摘要:

AbstractWe have compared the EGF responses of A431 cells when grown as monolayers at a variety of cell densities of as multicellular spheroids in order to investigate the effects of cell contact and 3‐dimensional structure on signal transduction. Proliferation of the A431 squamous carcinoma cell line grown in our laboratory was unaffected by EGF when grown in monolayer culture. As 3‐dimensional, multicellular spheroids, however, growth was stimulated by EGF. The maximum volume attainable in the presence of EGF was more than 30 times that in its absence. EGF‐dependent tyrosine phosphorylation was compared under these conditions by immunohistochemistry and Western blotting. In initial experiments using published procedures, tyrosine phosphorylation was density‐dependent in monolayers and undetectable in spheroids. However, the density‐dependence was abolished by the addition of high concentrations of protein tyrosine phosphatase inhibitors (1 mM Zn++and VO43−). The density dependence of EGF‐stimulated tyrosine phosphorylation in monolayers was, therefore, largely the result of changes in phosphatase activity rather than kinase. Using high concentrations of phosphatase inhibitors, phosphotyrosine was clearly visible by immunohistochemistry in the outermost cells of spheroids, but it was still not visible in the spheroid center. The lack of response within the spheroid was not related to the presence of EGF receptor nor diffusion of EGF. In companion experiments, we showed that staining for EGF receptor was present homogeneously throughout the spheroid and that EGF penetrated to its center under the conditions of the experiment. Thus, although an increase in tyrosine phosphatase activity was a major factor affecting tyrosine phosphorylation in the outer cells, other factors were important in the inner cells. We concluded that an increase of tyrosine phosphatase activity was the most important component of the adaptation of the EGF signal transduction system to high cell density in monolayer cultures. In spheroids, tyrosine phosphatases are also enhanced, but other factors, such as autocrine synthesis of TGF‐α and possibly the cellular distribution of EGF receptors and cell shape, play a role. © 199

ISSN:0021-9541    

DOI:10.1002/jcp.1041510302

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

年代:1992

数据来源: WILEY

摘要:

AbstractThe migration of human keratinocytes over the wound bed plays an important role in the re‐epithelialization of cutaneous wounds. Fibronectin, a large glycoprotein matrix component that is abundant within cutaneous wound beds, promotes keratinocyte migration. However, the mechanisms by which keratinocytes migrate over fibronectin are unknown. In this study, we sought to identify specific sites within the fibronectin molecule that induce keratinocyte locomotion and to characterize the cell surface receptors involved. The data show that the domain within the fibronectin molecule that induces human keratinocyte migration is the 120 kD cell‐binding domain close to the carboxyl terminus. The 40 kD heparin‐binding domain near the carboxyl terminus and the 45 kD gelatin‐binding domain near the amino terminus did not promote keratinocyte migration. In addition, keratinocyte migration on both fibronectin and the 120 kD cell‐binding domain was completely inhibited by the presence of GRGDSP peptide, suggesting that keratinocyte migration on fibronectin is mediated by recognizing the RGD sequence located within the cell‐binding domain of fibronectin. Furthermore, keratinocytes were able to migrate directly on immobilized RGD substratum. Cell migration on fibronectin is mediated by the α5β1 integrin since antibodies blocking the α5 and the β1 subunits completely inhibited keratinocyte migration on fibronectin. In addition, we demonstrate that human keratinocytes express α5β1 integrin in culture by flow cytometry. © 19

ISSN:0021-9541    

DOI:10.1002/jcp.1041510303

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

年代:1992

数据来源: WILEY

摘要:

AbstractExpression of apical cell surface proteins and glycoproteins was examined in polarized primary cultures of mouse uterine epithelial cells (UEC). Lectin‐gold cytochemistry revealed that wheat germ agglutinin (WGA) bound specifically to the components of the apical glycocalyx as well as intracellular vesicles. Double labeling with the pH sensitive dye 3‐(2,4‐dinitroanilino)‐3′amino‐N‐methyldipropylamine (DAMP) demonstrated the acidic nature of the WGA‐staining intracellular vesicles. The enzymatic and chemical sensitivities of the WGA binding sites on the apical cell surface were monitored both by WGA‐gold staining as well as by125I‐WGA binding assays. In thin sections, a large fraction of these sites were removed by pronase; however, application of a wide variety of proteases, glycosidases, or chemical treatments to the apical surface of intact UEC failed to reduce WGA binding. In no case did treatments designed to remove sialic acids reduce125I‐WGA binding more than 12%. In contrast, endo‐β‐galactosidase as well as a combination of β‐galactosidase with β‐hexosaminidase succeeded in removing 28% and 77% of these sites, respectively. These studies suggested that the majority of the apically disposed WGA binding sites involved N‐acetylglucosamine residues rather than sialic acids and included lactosaminoglycans. Many of the proteins detected at the apical cell surface by lactoperoxidase‐catalyzed radioiodination were WGA‐binding glycoproteins. A major class of these glycoproteins displayed Mr>200 kDa by SDS‐PAGE and was heavily labeled metabolically by3H‐glucosamine or by vectorial labeling at the apical cell surface with galactosyl transferase and UDP‐3H‐galactose. Analyses of the3H‐labeled oligosaccharides labeled by either procedure indicated that a large fraction of the apically disposed WGA‐binding oligosaccharides consisted of neutral, O‐linked mucin‐type structures with median MW of approximately 1,500. Oligosaccharides in this fraction were partially (15%) sensitive to endo‐β‐galactosidase digestion and bound toDatura stramoniumagglutinin (68%), demonstrating the presence of lactosaminoglycan sequences. UEC were an extremely effective barrier to attachment or invasion by either a highly invasive melanoma cell line, B16‐BL6, or implantation‐competent mouse blastocysts. In contrast, neither uterine stromal cells nor a non‐polarizing UEC cell line, RL95, prevented B16‐BL6 attachment. Treatment of UEC with 2‐acetamido‐2‐deoxy‐α‐D‐galactopyranoside (GalNAcide), an inhibitor of mucin biosynthesis, decreased apical binding of125I‐WGA by 66%, increased accessibility of apically disposed tryptic sites by 1.75‐fold, and increased apical expression of3H‐heparin binding sites by almost threefold; however, Gal‐NAcide‐treated UEC remained resistant to invasion by either B16‐BL6 cells or mouse blastocysts. Collectively, these data indicate that apical mucin glycoproteins of UEC provide an enzymatically resistant barrier which can limit accessibility to other lumenally disposed agents. Drastic reduction of mucin expression is likely to be

ISSN:0021-9541    

DOI:10.1002/jcp.1041510304

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

年代:1992

数据来源: WILEY

摘要:

AbstractWe have shown that human spermatozoa generate and release reactive oxygen species that can be detected by chemiluminescence techniques. Analysis of the cellular mechanisms responsible for this activity suggests that the probe, luminol, undergoes an intracellular dioxygenation reaction mediated by hydrogen peroxide and a sperm peroxidase located within the acrosome. Support for this model included the following observations: (1) the luminol‐dependent signal could be suppressed with peroxidase inhibitors, phenylhydrazine and sodium azide; (2) this suppression could be reversed by the addition of an azide‐insensitive peroxidase, horse radish peroxidase (HRP); (3) inhibition of intracellular superoxide dismutase (SOD) with potassium cyanide (KCN) suppressed the luminol signal; (4) peroxidase activity could be detected in purified populations of human spermatozoa with 3,3′,5,5′ tetramethylbenzidine (TMB); (5) this peroxidase was active at the pH prevailing within the acrosomal vesicle; and (6) peroxidase activity and luminol‐dependent chemiluminescence were minimal in spermatozoa exhibiting a congenital absence of acrosomes. Human spermatozoa could also generate lucigenin‐dependent chemiluminescent signals that could neither be suppressed with peroxidase inhibitors nor enhanced by the addition of peroxidase. However, these signals could be enhanced by suppression of intracellular SOD with KCN or inhibited by exogenous SOD, suggesting that lucigenin was responding to superoxide anion released into the extracellular space. The ability of chemiluminescent techniques to detect and discriminate the production of superoxide and hydrogen peroxide by spermatozoa should facilitate the further analysis of reactive oxygen species as mediators of normal and abnormal human sperm function. © 1992 Wil

