scholarly journals Fibronectin expression is determined by the genotype of the transformed parental cells in heterokaryons between normal and transformed fibroblasts.

1979 ◽  
Vol 80 (1) ◽  
pp. 118-127 ◽  
Author(s):  
P Laurila ◽  
J Wartiovaara ◽  
S Stenman
Keyword(s):  
Cell Surface ◽  
Minimal Surface ◽  
Human Fibroblasts ◽  
Transformed Cells ◽  
Transformed Cell ◽  
Gene Dose ◽  
Fibronectin Expression ◽  
A Cell ◽  
Dose Dependent ◽  
Was Gene

The expression of fibronectin, a cell surface-associated transformation-sensitive glycoprotein, was studied in hetero- and homokaryons of normal and SV40-transformed human fibroblasts. In immunofluorescence, fibroblast homokaryons had an intense surface-associated and intracelluar fibronectin fluorescence similar to that of normal fibroblasts. Transformed cells and their homokaryons had a minimal surface-associated and a weak intracellular fibronectin fluorescence. In heterokaryons formed between transformed and normal fibroblasts, the expression of fibronectin fell within 24 h to the level of the transformed cell homokaryons. The change was detectable already at 3 h after fusion and was gene-dose dependent. These results show that the transformed genotype determines fibronectin expression in the heterokaryons.

scholarly journals Hyaluronate synthetase inhibition by normal and transformed human fibroblasts during growth reduction

1987 ◽  
Vol 104 (4) ◽  
pp. 1105-1115 ◽  
Author(s):  
K Matuoka ◽  
M Namba ◽  
Y Mitsui
Keyword(s):  
Cell Proliferation ◽  
Cell Density ◽  
Human Fibroblasts ◽  
Transformed Cells ◽  
Synthetase Activity ◽  
Intact Cells ◽  
Free System ◽  
Glycosaminoglycan Synthesis ◽  
Cell Free System ◽  
A Cell

To establish the relation of glycosaminoglycan synthesis to cell proliferation, we investigated the synthesis of individual glycosaminoglycan species by intact cells and in a cell-free system, using normal and transformed human fibroblasts under differing culture conditions. Reducing serum concentration brought about a marked decline in the synthesis of hyaluronate (HA) as well as cell proliferation on both normal and transformed cells. Both HA synthesis and proliferation decreased with increasing cell densities markedly (in inverse proportion to cell density) in normal cells but gradually in transformed cells. This noticeable congruity of the changes in HA synthesis and proliferation indicates that the change in HA synthesis is related primarily to cell proliferation rather than to cell density or cellular transformation. Examination of HA synthesis in a cell-free system demonstrated that the activity of HA synthetase also fluctuated in conjunction with cell proliferation. Furthermore, growth-reduced cells (except crowded transformed cells) inhibited cell-free HA synthesis and this inhibition was induced coincidentally with a decrease in both HA synthetase activity and proliferation. These findings suggest that the change in HA synthesis is significant in the regulation of cell proliferation.

scholarly journals Dopamine reduces cell surface Na+/H+ exchanger-3 protein by decreasing NHE3 exocytosis and cell membrane recycling

2017 ◽  
Vol 313 (4) ◽  
pp. F1018-F1025 ◽  
Author(s):  
Ming Chang Hu ◽  
I. Alexandru Bobulescu ◽  
Henry Quiñones ◽  
Serge M. Gisler ◽  
Orson W. Moe
Keyword(s):  
Cell Membrane ◽  
Cell Surface ◽  
Proximal Tubule ◽  
Apical Cell ◽  
Brefeldin A ◽  
Dependent Manner ◽  
Culture Model ◽  
A Cell ◽  
The Many ◽  
Dose Dependent

The intrarenal autocrine-paracrine dopamine (DA) system mediates a significant fraction of the natriuresis in response to a salt load. DA inhibits a number of Na+ transporters to effect sodium excretion, including the proximal tubule Na+/H+ exchanger-3 (NHE3). DA represent a single hormone that regulates NHE3 at multiple levels, including translation, degradation, endocytosis, and protein phosphorylation. Because cell surface NHE3 protein is determined by the balance between exocytotic insertion and endocytotic retrieval, we examined whether DA acutely affects the rate of NHE3 exocytosis in a cell culture model. DA inhibited NHE3 exocytosis at a dose-dependent manner with a half maximal around 10−6 M. The DA effect on NHE3 exocytosis was blocked by inhibition of protein kinase A and by brefeldin A, which inhibits endoplasmic reticulum-to-Golgi transport. NHE3 directly interacts with the ε-subunit of coatomer protein based on yeast-two-hybrid and coimmunoprecipitation. Because NHE3 has been shown to be recycled back to the cell membrane after endocytosis, we measured NHE3 recycling using a biochemical reinsertion assay and showed that reinsertion of NHE3 back to the membrane is also inhibited by DA. In conclusion, among the many mechanisms by which DA reduces apical membrane NHE3 and induces proximal tubule natriuresis, one additional mechanism is inhibition of exocytotic insertion and reinsertion of NHE3 in the apical cell surface.

