Morphology, Gap Junctions and Phospholipase A2 Activation in TNF-Susceptible Cell Lines and Their TNF-Resistant Sublines1

2015 ◽  
pp. 82-86
Author(s):  
N. Matthews ◽  
R. A. Fiera ◽  
M. L. Neale
Keyword(s):  
Gap Junctions ◽  
Phospholipase A2 ◽  
Cell Lines ◽  
Susceptible Cell

scholarly journals Lack of phospholipase A2 mutations in neuroblastoma, melanoma and colon-cancer cell lines

1997 ◽  
Vol 72 (2) ◽  
pp. 337-339 ◽  
Author(s):  
Frank G. Haluska ◽  
Carol Thiele ◽  
Alisa Goldstein ◽  
Hensin Tsao ◽  
Eric P. Benoit ◽  
...  
Keyword(s):  
Colon Cancer ◽  
Phospholipase A2 ◽  
Cell Lines ◽  
Cancer Cell ◽  
Cancer Cell Lines ◽  
Colon Cancer Cell ◽  
Colon Cancer Cell Lines

scholarly journals Orai1 and Ca2+-independent phospholipase A2 are required for store-operated Icat-SOC current, Ca2+ entry, and proliferation of primary vascular smooth muscle cells

2012 ◽  
Vol 302 (5) ◽  
pp. C748-C756 ◽  
Author(s):  
Bo Yang ◽  
Tomasz Gwozdz ◽  
Joanna Dutko-Gwozdz ◽  
Victoria M. Bolotina
Keyword(s):  
Smooth Muscle ◽  
Vascular Smooth Muscle ◽  
Smooth Muscle Cells ◽  
Dna Synthesis ◽  
Phospholipase A2 ◽  
Cell Lines ◽  
Vascular Smooth Muscle Cells ◽  
Muscle Cells ◽  
Multiple Functions

Store-operated Ca2+ entry (SOCE) is important for multiple functions of vascular smooth muscle cells (SMC), which, depending of their phenotype, can resemble excitable and nonexcitable cells. Similar to nonexcitable cells, Orai1 was found to mediate Ca2+-selective (CRAC-like) current and SOCE in dedifferentiated cultured SMC and smooth muscle-derived cell lines. However, the role of Orai1 in cation-selective store-operated channels (cat-SOC), which are responsible for SOCE in primary SMC, remains unclear. Here we focus on primary SMC, and assess the role of Orai1 and Ca2+-independent phospholipase A2 (iPLA2β, or PLA2G6) in activation of cat-SOC current ( Icat-SOC), SOCE, and SMC proliferation. Using molecular, electrophysiological, imaging, and functional approaches, we demonstrate that molecular knockdown of either Orai1 or iPLA2β leads to similar inhibition of the whole cell cat-SOC current and SOCE in primary aortic SMC and results in significant reduction in DNA synthesis and impairment of SMC proliferation. This is the first demonstration that Orai1 and iPLA2β are equally important for cat-SOC, SOCE, and proliferation of primary aortic SMC.

scholarly journals Characterization of phospholipase A2 in monocytic cell lines. Functional and biochemical aspects of membrane association

1991 ◽  
Vol 276 (3) ◽  
pp. 631-636 ◽  
Author(s):  
W Rehfeldt ◽  
R Hass ◽  
M Goppelt-Struebe
Keyword(s):  
Substrate Specificity ◽  
Phospholipase A2 ◽  
Cell Lines ◽  
Cytosolic Phospholipase A2 ◽  
Biologically Active ◽  
Ph Optimum ◽  
Monocytic Cell ◽  
Cytosolic Phospholipase ◽  
Membrane Bound ◽  
Phospholipase A2 Activity

Phospholipase A2 activity was characterized in the human monocytic tumour-cell lines U937 and THP1. The enzyme showed an alkaline pH optimum and substrate specificity for arachidonoyl-phosphatidylcholine. The activation of phospholipase A2 required bivalent cations (Ca2+ greater than Mg2+ = Sr2+ greater than Ba2+). Investigation of the subcellular distribution of the enzyme revealed that the phospholipase A2 activity was shifted to the cytosol in the presence of EDTA, indicating that the association of the enzyme with the cellular membranes is Ca2+ (bivalent-cation)-dependent. Stimulation of THP1 cells for 2-4 h with the phorbol ester phorbol 12-myristate 13-acetate (PMA) activated cytosolic and membrane-bound phospholipase A2. At this time, no effect of PMA on phospholipase A2 activity was observed in the less mature U937 cells. However, when both cell lines were induced to differentiate along the monocytic pathway by a 2-3-day treatment with PMA, the cells released significant amounts of arachidonic acid and prostanoids. Compared with undifferentiated control cells, these PMA-differentiated cells showed a decrease in cytosolic phospholipase A2 activity and an increase in membrane-bound activity. Membrane-bound and cytosolic enzyme showed the same pH optimum, Ca(2+)-dependency and substrate specificity. These data indicate that membrane-bound and cytosolic phospholipase A2 activities represent one enzyme and that the membrane-bound form is the biologically active phospholipase A2.