ISSN:0021-9541    

DOI:10.1002/jcp.1041510305

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

年代:1992

数据来源: WILEY

摘要:

AbstractThe possible involvement of DNA topoisomerase I in cell transition from G0to G1and in progression through the cell cycle was studied by estimating the ability of human peripheral blood lymphocytes to undergo mitogenic stimulation in the presence of the topoisomerase I inhibitor camptothecin (CAM). Exposure of quiescent G0lymphocytes to up to 3 μM CAM for 24 h had no significant effect on their ability to subsequently undergo mitogenic stimulation in the presence of phytohemagglutinin (PHA); higher doses of CAM, although not immediately cytotoxic, impaired the mitogenic response. Stimulation of lymphocytes with PHA in the presence of ≤ 1.5 μM CAM resulted in unperturbed transition of these cells from G0to G1characterized as an increase in cellular rRNA content, appearance of interleukin‐2 receptor, and, after removal of CAM, response to interleukin‐2 by entering S phase of the cell cycle. However, lymphocytes were prevented from entering S phase in the presence of CAM at a concentration of ≥30 nM, and their rate of progression through S was minimal even at CAM concentration as low as 3 nM. When cycling lymphocytes (48 h after stimulation by PHA) were treated with CAM, the cell progression through S and G2was also very sensitive to the inhibitor: the cells were “frozen” in S and G2at ≥ 6 nM CAM. These cells died within 24 h; their selective loss from the cultures (with only G0/G1cells remaining) coincided with the appearance of cells with fractional DNA content, typical of apoptotic cells. Human lymphocytic leukemic MOLT‐4 cells were arrested in S and G2at ≥ 7.5 nM CAM. Thus, progressions through S and G2of both normal and leukemic lymphocytes were perturbed at approximately two orders of magnitude lower CAM concentration than the G0to G1transition. These data suggest that DNA replication and chromosomal events during G2are more sensitive to inhibition of DNA topoisomerase I, compared with the early events of lymphocyte stimulation, which involve activation and transcription of numerous genes associated with the G0to G1transition. The antitumor properties of CAM may be related to its high cytostatic/cytotoxic activity toward cycling cells and relative resistance of cells in G0or undergoing transition from G0to G1. ©

ISSN:0021-9541    

DOI:10.1002/jcp.1041510306

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

年代:1992

数据来源: WILEY

摘要:

AbstractErythropoietin (EPO) retards DNA breakdown characteristic of programmed cell death (apoptosis) and promotes survival in erythroid progenitor cells. The mechanism by which EPO inhibits programmed death is unknown. In the well‐characterized model of glucocorticoid‐treated thymocytes, activation of a Ca2+/Mg2+‐sensitive endonuclease and new protein and RNA syntheses have been found necessary for apoptosis. We examined the effects of EPO on the free intracellular calcium ion concentration ([Ca2+]i), and the roles of Ca2+and RNA and protein syntheses on DNA cleavage in erythroid progenitor cells. The murine model of erythroid differentiation using Friend leukemia virus‐infected proerythroblasts (FVA cells) was used. EPO did not affect the [Ca2+]iin FVA cells. Decreasing [Ca2+]iby extracellular Ca2+chelation with EGTA facilitated DNA breakdown. Increasing [Ca2+]iwith the calcium ionophore 4‐bromo‐A23187 increased DNA cleavage; however, DNA fragments generated by high [Ca2+]iwere much larger than those seen in the absence of EPO or presence of EGTA. Increased [Ca2+]ialso inhibited DNA breakdown to small oligonucleosomal fragments characteristic of cells cultured without EPO. However no concentration of ionophore protected the high molecular weight DNA as did EPO. Cycloheximide inhibited DNA breakdown in a dose dependent manner in cultures lacking EPO, but two other protein synthesis inhibitors, pactamycin and puromycin, did not prevent DNA breakdown. Inhibition of RNA synthesis with actinomycin D did not prevent DNA breakdown. Cells with morphological characteristics similar to those reported in other cells undergoing programmed death accumulated in EPO‐deprived cultures. These studies demonstrate that although DNA cleavage and morphological changes are common to apoptotic cells, the roles for Ca2+and protein and RNA syntheses are not universal and suggest that apoptosis can be regulated by different biochemical mechanisms in different cell types. © 1992 W

