Intracellular redistribution of Ku immunoreactivity in response to cell-cell contact and growth modulating components in the medium

1996 ◽  
Vol 109 (7) ◽  
pp. 1937-1946 ◽  
Author(s):  
J.W. Fewell ◽  
E.L. Kuff
Keyword(s):  
Mammalian Cells ◽  
Culture Medium ◽  
Primary Cultures ◽  
Human Keratinocytes ◽  
Inhibitory Effect ◽  
Cell Contact ◽  
Nuclear Component ◽  
Confluent Cell ◽  
Cell Cell

Ku is a heterodimeric protein first recognized as a human autoantigen but now known to be widely distributed in mammalian cells. Analysis of repair-deficient mutant cells has shown that Ku is required for DNA repair, and roles in DNA replication and transcription have also been suggested on the basis of in vitro observations. Ku is generally regarded as a nuclear component. However, in the present paper, we show that a quantitatively significant fraction (half or more) of Ku is located in the cytoplasm of cultured primate cells, and that major changes in epitope accessibility of both nuclear and cytoplasmic Ku components are associated with the transition from sparse to confluent cell densities. The same changes in immunoreactivity were seen in HeLa, 293, CV-1 (monkey) and HPV-transformed keratinocyte cell lines, and in primary cultures of human keratinocytes. The immunostaining pattern of sparsely grown cells could be converted to the ‘confluent’ configuration by re-plating them at the same low density on a monolayer of mouse 3T3 cells. The confluent antigen pattern could also be induced in sparse cells within 15–30 minutes by exposure of the cells to serum- or Ca(2+)-free medium or overnight with 2 mM hydroxyurea. Somatostatin at 0.12 mM blocked the effects of serum/Ca2+ deprivation of Ku p70 antigen distribution in sparse CV-1 cells, and in confluent cultures reversed the usual nuclear concentration of p70 immunoreactivity. However, somatostatin did not alter the expected immunostaining patterns of p86. Preliminary studies indicate that sparse CV-1 cells, but not HeLa cells, respond to as little as 1 pM of TGF-beta 1 in the culture medium by the rapid appearance of nuclear immunoreactivity. TGF-alpha had no apparent effect. These findings are consistent with the participation of Ku in a signal transduction system responsive to the inhibitory effect of cell-cell contact on the one hand and to cytokines and growth-supportive components of the culture medium on the other.

scholarly journals The Catalytic Activity of the Src Family Kinases Is Required to Disrupt Cadherin-dependent Cell–Cell Contacts

2000 ◽  
Vol 11 (1) ◽  
pp. 51-64 ◽  
Author(s):  
Dewi W. Owens ◽  
Gordon W. McLean ◽  
Anne W. Wyke ◽  
Christos Paraskeva ◽  
E. Kenneth Parkinson ◽  
...  
Keyword(s):  
Catalytic Activity ◽  
Epithelial Cell ◽  
Kinase Inhibitor ◽  
Human Keratinocytes ◽  
Src Family Kinases ◽  
Cell Contact ◽  
Cell Contacts ◽  
Intercellular Contacts ◽  
Cell Cell

Despite the importance of epithelial cell contacts in determining cell behavior, we still lack a detailed understanding of the assembly and disassembly of intercellular contacts. Here we examined the role of the catalytic activity of the Src family kinases at epithelial cell contacts in vitro. Like E- and P-cadherin, Ca2+ treatment of normal and tumor-derived human keratinocytes resulted in c-Yes (and c-Src and Fyn), as well as their putative substrate p120CTN, being recruited to cell–cell contacts. A tyrosine kinase inhibitor with selectivity against the Src family kinases, PD162531, and a dominant-inhibitory c-Src protein that interferes with the catalytic function of the endogenous Src kinases induced cell–cell contact and E-cadherin redistribution, even in low Ca2+, which does not normally support stable cell–cell adhesion. Time-lapse microscopy demonstrated that Src kinase inhibition induced stabilization of transiently formed intercellular contacts in low Ca2+. Furthermore, a combination of E- and P-cadherin-specific antibodies suppressed cell–cell contact, indicating cadherin involvement. As a consequence of contact stabilization, normal cells were unable to dissociate from an epithelial sheet formed at high density and repair a wound in vitro, although individual cells were still motile. Thus, cadherin-dependent contacts can be stabilized both by high Ca2+ and by inhibiting Src activity in low (0.03 mM) Ca2+ in vitro.