scholarly journals Folates and Anti-folates � Chemical Perspectives, in vitro and in ovo Assessments

2020 ◽  
Vol 71 (7) ◽  
pp. 481-490
Author(s):  
Bogdan Sorop ◽  
Vlad Laurentiu David ◽  
Alina Heghes ◽  
Delia Berceanu-Vaduva ◽  
Lavinia Balan ◽  
...  
Keyword(s):  
Human Fibroblasts ◽  
Acid Synthesis ◽  
Enzymatic Reactions ◽  
Dependent Manner ◽  
Cam Assay ◽  
In Ovo ◽  
Amino Acid Synthesis ◽  
A Cell ◽  
Dose Dependent

Folates participate in DNA replication reactions, act as a substrate in enzymatic reactions related to amino acid synthesis and vitamin metabolism while antifolates participate in reactions that inhibit the formation of tetrahydrofolate with consequences on protein and nucleic acid synthesis and implicitly on growth and development both types of cells, healthy and diseased. In the present study, the viability of healthy cells, keratinocytes and human fibroblasts was evaluated in the presence of three folates (folic, dihydrofolic and tetrahydrofolic acids), one antifolate (methotrexate) and combinations between them by Alamar blue assay. The antiangiogenic potential was also evaluated by in ovo technique, CAM assay. Cell viability was influenced in a cell-dependent and dose-dependent manner, fibroblasts being more sensitive to the action of the test compounds, especially the combination of metrotrexate and dihydrofolate. Data related to CAM assay showed that methotrexate revealed a slightly higher vessel density, but without inducing toxicity on vascular architecture and functionality. The data obtained highlight the greater sensitivity of the viability of fibroblasts in the presence of metrotrexate and its combinations with folates used in the study.

Activation, processing and trafficking of extracellular heparanase by primary human fibroblasts

2002 ◽  
Vol 115 (10) ◽  
pp. 2179-2187 ◽  
Author(s):  
Liat Nadav ◽  
Amiram Eldor ◽  
Oron Yacoby-Zeevi ◽  
Eli Zamir ◽  
Iris Pecker ◽  
...  
Keyword(s):  
Cell Surface ◽  
Heparan Sulfate ◽  
Metastatic Cancer ◽  
Human Fibroblasts ◽  
Basement Membranes ◽  
Prolonged Storage ◽  
Cellular Activation ◽  
Proteolytic Activation ◽  
Highly Active ◽  
A Cell

Heparanase is a heparan-sulfate-degrading endoglycosidase that has important roles in various biological processes, including angiogenesis, wound healing and metastatsis. Human heparanase is synthesized as a 65 kDa latent precursor, which is proteolytically processed into a highly active 50 kDa form. Extracellular heparanase is found in various tissues and is utilized by both normal cells and metastatic cancer cells to degrade heparan sulfate moieties in basement membranes and extracellular matrices. This study characterizes the processing and trafficking events associated with cellular activation of extracellular heparanase. We show that primary human fibroblasts are capable of binding and converting the 65 kDa heparanase precursor into its highly active 50 kDa form, concomitantly with its cytoplasmic accumulation. Heparanase uptake depends on the actin cytoskeleton integrity, resulting in a prolonged storage of the enzyme, mainly in endosomal structures. Heparanase endocytosis and its proteolytic activation are independent processes,indicating that heparanase cleavage is a cell surface event. Heparin completely inhibits heparanase endocytosis but only partially inhibits its association with the cells, suggesting that cell surface heparan sulfate moieties play a specific role in its endocytosis. Cellular binding and uptake of extracellular heparanase control its activation, clearance rate and storage within the cells.

scholarly journals Intracellular ionic changes in normal and transformed human fibroblasts after extracellular Ca2+ deprivation

1981 ◽  
Vol 194 (3) ◽  
pp. 707-711 ◽  
Author(s):  
B J Hazelton ◽  
J T Tupper
Keyword(s):  
Growth Response ◽  
Human Fibroblasts ◽  
Growth Arrest ◽  
Fold Increase ◽  
Transformed Cells ◽  
Transformed Cell ◽  
Permeability Changes ◽  
Na Efflux ◽  
Ionic Changes ◽  
G0 Phase

The lowering of extracellular Ca2+ concentration in the growth medium reversibly blocks normal, but not SV40-transformed WI38 diploid fibroblasts in the early G1/G0 phase of the cell cycle. This growth response is characterized by specific changes in ionic content and transport. Ca2+ deprivation (0.03 mM) has little effect on the K+ content of either normal or transformed cells. Na+ content, however, is increased nearly 2-fold in the normal cells. This increase is presumably due to a 3-fold increase in unidirectional Na+ influx in Ca2+-deprived cells. The increased intracellular Na+ also gives rise to a nearly 3-fold enhancement of the active (ouabain-sensitive) Na+ efflux. Ca2+ deprivation causes only slight increases in Na+ influx, ouabain-sensitive Na+ efflux and intracellular Na+ in the transformed cell. In contrast, the transformed cells lose nearly 60% of their intracellular Ca2+ on deprivation, whereas normal WI38 cells lose only 10%. The data suggest that the growth arrest exhibited by the normal cell but not the transformed cell may be related to different membrane-transport and permeability changes in response to Ca2+ deprivation.

scholarly journals Downregulation of peptide transporter genes in cell lines transformed with the highly oncogenic adenovirus 12.