Down-regulation by butylated hydroxytoluene of the number and function of gap junctions in epithelial cell lines derived from mouse lung and rat liver

1995 ◽  
Vol 16 (10) ◽  
pp. 2575-2582 ◽  
Author(s):  
X. Guan ◽  
J. Hardenbrook ◽  
M.J. Fernstrom ◽  
R. Chaudhuri ◽  
A.M. Malkinson ◽  
...  
Keyword(s):  
Epithelial Cell ◽  
Gap Junctions ◽  
Cell Lines ◽  
Rat Liver ◽  
Butylated Hydroxytoluene ◽  
Mouse Lung ◽  
Down Regulation ◽  
Epithelial Cell Lines ◽  
And Function

scholarly journals Differential phosphorylation of the gap junction protein connexin43 in junctional communication-competent and -deficient cell lines.

1990 ◽  
Vol 111 (5) ◽  
pp. 2077-2088 ◽  
Author(s):  
L S Musil ◽  
B A Cunningham ◽  
G M Edelman ◽  
D A Goodenough
Keyword(s):  
Cell Adhesion ◽  
Gap Junctions ◽  
Gap Junction ◽  
Cell Lines ◽  
Gap Junctional Communication ◽  
Competent Cells ◽  
Deficient Cell ◽  
Junctional Communication ◽  
Gap Junctional ◽  
Cell Cell

Connexin43 is a member of the highly homologous connexin family of gap junction proteins. We have studied how connexin monomers are assembled into functional gap junction plaques by examining the biosynthesis of connexin43 in cell types that differ greatly in their ability to form functional gap junctions. Using a combination of metabolic radiolabeling and immunoprecipitation, we have shown that connexin43 is synthesized in gap junctional communication-competent cells as a 42-kD protein that is efficiently converted to a approximately 46-kD species (connexin43-P2) by the posttranslational addition of phosphate. Surprisingly, certain cell lines severely deficient in gap junctional communication and known cell-cell adhesion molecules (S180 and L929 cells) also expressed 42-kD connexin43. Connexin43 in these communication-deficient cell lines was not, however, phosphorylated to the P2 form. Conversion of S180 cells to a communication-competent phenotype by transfection with a cDNA encoding the cell-cell adhesion molecule L-CAM induced phosphorylation of connexin43 to the P2 form; conversely, blocking junctional communication in ordinarily communication-competent cells inhibited connexin43-P2 formation. Immunohistochemical localization studies indicated that only communication-competent cells accumulated connexin43 in visible gap junction plaques. Together, these results establish a strong correlation between the ability of cells to process connexin43 to the P2 form and to produce functional gap junctions. Connexin43 phosphorylation may therefore play a functional role in gap junction assembly and/or activity.

scholarly journals High-throughput measurement of gap junctional intercellular communication

2014 ◽  
Vol 306 (12) ◽  
pp. H1708-H1713 ◽  
Author(s):  
Jun Liu ◽  
Vinayakumar Siragam ◽  
Jun Chen ◽  
Michael D. Fridman ◽  
Robert M. Hamilton ◽  
...  
Keyword(s):  
Gap Junctions ◽  
Gap Junction ◽  
Cell Lines ◽  
High Throughput ◽  
Intercellular Communication ◽  
Dye Transfer ◽  
Gap Junctional Intercellular Communication ◽  
Cell Injection ◽  
Operation Speed ◽  
Gap Junctional

Gap junctional intercellular communication (GJIC) is a critical part of cellular activities and is necessary for electrical propagation among contacting cells. Disorders of gap junctions are a major cause for cardiac arrhythmias. Dye transfer through microinjection is a conventional technique for measuring GJIC. To overcome the limitations of manual microinjection and perform high-throughput GJIC measurement, here we present a new robotic microinjection system that is capable of injecting a large number of cells at a high speed. The highly automated system enables large-scale cell injection (thousands of cells vs. a few cells) without major operator training. GJIC of three cell lines of differing gap junction density, i.e., HeLa, HEK293, and HL-1, was evaluated. The effect of a GJIC inhibitor (18-α-glycyrrhetinic acid) was also quantified in the three cell lines. System operation speed, success rate, and cell viability rate were quantitatively evaluated based on robotic microinjection of over 4,000 cells. Injection speed was 22.7 cells per min, with 95% success for cell injection and >90% survival. Dye transfer cell counts and dye transfer distance correlated with the expected connexin expression of each cell type, and inhibition of dye transfer correlated with the concentration of GJIC inhibitor. Additionally, real-time monitoring of dye transfer enables the calculation of coefficients of molecular diffusion through gap junctions. This robotic microinjection dye transfer technique permits rapid assessment of gap junction function in confluent cell cultures.