Pulsatile GnRH secretion from primary cultures of sheep olfactory placode explants

Reproduction ◽  
2000 ◽  
pp. 391-396 ◽  
Author(s):  
AH Duittoz ◽  
M Batailler
Keyword(s):  
Culture Medium ◽  
Primary Cultures ◽  
Pulsatile Secretion ◽  
Olfactory Placode ◽  
Gnrh Secretion ◽  
Individual Culture ◽  
Pulsatile Gnrh

The aim of this study was to investigate the development of pulsatile GnRH secretion by GnRH neurones in primary cultures of olfactory placodes from ovine embryos. Culture medium was collected every 10 min for 8 h to detect pulsatile secretion. In the first experiment, pulsatile secretion was studied in two different sets of cultures after 17 and 24 days in vitro. In the second experiment, a set of cultures was tested after 10, 17 and 24 days in vitro to investigate the development of pulsatile GnRH secretion in each individual culture. This study demonstrated that (i) primary cultures of GnRH neurones from olfactory explants secreted GnRH in a pulsatile manner and that the frequency and mean interpulse duration were similar to those reported in castrated ewes, and (ii) pulsatile secretion was not present at the beginning of the culture but was observed between 17 and 24 days in vitro, indicating the maturation of individual neurones and the development of their synchronization.

scholarly journals Bone marrow mesenchymal stem cells promote prostate cancer cell stemness via cell–cell contact to activate the Jagged1/Notch1 pathway

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Ji-wen Cheng ◽  
Li-xia Duan ◽  
Yang Yu ◽  
Pu Wang ◽  
Jia-le Feng ◽  
...  
Keyword(s):  
Prostate Cancer ◽  
Stem Cells ◽  
Mesenchymal Stem Cells ◽  
Western Blot ◽  
Cell Contact ◽  
Activity Levels ◽  
Tumor Resistance ◽  
Cell Cell

Abstract Background Mesenchymal stem cells (MSCs) play a crucial role in cancer development and tumor resistance to therapy in prostate cancer, but the influence of MSCs on the stemness potential of PCa cells by cell–cell contact remains unclear. In this study, we investigated the effect of direct contact of PCa cells with MSCs on the stemness of PCa and its mechanisms. Methods First, the flow cytometry, colony formation, and sphere formation were performed to determine the stemness of PCaMSCs, and the expression of stemness-related molecules (Sox2, Oct4, and Nanog) was investigated by western blot analysis. Then, we used western blot and qPCR to determine the activity levels of two candidate pathways and their downstream stemness-associated pathway. Finally, we verified the role of the significantly changed pathway by assessing the key factors in this pathway via in vitro and in vivo experiments. Results We established that MSCs promoted the stemness of PCa cells by cell–cell contact. We here established that the enhanced stemness of PCaMSCs was independent of the CCL5/CCR5 pathway. We also found that PCaMSCs up-regulated the expression of Notch signaling-related genes, and inhibition of Jagged1-Notch1 signaling in PCaMSCs cells significantly inhibited MSCs-induced stemness and tumorigenesis in vitro and in vivo. Conclusions Our results reveal a novel interaction between MSCs and PCa cells in promoting tumorigenesis through activation of the Jagged1/Notch1 pathway, providing a new therapeutic target for the treatment of PCa.

Proliferation and agglutinability of primary and transformed human epithelial cells in culture

1976 ◽  
Vol 21 (3) ◽  
pp. 553-561
Author(s):  
M.A. Ricard ◽  
R.J. Hay
Keyword(s):  
Culture Medium ◽  
Monolayer Culture ◽  
Primary Cultures ◽  
Short Term ◽  
Chloromethyl Ketone ◽  
Normal Populations ◽  
Short Term Exposure ◽  
Cell Strains ◽  
Human Placentae

Primary epithelial populations (HAM) were obtained by dissociation of the amniotic membrane stripped from human placentae. Agglutinability of cells from such normal populations and of cells from the transformed epithelial line WISH was then compared using concavanalin A as mediator. Extensive similar studies have previously been reported with cell strains isolated from other species. Freshly dissociated HAM cells from primary cultures agglutinated much less readily than did cells from WISH populations. Furthermore, the former exhibited a drastic decline in agglutinability as a function of time in suspension culture after trypsinization. Short-term exposure (60 h) of HAM cells in monolayer culture to 5-bromodeoxyuridine (BrdU) elicited heightened agglutinability detectable through 22 days in vitro. Addition of the protease inhibitors n-tosyl-L-lysyl-chloromethyl ketone (TLCK) or p-tosyl-L-arginine-methyl ester (TAME) to the culture medium inhibited proliferation of the WISH line by 40–50% while effecting only a 10–15% inhibition of HAM cells. These results also confirm data with other cell species indicating that high proteolytic activity at the surface of transformed cells may be related to the rapid proliferation rate.