1994 ◽  
Vol 180 (2) ◽  
pp. 477-488 ◽  
Author(s):  
R Rotem-Yehudar ◽  
S Winograd ◽  
S Sela ◽  
J E Coligan ◽  
R Ehrlich
Keyword(s):  
Cell Surface ◽  
Cell Lines ◽  
Surface Expression ◽  
Class I ◽  
Cell Surface Expression ◽  
Peptide Transporter ◽  
Transformed Cells ◽  
Mrna Levels ◽  
Transformed Cell ◽  
I Gene

The expression of class I major histocompatibility complex antigens on the surface of cells transformed by adenovirus 12 (Ad12) is generally very low, and correlates with the high oncogenicity of this virus. In primary embryonal fibroblasts from transgenic mice that express both endogenous H-2 genes and a miniature swine class I gene (PD1), Ad12-mediated transformation results in suppression of cell surface expression of all class I antigens. Although class I mRNA levels of PD1 and H-2Db are similar to those in nonvirally transformed cells, recognition of newly synthesized class I molecules by a panel of monoclonal antibodies is impaired, presumably as a result of inefficient assembly and transport of the class I molecules. Class I expression can be partially induced by culturing cells at 26 degrees C, or by coculture of cells with class I binding peptides at 37 degrees C. Analysis of steady state mRNA levels of the TAP1 and TAP2 transporter genes for Ad12-transformed cell lines revealed that they both are significantly reduced, TAP2 by about 100-fold and TAP1 by 5-10-fold. Reconstitution of PD1 and H-2Db, but not H-2Kb, expression is achieved in an Ad12-transformed cell line by stable transfection with a TAP2, but not a TAP1, expression construct. From these data it may be concluded that suppressed expression of peptide transporter genes, especially TAP2, in Ad12-transformed cells inhibits cell surface expression of class I molecules. The failure to fully reconstitute H-2Db and H-2Kb expression indicates that additional factors are involved in controlling class I gene expression in Ad12-transformed cells. Nevertheless, these results suggest that suppression of peptide transporter genes might be an important mechanism whereby virus-transformed cells escape immune recognition in vivo.

scholarly journals Reduced Phosphorylation of p50 Is Responsible for Diminished NF-κB Binding to the Major Histocompatibility Complex Class I Enhancer in Adenovirus Type 12-Transformed Cells

1999 ◽  
Vol 19 (3) ◽  
pp. 2169-2179 ◽  
Author(s):  
David B. Kushner ◽  
Robert P. Ricciardi
Keyword(s):  
Major Histocompatibility Complex ◽  
Cell Surface ◽  
Major Histocompatibility Complex Class ◽  
Adenovirus Type ◽  
Class I ◽  
Transformed Cells ◽  
Complex Class ◽  
Transformed Cell ◽  
Histocompatibility Complex ◽  
Nuclear Extracts

ABSTRACT Reduced cell surface levels of major histocompatibility complex class I antigens enable adenovirus type 12 (Ad12)-transformed cells to escape immunosurveillance by cytotoxic T lymphocytes (CTL), contributing to their tumorigenic potential. In contrast, nontumorigenic Ad5-transformed cells harbor significant cell surface levels of class I antigens and are susceptible to CTL lysis. Ad12 E1A mediates down-regulation of class I transcription by increasing COUP-TF repressor binding and decreasing NF-κB activator binding to the class I enhancer. The mechanism underlying the decreased binding of nuclear NF-κB in Ad12-transformed cells was investigated. Electrophoretic mobility shift assay analysis of hybrid NF-κB dimers reconstituted from denatured and renatured p50 and p65 subunits from Ad12- and Ad5-transformed cell nuclear extracts demonstrated that p50, and not p65, is responsible for the decreased ability of NF-κB to bind to DNA in Ad12-transformed cells. Hypophosphorylation of p50 was found to correlate with restricted binding of NF-κB to DNA in Ad12-transformed cells. The importance of phosphorylation of p50 for NF-κB binding was further demonstrated by showing that an NF-κB dimer composed of p65 and alkaline phosphatase-treated p50 from Ad5-transformed cell nuclear extracts could not bind to DNA. These results suggest that phosphorylation of p50 is a key step in the nuclear regulation of NF-κB in adenovirus-transformed cells.

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