摘要:

AbstractZirc(II) accumulated by platelets has profound effects on platelet activity. This study is focused on the distribution of Zn(II) between human platelet subcellular compartments. After incubation with86Rb+and platelet lysis, the organelles were separated by sucrose density gradient centrifugation. Fibrinogen served as a marker for α‐granules.86Rb+and factor XIII served as markers for the cytoplasmic fractions. Zn(II) was found to be distributed between the cytoplasm and the α‐granules, with variations between different individual units. The total platelet Zn concentration and its relative subcellular distribution were dependent on its extracellular level. Incubation of platelets with 100 μM Zn(II) resulted in a twofold increase of its level in the cytoplasm and by one order of magnitude in the α‐granules. In addition to the anticipated factor XIII activity in the cytoplasmic pool fraction, we found thrombin‐inducible factor XIII activity within the α‐granules. Immunoblotting confirmed the presence of both the a and b subunits of plasma factor XIII (a2b2form) in the α‐granules. As fibrinogen is not synthesized in the platelet, we propose that by virtue of their mutual binding, fibrinogen, Zn(II) and plasma factor XIII‐a2b2are simultaneously taken up into the α‐granules by endocytosis, presumably through the vehicle of the GPIIb/IIIa fibrinogen receptor. A rationale for copackaging these components within the α‐granules is that Zn(II) inhibits factor XIII activity and thereby prevents the premature cross‐linking of the concentrated fibrinogen prior to platelet activation and secretion. By contrast, cytoplasmic Zn(II) may increase platelet responsiveness to agonists due to its interaction with cytoplasmic modulators of platelet activ

ISSN:0021-9541    

DOI:10.1002/jcp.1041560302

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

年代:1993

数据来源: WILEY

摘要:

AbstractHepatocytes maintained in collagen gels remain differentiated for prolonged periods of time compared to cells maintained on conventional cultures. Previous studies with other culture systems in which chemical supplements or substratum modifications enhanced hepatocyte differentiation showed that in all of these systems hepatocytes do not respond to mitogens. In this study it is shown that hepatocytes maintained between two layers of collagen gels respond to mitogens HGF (also known as scatter factor (HGF/SF)) and epidermal growth factor (EGF). Cell density did not affect the responsiveness to mitogens as in conventional cultures. In addition both mitogens (HGF more pronounced) induce characteristic morphogenic changes in which hepatocytes form processes and join in formation of cords. Hepatocytes respond to mitogens for up to 6 days in culture at which point they become refractory to further mitogenic stimulation. This occurs despite electron microscopic evidence that these cells are fully viable when they become refractory to mitogenesis. The refractory state is not modified by substitution of one growth factor for the other or by addition of growth factors at different times. Hepatocytes in the refractory state become again responsive to mitogens when the collagen gels are dispersed by collagenase and the cells are replated on conventional substrates. © 1993 Wiley‐Liss, I

ISSN:0021-9541    

DOI:10.1002/jcp.1041560303

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

年代:1993

数据来源: WILEY

摘要:

AbstractThe insulin‐like growth factors (IGFs) have paradoxical effects on skeletal myoblast differentiation. While low concentrations of IGF stimulate myoblast differentiation, high concentrations of IGF induce a progressive decrease in myoblast differentiation. The mechanism of this inhibition is unknown. Using a retroviral expression vector, we developed a subline of mouse P2 mouse myoblasts (P2‐LISN) which expressed 7.5 times higher levels of type‐1 IGF receptors than control (P2‐LNL6) myoblasts, which were infected with a virus lacking the type‐1 IGF receptor sequence. Overexpression of the type‐1 IGF receptor caused the IGF dose‐response curves of stimulation and progressive inhibition of differentiation to shift to the left. Additionally, at high insulin and IGF‐I concentrations, complete inhibition of P2‐LISN myoblast differentiation occurred. These results suggest that inhibition of differentiation at high ligand concentrations was not due to the primary involvement of other species of receptors for IGF. Type‐1 IGF receptor downregulation as a mechanism for inhibition of differentiation was also ruled out since P2‐LISN myoblasts constitutively expressed high levels of type‐1 IGF receptors. Additionally, inhibition of differentiation at high concentrations of IGF‐I was not correlated with overt stimulation of proliferation or with IGF binding protein (IGF‐BP) release into the culture medium. These results indicate that the type‐1 IGF receptor mediates two conflicting signal pathways in myogenic cells, differentiation‐inducing and differentiation‐inhibitory, which predominate at different ligand conce

ISSN:0021-9541    

DOI:10.1002/jcp.1041560304

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

年代:1993

数据来源: WILEY

摘要:

AbstractIn the present study, we have sought to determine whether a given signal transduction pathway can have diverse effects on subpopulations of cells of a lineage depending upon the stage of differentiation. To test this hypothesis, we selected the cyclic adenosine monophosphate (cAMP) signal transduction pathway because of its recognized importance in mediating the actions of many hormones, e.g., parathyroid hormone which acts on the bone‐forming cells, the osteoblasts. Subpopulations of human osteosarcoma SaOS‐2 cells with low (LSaOS) and high (HSaOS) alkaline phosphatase (ALP) content were chosen as model systems for preosteoblasts (pre‐OB) and osteoblasts (OB), respectively. Dibutyryl cyclic AMP (DBcAMP) treatment of serum free cultures produced a differential effect on the proliferation of LSaOS cells (40 ± 5% of control at 1 mM DBcAMP,P<0.001) compared with HSaOS cells (no statistically significant effect). The finding supports the hypothesis. Next, we sought evidence for mediation, at least in part, by the insulin‐like growth factor (IGF)‐II regulatory system. We report that the basal expression of IGF‐II, IGF binding protein (IGFBP)‐3, and IGFBP‐4 was higher in LSaOS cells than in HSaOS cells with the opposite true for type I IGF receptor. DBcAMP treatment of LSaOS cells decreased IGF‐II and IGFBP‐3 but increased IGFBP‐4 and type I IGF receptor; no effect was observed for the type II IGF receptors. DBcAMP treatment of HSaOS cells had no detectable effect on IGF‐II; IGFBP‐3, or type I and type II IGF receptor expression; only IGFBP‐4 expression increased with DBcAMP. These observations suggest that the differential regulation of cell proliferation by the cAMP signal transduction pathway may be mediated, at least in part, by the IGF‐II regulatory

ISSN:0021-9541    

DOI:10.1002/jcp.1041560305

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

年代:1993

数据来源: WILEY

摘要:

AbstractPost‐confluent populations of LLC‐PK1cells express many glycoproteins at the surface, including glycoproteins which bind the lectin Dolichos biflorus agglutinin (DBA). DBA‐binding glycoproteins are localized preferentially to the apical cell surface and exhibit molecular weights ranging from less than 30 kDa to greater than 200 kDa. Subconfluent cell populations exhibit little surface DBA binding. Upon attaining confluence DBA binding capacity increases progressively over several days. This correlates roughly with increasing activities of several differentiated apical membrane functions. Expression of DBA binding sites at the surface occurs on a cell‐by‐cell basis. An increasing level of surface DBA binding in the cell population corresponds to an increasing proportion of the cell population expressing binding sites. Activation of cAMP‐dependent protein kinase in post‐confluent cell populations, and to a lesser extent in subconfluent populations, increases surface DBA binding sites by a protein synthesis‐dependent mechanism after a lag period of at least 24 h. This results from increases in both the proportion of cells expressing surface binding sites and the level of binding sites on individual cells. © 1993

ISSN:0021-9541    

DOI:10.1002/jcp.1041560306

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

年代:1993

数据来源: WILEY

摘要:

AbstractConfluent cultures of primary hamster tracheal surface epithelial (HTSE) cells grown on a thick collagen gel are highly enriched with secretory cells and constitutively release mucins. In the present experiment, we examined the possible effect of mechanical strain of cultured HTSE cells on the release of mucin. The mechanical strain of cells was accomplished by several methods: 1) by floating the gel from the culture dish by rimming; 2) by treatment with EGTA which interrupts intercellular tight junctions; 3) by treatment with collagenase which disrupts the cell‐matrix adhesion; and 4) by mechanically flexing the collagen gel matrix. All these conditions caused increases of mucin release without damage on the plasma membrane. We conclude that a number of mechanical strains which might alter cell shape can stimulate mucin release from cultured HTSE cells. Such a mechanism might be operative in the physiological regulation of airway goblet cell mucin secretion where mechanical strains may be induced on epithelial cells by underlying smooth muscles. © 1993 Wiley‐Liss,

ISSN:0021-9541    

DOI:10.1002/jcp.1041560307

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

年代:1993

数据来源: WILEY

摘要:

AbstractThe involvement of protein kinase C (PKC) in epidermal growth factor (EGF)‐induced human keratinocyte migration was studied with the phagokinetic assay. It was concluded that PKC activation does not mediate, but rather inhibits, EGF‐induced keratinocyte migration. The following experimental observations support these conclusions: 1) The PKC inhibitor H‐7 did not inhibit EGF‐induced migration but instead led to a modest enhancement. 2) PKC activators such as phorbol‐12‐myristate‐13‐acetate (PMA), phorbol‐12,13‐dibutyrate (PDBu), and 1,2‐dioctanoly‐sn‐glycerol inhibited migration, but biologically inactive 4α‐PMA had no effect. 3) PMA did not inhibit keratinocyte attachment and spreading but blocked migration almost immediately after addition. 4) Migration of PKC‐depleted cells, which were produced by prolonged treatment with PDBu, was enhanced similarly to normal cells by EGF. 5) PKC‐depleted cells were not susceptible to the inhibitory effects of phorbol esters on migration. Additional experiments, in which cells were preactivated with EGF, suggested that PKC inhibits the EGF effect at a post‐receptor level. The inhibitory effect of PKC on keratinocyte migration was not restricted to EGF‐induced migration; PKC activation also inhibited keratinocyte migration induced by bovine pituitary extract, insulin, insulin‐like growth factor‐1, and keratinocy

ISSN:0021-9541    

DOI:10.1002/jcp.1041560308

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

年代:1993

数据来源: WILEY

摘要:

AbstractTo ascertain if 17β‐estradiol (E2)‐induced proliferation could be attenuated by blocking the expression of endogenous transforming growth factor α (TGFα), estrogen receptor (ER)‐positive, estrogen‐responsive MCF‐7 or ZR‐75‐1 cells and ER‐negative, estrogen‐nonresponsive MDA‐MB‐468 or HS‐578T cells were infected with a recombinant amphotropic, replication‐defective retroviral expression vector containing a 435 base pair (bp)Apa1‐EcoR1 coding fragment of the human TGFα cDNA oriented in the 3′ to 5′ direction and under the transcriptional control of an internal heavy metal‐inducible mouse metallothionein (MT‐1) promoter and containing the neomycin (neo) resistance gene. E2‐stimulated expression of endogenous TGFα mRNA was inhibited by 4–5‐fold, and the production of TGFα protein was inhibited by 50–80% when M‐1 mass‐infected MCF‐7 or MZ‐1 mass‐infected ZR‐75‐1 cells were treated with 0.75‐1 μM CdCl2, whereas in comparably treated parental MCF–7 or ZR‐75‐1 cells there was no significant effect upon these parameters. E2‐stimulated anchorage‐dependent growth (ADG) and anchorage‐independent growth (AIG) of the M‐1 or MZ‐1 cells was inhibited by 60–90% following CdCl2treatment. In contrast, neither the ADG nor AIG of the parental noninfected MCF‐7 or ZR‐75‐1 cells that were maintained in the absence or presence of E2 was affected by comparable concentrations of CdCl2. The ADG and AIG of TGFα antisense MD‐1 mass‐infected MDA‐MB‐468 cells that express high levels of endogenous TGFα mRNA were also inhibited by 1 μM CdCl2, whereas the ADG and AIG of MH‐1 mass‐infected HS‐578T cells, a TGFα‐negative cell line, were unaffected by CdCl2treatment. These results suggest that TGFα may be

ISSN:0021-9541    

DOI:10.1002/jcp.1041560309

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

年代:1993

数据来源: WILEY

摘要:

AbstractPrevious studies have reported that the proliferation of A431 cells, a human squamous cell carcinoma cell line, was stimulated by picomolar epidermal growth factor (EGF) but inhibited by nanomolar EGF. This biphasic dose‐response phenomenon is not observed in normal human epithelial cells where nanomolar EGF is usually mitogenic. We have examined the effects of inhibitory and stimulatory concentrations of EGF on the growth and differentiation of A431 cells. In the presence of 100 pM EGF, A431 cells showed a mild increase in growth rate (129% of control) compared to cells grown in the absence of EGF. At 10 nM EGF, growth inhibition to 63% of control was observed. EGF at 10 nM stimulates a twofold increase both in cornified envelope formation and in epidermal transglutaminase activity, suggesting that high concentrations of EGF induce terminal differentiation in A431 cells. Mitogenic concentrations of EGF (100 pM) had no significant effect on these differentiation markers. Chronic exposure of A431 cells to 20 or 50 nM EGF resulted in EGF‐resistant A431 variants that are neither growth arrested nor induced to terminally differentiate by 10 nM EGF. Removal of EGF from the growth medium of the EGF‐resistant cells resulted in the reversion of these cells back to the wild‐type A431 biphasic response pattern within 2 weeks. Our results suggest that A431 cells have the capacity to non‐mutatively alter their response pattern to EGF in vitro to maintain themselves in a state of optimum proliferation and away from terminal differentiation. © 1993 Wiley

ISSN:0021-9541    

DOI:10.1002/jcp.1041560310

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

年代:1993

数据来源: WILEY

摘要:

AbstractAdult rat hepatocytes aggregated to form floating multicellular spheroids when cultured in Primaria dishes, which have a positively charged surface, in serum‐free Williams' medium E (WE) supplemented with insulin and epidermal growth factor (EGF). These hormones were essential for maintenance of the spheroids, whereas the size of the spheroids depended on the inoculum cell density. The spheroids retained in vivo levels of expressions of albumin and glucokinase and synthesized scarcely any DNA even in the presence of insulin and EGF. On transfer to type I collagen‐coated dishes, the spheroids gradually disaggregated and the cells formed monolayers, in which the expressions of albumin and glucokinase were suppressed and DNA synthesis and hexokinase activity were increased. DNA synthesis of hepatocytes in monolayer culture was maximal 24 hr after transfer of the spheroids, ∼80% of the hepatocyte nuclei were labelled with bromodeoxyuridine during culture for 48 hr, and the mitotic index was ∼70% after 60 hr. These results suggest that, in spheroids, hepatocytes remained in the G0phase, but that when they formed monolayers, they progressed to the G1phase and proceeded through the cell cycle in the presence of insulin and EGF. This work shows that the cell cycle of hepatocytes in culture can be manipulated by providing conditions for quiescence as spheroids or growth as monolayers and that the shape of hepatocytes is important for regulating their growth and liver‐specific functions. © 1993 Wiley

ISSN:0021-9541    

DOI:10.1002/jcp.1041560311

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

年代:1993

数据来源: WILEY