scholarly journals SyNPL: Synthetic Notch pluripotent cell lines to monitor and manipulate cell interactions in vitro and in vivo

2021 ◽  
Author(s):  
Mattias Malaguti ◽  
Rosa Portero Migueles ◽  
Jennifer Annoh ◽  
Daina Sadurska ◽  
Guillaume Blin ◽  
...  
Keyword(s):  
Cell Fate ◽  
Cell Lines ◽  
Modular Design ◽  
Cell Interactions ◽  
Cell Populations ◽  
Cell Contact ◽  
Pluripotent Cells ◽  
Cell Fate Decisions ◽  
Cell Cell

ABSTRACTCell-cell interactions govern differentiation and cell competition in pluripotent cells during early development, but the investigation of such processes is hindered by a lack of efficient analysis tools. Here we introduce SyNPL: clonal pluripotent stem cell lines which employ optimised Synthetic Notch (SynNotch) technology to report cell-cell interactions between engineered “sender” and “receiver” cells in cultured pluripotent cells and chimaeric mouse embryos. A modular design makes it straightforward to adapt the system for programming differentiation decisions non-cell-autonomously in receiver cells in response to direct contact with sender cells. We demonstrate the utility of this system by enforcing neuronal differentiation at the boundary between two cell populations. In summary, we provide a new tool which could be used to identify cell interactions and to profile changes in gene or protein expression that result from direct cell-cell contact with defined cell populations in culture and in early embryos, and which can be adapted to generate synthetic patterning of cell fate decisions.

scholarly journals Lysophosphatidic acid and bFGF control different modes in proliferating myoblasts.

1996 ◽  
Vol 132 (1) ◽  
pp. 181-193 ◽  
Author(s):  
S Yoshida ◽  
A Fujisawa-Sehara ◽  
T Taki ◽  
K Arai ◽  
Y Nabeshima
Keyword(s):  
Lysophosphatidic Acid ◽  
Intracellular Signaling ◽  
Myogenic Differentiation ◽  
Mrna Level ◽  
Biological Significance ◽  
Cell Contact ◽  
C2c12 Myoblast ◽  
Bhlh Proteins ◽  
Cell Cell

Myogenic cells provide excellent in vitro models for studying the cell growth and differentiation. In this study we report that lysophosphatidic acid (LPA), a bioactive phospholipid contained in serum, stimulates the growth and inhibits the differentiation of mouse C2C12 myoblast cells, in a distinct manner from basic fibroblast growth factor (bFGF) whose mitotic and anti-differentiation actions have been well investigated. These actions of LPA were both blocked by pertussis toxin, suggesting the involvement of Gi class of G proteins, whereas bFGF acts through receptor tyrosine kinases. Detailed analysis revealed that LPA and bFGF act differently in regulating the myogenic basic helix-loop-helix (bHLH) proteins, the key players in myogenic differentiation process. LPA stimulates the proliferation of undifferentiated myoblasts allowing the continued expression of MyoD, but in contrast, bFGF does so with the MyoD expression suppressed at the mRNA level. Both compounds maintain the myf-5 expression, and suppress the myogenin expression. In addition, while LPA did not inhibit cell-cell contact-induced differentiation, bFGF strongly inhibited this process. Furthermore, LPA and bFGF act cooperatively in their mitogenic and anti-differentiation abilities. These findings indicate that LPA and bFGF differently stimulate intracellular signaling pathways, resulting in proliferating myoblasts each bearing a distinct expression pattern of myogenic bHLH proteins and distinct differentiation potentials in response to cell-cell contact, and illustrate the biological significance of Gi-mediated and tyrosine kinase-mediated signals.

scholarly journals Preparation and Evaluation of Collagen-Based Patches as Curcumin Carriers