ISSN:0021-9541    

DOI:10.1002/jcp.1041510307

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

年代:1992

数据来源: WILEY

摘要:

AbstractStudies were carried out to analyze how different extracellular matrix (ECM) molecules regulate hepatocyte growth and differentiation. Freshly isolated rat hepatocytes were cultured on non‐adhesive plastic dishes that were pre‐coated with defined densities of either laminin, fibronectin, type I collagen, or type IV collagen. Sparse cell plating densities were used to minimize cell‐cell contact formation and all studies were carried out in chemically defined medium that contained a saturating amount of soluble growth factors. Dishes coated with a low ECM density (1 ng/cm2) supported hepatocyte attachment, but did not promote cell spreading or growth. Computerized image analysis confirmed that over 80% of cells remained free of contact with other cells under these conditions. Yet, these round cells maintained high levels of albumin gene expression as well as elevated secretion rates for multiple liver‐specific proteins (albumin, transferrin, and fibrinogen), regardless of the type of ECM molecule used for cell attachment. When ECM coating densities were raised from 1 to 1,000 ng/cm2, cell spreading, expression of histone mRNA, DNA synthesis, and cell proliferation all increased in parallel. Activation of growth by high ECM densities was also accompanied by a concomitant down‐regulation of differentiated functions and again, dishes coated with all four types of ECM molecules produced similar effects. Thus, the ability to switch hepatocytes from differentiation to growth (i.e., between different genetic programs) is not limited to a single ECM molecule, a distinct three dimensional ECM geometry, or due to alteration of cell‐cell interactions. Rather, the regulatory signals conveyed by immobilized ECM molecules depend on the density at which they are presented and thus, on their ability to either prohibit or support cell spreading. © 1992 Wil

ISSN:0021-9541    

DOI:10.1002/jcp.1041510308

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

年代:1992

数据来源: WILEY

摘要:

AbstractTo determine whether endothelium‐derived relaxing factor (EDRF) contributes to the regulation of endothelial permeability, the transendothelial flux of14C‐su‐crose, a marker for the paracellular pathway across endothelial monolayers (Oliver,J. Cell. Physiol.145:536–548, 1990), was examined in monolayers of bovine aortic endothelial cells grown on collagen‐coated filters. The permeability coefficient of14C‐sucrose was significantly decreased by 10−3M 8‐Bromoguanosine 3′,5′‐cyclic monophosphate or by 5 × 10−6M glyceryl trinitrate, an activator of soluble guanylate cyclase. Depletion of L‐arginine from endothelial monolayers increased14C‐sucrose permeability from 3.21 ± 0.59 to 3.88 ± 0.50 × 10−5cm · sec−1(mean ± SEM; n = 6;P<0.05). The acute administration of 5 × 10−4M L‐arginine to monolayers depleted of this amino acid decreased14C‐sucrose permeability from 2.91 ± 0.27 to 2.52 ± 0.26 × 10−5cm · sec−1(n = 11;P<0.05).14C‐sucrose permeability was increased by 10−7M bradykinin and this effect was enhanced by the presence of each one of the following compounds: 10−5M methylene blue, 4 × 10−6M oxyhemoglobin, 5 × 10−4M NG‐methyl‐L‐arginine or 5 × 10−4M Nω‐nitro‐L‐arginine. These results suggest that EDRF contributes to the sealing of the endothelial monolayer and that EDRF released by bradykinin acts as a feedback inhibitor attenuating the increase in endothelial permeability induced by this peptide. Because endothelial cells have the ability to contract and relax and possess guanylate cyclase responsive to nitric oxide, our results suggest that EDRF decreases14C‐sucrose permeability by relaxing endothelial c

