSeveral peptide growth factors influence the growth and differentiation of neural cells. To investigate further the growth‐promoting effects of the somatomedins on cells of neural origin, the authors characterized the binding and mitogenic effects of insulin‐like growth factor‐1 (IGF‐1) on a functionally differentiated rat neuronal cell line (B104). Specific, high‐affinity (Kd ≅ 10−9M) receptors for IGF‐1 were abundant (approximately 124,000 binding sites/B104 cell). These IGF‐1 receptors were similar to those of non‐neural tissue in that they contained 135,000 dalton binding subunits (demonstrated by affinity labeling and autoradiography) and recognized insulin at high concentrations. IGF‐1 was more potent than insulin at stimulating B 104 cell replication in serum‐free medium and, at an initial concentration of 100 ng/ml, was the only exogenous growth factor needed to maintain growth through several cell divisions. Furthermore, cells of later passage were found to secrete specific IGF binding proteins that produced an unusual, biphasic binding curve in radioligand displacement studies. These binding proteins apparently sequester IGF‐1, limiting its access to the cell. Experiments with B 104 cells may provide useful information about the role of IGFs and their binding proteins as potential regulators of growth and differentiation of

ISSN:0021-9541    

DOI:10.1002/jcp.1041390304

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

年代:1989

数据来源: WILEY

摘要:

AbstractCell‐cycle regulation of human diploid fibroblasts (HDF) is located in the proximal half of G1, designated G1‐pm (G1‐postmitosis). In order to traverse this subphase, cells require serum factors or PDGF. However, when cells have traversed into the distal half of G1, designated G1‐ps (G1‐pre‐DNA synthesis), they become independent of serum or PDGF and progress through the remainder of the cell cycle at an invariable rate. From this, it follows that a specific G1‐pm block can be induced by serum depletion. A similar G1‐pm block could also be induced by a moderate inhibition of overall protein synthesis following treatment with CHM. Even this block could be prevented by the addition of PDGF, suggesting that a high level of protein synthesisin itselfis not necessary for sustaining cell‐cycle traverse. Nevertheless, a critical accumulation of some specific proteins might be required for the G1‐pm/G1‐ps‐transition. However, the underlying mechanisms of modulation of the accumulation of such proteins by PDGF must involve alternative regulatory events (e.g., gene expression, protein stabilization) rather than protein synthesis. Among the possible cell cycleregulatory proteins, the present study focused on 3‐hydroxy‐3‐methylglutaryl coenzyme A (HMG CoA) reductase. This enzyme is regulated by various kinds of control mechanisms and regulates the biosynthesis of sterols and nonsterol isoprenes, some of which are proposed to be necessary for mammalian cell growth (Brown and Goldstein, 1980). The present results suggest that regulation of HMG CoA reductase may be involved in the control of the G

ISSN:0021-9541    

DOI:10.1002/jcp.1041390305

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

年代:1989

数据来源: WILEY

摘要:

AbstractWe have derived serum‐free mouse embryo (SFME) cultures in a basal nutrient medium supplemented with insulin, transferrin, epidermal growth factor (EGF), high‐density lipoprotein (HDL), and fibronectin. These cells are nontumorigenic, lack gross chromosomal aberrations, and exhibit several other unique properties, including dependence on EGF for survival and growth inhibition by serum. We have examined the concentration dependence of the growth stimulatory effects of protein supplements used in the SFME medium formulation and surveyed other supplements that might act as alternative or complementary additions to the culture medium. Insulin could be replaced by insulin‐like growth factor I and EGF could be replaced by transforming growth factor alpha in the same concentration range. Transferrin could be replaced by higher concentrations of lactoferrin. Deterioration of cultures in the absence of EGF began within 8 hours of the removal of the growth factor, and could be prevented by the addition of fibroblast growth factor/heparin‐binding growth factor. Attachment proteins other than fibronectin were effective on SFME cells, but limited success was obtained when substituting other lipid preparations for HDL. These data introduce a precise system for exploring the unusual characteristics of SFME cells and contribute additional information that may be useful in the extension of these approaches to other cell types and