Polymers ◽  
2020 ◽  
Vol 12 (10) ◽  
pp. 2393
Author(s):  
Zoi Terzopoulou ◽  
Anna Michopoulou ◽  
Artemis Palamidi ◽  
Elena Koliakou ◽  
Dimitrios Bikiaris
Keyword(s):  
Topical Treatment ◽  
Chitosan Nanoparticles ◽  
In Vitro Release ◽  
Human Keratinocytes ◽  
Inhibitory Effect ◽  
Psoriatic Skin ◽  
Burst Release ◽  
Swelling Ratio ◽  
Natural Polyphenol

Patients with psoriasis are dissatisfied with the standard pharmacological treatments, whether systemic or topical, with many of them showing interest in complementary and alternative medicine. Curcumin (Cur), a natural polyphenol derived from turmeric, has recently gained attention for skin-related diseases because of its proven anti-inflammatory action. However, topical treatment with Cur would be inadequate because of its hydrophobicity, instability, and low bioavailability. In addition, hyperkeratosis and lack of moisture in psoriatic skin result in low penetration that would prevent actives from permeating the stratum corneum. In this work, a polymer-based formulation of Cur for the topical treatment of psoriasis is reported. To improve the physicochemical stability of Cur, it was first encapsulated in chitosan nanoparticles. The Cur-loaded nanoparticles were incorporated in a hydrophilic, biocompatible collagen-based patch. The nanoparticle-containing porous collagen patches were then chemically cross-linked. Morphology, chemical interactions, swelling ratio, enzymatic hydrolysis, and Cur release from the patches were evaluated. All patches showed excellent swelling ratio, up to ~1500%, and after cross-linking, the pore size decreased, and their hydrolysis rates decelerated. The in vitro release of Cur was sustained with an initial burst release, reaching 55% after 24 h. Cur within the scaffolds imparted a proliferation inhibitory effect on psoriatic human keratinocytes in vitro.

Stimulation of in vitro hematopoiesis by a murine fetal hepatocyte clone through cell?cell contact

1994 ◽  
Vol 160 (3) ◽  
pp. 445-454 ◽  
Author(s):  
Masanobu Nanno ◽  
Masahiro Hata ◽  
Hideki Yagi ◽  
Tsunetoshi Itoh ◽  
Hideyuki Doi ◽  
...  
Keyword(s):  
Cell Contact ◽  
Fetal Hepatocyte ◽  
Stimulation Of ◽  
Cell Cell

scholarly journals The role of integrins in the maintenance of endothelial monolayer integrity.

1991 ◽  
Vol 112 (3) ◽  
pp. 479-490 ◽  
Author(s):  
M G Lampugnani ◽  
M Resnati ◽  
E Dejana ◽  
P C Marchisio
Keyword(s):  
Umbilical Vein ◽  
Dermal Fibroblasts ◽  
Collagen Type Iv ◽  
Cell Contact ◽  
Endothelial Monolayer ◽  
Integrin Receptors ◽  
Endothelial Lining ◽  
Type Iv ◽  
Cell Cell

This paper shows that, in confluent human umbilical vein endothelial cell (EC) monolayers, the integrin heterodimers alpha 2 beta 1 and alpha 5 beta 1, but not other members of the beta 1 subfamily, are located at cell-cell contact borders and not at cellular free edges. Also the alpha v chain, but not its most common partner beta 3, that is widely expressed in EC cell-matrix junctions, is found at cell-cell borders. In EC monolayers, the putative ligands of alpha 2 beta 1 and alpha 5 beta 1 receptors, i.e., laminin, collagen type IV, and fibronectin, are also organized in strands corresponding to cell-cell borders. The location of the above integrin receptors is not an artifact of in vitro culture since it has been noted also in explanted islets of the native umbilical vein endothelium. The integrins alpha 2 beta 1 and alpha 5 beta 1 play a role in the maintenance of endothelial monolayer continuity in vitro. Indeed, specific antibodies to alpha 2 beta 1, alpha 5 beta 1, and the synthetic peptide GRGDSP alter its continuity without any initial cell detachment. Moreover, antibodies to alpha 5 beta 1 increase the permeation of macromolecules across confluent EC monolayers. In contrast beta 3 antibodies were ineffective. It is suggested that the relocation of integrins to cell-cell borders is a feature of cells programmed to form polarized monolayers since integrins have a different distribution in nonpolar confluent dermal fibroblasts. The conclusion is that some members of the integrin superfamily collaborate with other intercellular molecules to form lateral junctions and to control both the monolayer integrity and the permeability properties of the vascular endothelial lining. This also suggest that integrins are adhesion molecules provided with a unique biochemical adaptability to different biological functions.

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