ISSN:0021-9541    

DOI:10.1002/jcp.1041510309

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

年代:1992

数据来源: WILEY

摘要:

AbstractIn Swiss 3T3, murine fibroblasts, interleukin 1 (IL‐1) and bradykinin stimulate prostaglandin E2(PGE2) synthesis. However, in the present study, we found that neither agonist stimulated PGE2synthesis in BALB/c 3T3 murine fibroblasts, this in spite of expression of similar numbers of receptors for each agonist compared to Swiss 3T3 cells. When BALB/c 3T3 cells were preincubated with cAMP analogs, both IL‐1 and bradykinin stimulated PGE2synthesis to levels similar to those observed in Swiss 3T3 cells. Similarly, when the cells were preincubated with forskolin, which activates the catalytic subunit of adenylate cyclase directly, or NECA, which stimulates cellular cAMP accumulation by activating adenosine receptors, IL‐1 and bradykinin stimulated PGE2synthesis. Rp‐cAMPS, an inhibitor of cAMP‐dependent protein kinase, blocked the ability of cAMP or NECA to render cells responsive to IL‐1 and bradykinin. In basal BALB/c 3T3 cells, bradykinin and IL‐1 stimulated arachidonate release in the absence of cAMP, but little conversion of released arachidonate to PGE2occurred. cAMP, forskolin, and NECA all increased cyclooxygenase activity in the cells. SV‐T2is a clonal line originating from BALB/c 3T3 transformed with SV‐40. In these cells, IL‐1 and bradykinin stimulated PGE2svnthesis despite basal intracellular cAMP concentrations similar to BALB/c, and cAMP only modestly potentiated the response. In summary, cyclooxygenase expression appears to be regulated by cAMP in BALB/c 3T3 cells, and SV‐40 transformation results in increased cyclooxygenase expression, apparently independent of cAMP.

ISSN:0021-9541    

DOI:10.1002/jcp.1041510310

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

年代:1992

数据来源: WILEY

摘要:

AbstractExposure of mammalian cells to hyperthermia is known to cause protein aggregation in the nucleus. The presence of such aggregates has been detected as the relative increase in the protein mass that is associated with nuclei isolated from heated cells. We have characterized these excess nuclear proteins from the nuclei of heated HeLa cells by two‐dimensional gel electrophoresis. The abundance of cytoskeletal elements which co‐purify with the nuclei did not increase with exposure to hyperthermia, indicating that these proteins are not part of the excess nuclear proteins. In contrast, several specific polypeptides become newly bound or increase in abundance in nuclei isolated from heated cells. Members of the hsp 70 family were identified as a major component of the excess nuclear proteins. Among the other excess nuclear proteins we identified ten that had apparent molecular weights of 130, 95, 75, 58, 53, 48, 46, 37, 28, and 26 kilodaltons. Since hsp 70 is mainly cytoplasmic in non‐heated cells, its association with nuclei in heated cells indicates that one mechanism accounting for the heat‐induced excess nuclear proteins is the movement of cytoplasmic proteins to the nucleus. We also obtained evidence that increased binding of nuclear proteins is another mechanism for this effect. No overall increase or decrease in the phosphorylation of nuclear proteins was found to be associated with such altered binding or movement from the cytoplasm to the nucleus. © 1992 Wiley

ISSN:0021-9541    

DOI:10.1002/jcp.1041510311

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

年代:1992

数据来源: WILEY