scholarly journals Apoptosis induced by polyclonal antilymphocyte globulins in human B- cell lines

Blood ◽  
1994 ◽  
Vol 83 (4) ◽  
pp. 1051-1059 ◽  
Author(s):  
N Bonnefoy-Berard ◽  
L Genestier ◽  
M Flacher ◽  
JP Rouault ◽  
G Lizard ◽  
...  
Keyword(s):  
B Cell ◽  
Cell Lines ◽  
Immunosuppressive Agents ◽  
Resistant Cell ◽  
Resistant Cell Line ◽  
Lymphoma Cell ◽  
Dna Breaks ◽  
Nuclear Condensation ◽  
Susceptible Cell ◽  
Barr Virus

Antilymphocyte and antithymocyte globulins (ALG) are currently used as immunosuppressive agents in clinical transplantation and for the treatment of severe aplastic anemia. ALG contain a mixture of antibodies that recognize T- and B-cell-specific antigens but mostly nonlineage-specific molecules. We reported previously that ALG could inhibit the proliferation of activated B cells and B cell lines (Bonnefoy-Berard et al, Blood 79:2164, 1992). We show here that ALG induce apoptosis of several human hematopoietic cell lines, as shown by nuclear condensation and fragmentation in fluorescence and electronic microscopy and by double-strand DNA breaks shown by DNA electrophoresis. Apoptosis was achieved without elevation of intracellular Ca2+ and requirement for mRNA and protein synthesis. Most of the B-cell lines tested (Epstein-Barr virus [EBV]-transformed lymphoblastoid cell lines, EBV-negative and groups I/III EBV-positive Burkitt's lymphoma cell lines, as well as other B-lymphoma cell lines) were susceptible to ALG-induced cytotoxicity. Myelomonocytic and T-cell lines were much less susceptible than B-cell lines. Susceptibility to ALG-induced cytotoxicity was not correlated with intracellular Bcl-2 level. Most cell lines that express high levels of Fas/Apo-1 antigen were susceptible to ALG. However, several lines of evidence support the conclusion that, in addition to Fas/Apo-1, other cell surface molecules can mediate ALG-induced apoptosis. The cytotoxic activity could be fully removed by adsorption on susceptible cell lines but not on a resistant cell line, indicating that it was mediated by antibodies specific for surface antigens expressed only on susceptible cell lines. Apoptosis was triggered by ALG F(ab')2 fragments as well as by intact ALG. This cytotoxic property of ALG may account for their antiproliferative effect and might contribute to some extent to the relatively lower risk of posttransplant lymphoproliferative disorders previously reported in ALG-treated patients.

Ruthenium red-positive surface layer, extracellular filamentous material and intercellular junctions in hybrids between tumour and normal cells: abundant gap junctions correlate with density-dependent inhibition of growth

1978 ◽  
Vol 31 (1) ◽  
pp. 323-339
Author(s):  
S.C. Stamatoglou ◽  
C.J. Marshall
Keyword(s):  
Gap Junctions ◽  
Cell Lines ◽  
Intercellular Junctions ◽  
Cell Types ◽  
Parental Cell ◽  
Ruthenium Red ◽  
Hybrid Cells ◽  
Inhibition Of Growth ◽  
Dependent Inhibition ◽  
Transformed Cell Lines

The cell surface and intercellular junctions of transformed and non-transformed hybrid cells, obtained from fusion of murine mammary adenocarcinoma cells (TA3B) and normal rat embryofibroblasts (REF), were compared with those of the parental cells, using ruthenium red (RR) staining of cells fixed in situ. An RR-positive layer of variable thickness was found on the surface of all cell types. Measurements of the thickness of this layer on the free surface of the cell cultures deomonstrated a significant difference between transformed and non-transformed cells. The thickness distribution of the RR layer was similar on the surface of REF and non-transformed hybrid cells, but there was significant variation among all the transformed cell lines. Extracellular filamentous RR-positive material, usually in direct contact with the cell surface, was present in confluent cultures of REF and non-transformed hybrid cells, but was absent in the transformed cell lines. Gap junctions were few in both parental cell lines and rare or absent in transformed hybrids; a large increase in number and size of gap junctions was found in non-transformed hybrids. Abundance of long gap junctions was correlated with density-dependent inhibition of growth, since non-transformed hybrids grew in single layers whereas both normal and tumour parental cell types, and transformed hybrids grew in multilayers. Tight junctions were frequently encountered in TA3B and transformed hybrid cells but were not seen in REF cells and only occasionally seen in non-transformed hybrids. Intermediate-type junctions occurred in all cells, but desmosomes were found only rarely in TA3B cells and never in the other cell types.

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