ISSN:0021-9541    

DOI:10.1002/jcp.1041390306

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

年代:1989

数据来源: WILEY

摘要:

AbstractAn apparatus has been designed to subject vascular cells grown on a compliant substrate in vitro to uniform, quantifiable levels of biaxial deformation. The system described can be controlled with respect to strain level, rate, and frequency to mimic the pulsatile force to which vascular cells are exposed in vivo under both physiologic and pathologic conditions. In the experiments presented here, bovine pulmonary artery endothelial cells were grown on a substrate of segmented polyurethane urea (MitrathaneTM). Cell growth and morphology on this substrate were compared with those of cells grown on standard tissue culture polystyrene with no difference noted between the two substrates. Primary cultures of pulmonary artery endothelial cells were seeded onto Mitrathane, which was then subjected to cyclic biaxial deformation‐producing strains of 0.78%, 1.76%, 4.9%, or 12.5% at a frequency of 1 sec−1and a duty cycle of 0.5 sec−1for 7 h. Cells subjected to deformations generating strains of either 4.9% or 12.5% secreted significantly less fibronectin than nondeformed cells. Similar results were obtained in experiments using cloned pulmonary artery endothelial cells on Mitrathane subjected to the 4.9% strain; however, total protein synthesis was increased. Cell viability and DNA synthesis were not affected by cyclic biaxial deformation in these experi

ISSN:0021-9541    

DOI:10.1002/jcp.1041390307

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

年代:1989

数据来源: WILEY

摘要:

AbstractThis study describes the alterations induced by Interleukin‐1α and ‐β (IL‐1α and IL‐1β) on fibroblast‐synthesized extracellular matrix. Fibroblasts were grown between pieces of dentin or in collagen‐coated Terasaki wells for 3 or 6–9 weeks to create 3‐dimensional cell‐containing matrices constituted primarily of proteoglycans and collagens, respectively. Following incubation with IL‐1α or IL‐1β (10−9M) at 37°C for 24 or 72 hr, samples were prepared for light and electron microscopy. Both IL‐1α and IL‐1β induced collapse of the extracellular matrix by 72 hr, as manifested by a decrease of the cross‐sectional area and an increased density of the matrices. Three‐week matrices were reduced 26% and 45% by using IL‐1α and IL‐1β, respectively. Comparable values obtained by using 6‐week matrices were 14% and 30%. Cells within the matrix, normally stellate in shape with numerous extended processes, attained a more rounded or spindle shape with few and reduced processes and showed apparent alterations at cell matrix attachment sites and rearrangement of the cytoskeleton. Elongated cells at the top of the matrix appeared more compressed. The alterations were more pronounced in cultures incubated with IL‐β than with IL‐1α. Immunocytochemistry of extracellular matrix components revealed a decrease in staining intensity of chondroitin and dermatan sulfate in the 3‐week matrix following IL‐1β incubation. There was also a decrease in collagen type I staining of 9‐week matrices treated with IL‐1α or IL‐1β. These studies show that IL‐1 has an effect on fibroblast‐synthesized extracellular matrix and indicate that the effects of IL‐1α and IL‐1β may differ. The resulting collapse of the matrix

ISSN:0021-9541    

DOI:10.1002/jcp.1041390308

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

年代:1989

数据来源: WILEY

摘要:

AbstractThe growth of MG63 human osteosarcoma cell line in 5% serum is stimulated by epidermal growth factor (EGF), platelet‐derived growth factor (PDGF), or heparin‐binding growth factor‐1 (HBGF‐1). The mitogenic effect of EGF and PDGF is completely blocked by TFG‐β at 1 ng per ml and the effect of HBGF‐1 is attenuated by 75–80%. Treatment of MG63 cells with TGF‐β reduces HBGF‐1 receptor binding affinity from 1.24 × 10–11 M to 3.51 × 10–11 M with no change on the receptor number (1.1. × 103 per cell). The receptor‐binding affinity of EGF and PDGF is not altered by TGF‐β treatment; however, the number of EGF receptor is increased by 25%. Both EGF and PDGF stimulate MG63 cellular tyrosine kinase activity, and such stimulation is inhibited by TGF‐β pretreatment. No change in the cellular protein tyrosine phosphorylation pattern can be detected in HBGF‐1‐stimulated cells with and without TGF‐β pretreatment. These data suggest that TGF‐β inhibits EGF and PDGF mitogenicity by blocking EGF‐ and PDGF‐stimulated tyrosine kinase activity and attenuates HBGF‐

ISSN:0021-9541    

DOI:10.1002/jcp.1041390309

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

年代:1989

数据来源: WILEY

摘要:

AbstractThe attachment of polymorphonuclear granulocytes (PMNs) to vascular endothe‐lial cells occurs continually in normal tissues; however, knowledge of the factors that control leukocyte margination is incomplete. In the present study, we used cell cultures of pulmonary artery endothelium to study their interaction with PMNs. Endothelial cells were seeded in Costar 24‐well plates following which PMNs were inoculated onto the endothelial monolayers and incubated for 2 to 20 hours. During this period, fibronectin synthesis by endothelial cells was estimated by ELISA. In wells to which PMNs had been added, supernatant fibronectin concentration was increased at all time points during the 20 hour incubation. At 20 hours, supernatants from wells to which PMNs had been added contained approximately 2 1/2 times the control level of fibronectin. Since the amount of fibronectin, as determined by ELISA, adsorbed onto the added PMNs was negligible, these data suggest that PMNs can modulate the synthesis of fibronectin by pulmonary artery cells. Pulse labeling experiments and measurements of endothelial intracellular fibronectin also suggest this possibility. The endothelial response does not appear to be owing to nonspecific physical interaction since similarly sized polystyrene beads did not cause any change in supernatant fibronectin levels while glutaraldehyde‐fixed PMNs caused only a 20–25% increase in fibronectin

ISSN:0021-9541    

DOI:10.1002/jcp.1041390310

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

年代:1989

数据来源: WILEY

摘要:

AbstractWe have examined the ability of epidermal growth factor (EGF) to regulate prostacyclin production by cultured A10 smooth muscle cells. EGF by itself had no effect on prostacyclin production, but it augmented the response to arg8‐vasopressin. An EGF stimulation of prostacyclin production was also observed in the presence of the calcium ionophore A23187; it therefore seemed likely that the key event required for EGF to stimulate prostacyclin production might be an increase in the available cellular Ca2+. Studies with 45Ca2+ showed that vasopressin both mobilised Ca2+ from intracellular stores and increased the influx of extracellular Ca2+ into A10 cells. The increase in prostacyclin production caused by vasopressin and the augmentation by EGF were both abolished by TMB‐8, an antagonist ofCa2+mobilisation, by EGTA, a chelator of Ca2+ ions, or by incubating cultures in the absence of added Ca2+. These results were consistent with a central role for Ca2+ in the responses and showed that both intracellular and extracellular sources of Ca2+ were important for the triggering of prostacyclin production. The increases in prostacyclin production were only marginally affected by nifedipine, and no responses were seen (either in the absence or presence of EGF) when KCL was used to depolarise the cell membrane. These data indicated that uptake of Ca2+ ions via voltage‐dependent channels was unlikely to be a major factor in the stimulation of prostanoid production. We conclude that the ability of EGF to stimulate prostacyclin production in A10 smooth muscle cells depends upon a concurrent stimulus that will increase available intracellular Ca2+ l

ISSN:0021-9541    

DOI:10.1002/jcp.1041390311

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

年代:1989

数据来源: WILEY