scholarly journals Biologically Active Fas Antigen and Its Cognate Ligand Are Expressed on Plasma Membrane-Derived Extracellular Vesicles

Blood ◽  
1998 ◽  
Vol 91 (10) ◽  
pp. 3862-3874 ◽  
Author(s):  
Joseph Albanese ◽  
Sarkis Meterissian ◽  
Maria Kontogiannea ◽  
Catherine Dubreuil ◽  
Arthur Hand ◽  
...  

Abstract Exfoliation of plasma membrane components is a directed process that consumes energy and requires active cell metabolism. Proteins involved in regulating the survival and proliferation of eukaryotic cells are released on exfoliated vesicles. We examine here whether the Fas receptor and its cognate ligand (FasL) are present on vesicles shed from high metastatic potential CX-1 cells and low metastatic potential MIP-101 cells and from HuT 78 cells, respectively. Rates of exfoliation at 2 hours and cumulative levels of extracellular vesicles in serum-free medium conditioned by CX-1 cells are increased by 1.8-fold and 1.6-fold, respectively, relative to that in medium conditioned by MIP-101 cells. Although vesicles shed from both cancer cell lines contain Fas antigen, the amount of Fas per vesicle and the percentage of vesicles containing Fas are increased for vesicles isolated from MIP-101 cells, relative to those from CX-1 cells, as determined by immunogold particle labeling and electron microscopy and by immunofluorescence microscopy and flow cytometry. Results of metabolic labeling with 35S-methionine indicate that Fas biosynthesis is reduced by up to 3.3-fold for CX-1 cells, relative to that of MIP-101 cells, consistent with the finding of decreased Fas on vesicles shed from the plasma membrane of CX-1 cells. Although mRNA for soluble Fas receptor is detectable in both cell lines, depletion of shed vesicles from serum-free medium by ultracentrifugation removes all detectable biological activity. FasL is detected on vesicles exfoliated from HuT 78 cells by immunoelectron microscopy and Western blot analysis. FasL-bearing vesicles induce apoptosis of Fas-expressing cancer cells at the same level as observed by treatment with monoclonal anti-Fas antibody. Furthermore, Fas-bearing extracellular vesicles from MIP-101 but not from CX-1 cells protect the CX-1 cell line from FasL-induced and anti-Fas–mediated apoptosis, indicating that Fas present on shed vesicles is biologically active. We conclude that the Fas antigen and its cognate ligand are exfoliated from the cell surface in a bioactive configuration. Exfoliation may provide a mechanism for long-range signal-directed apoptosis while maintaining Fas/FasL on a membrane surface.

Blood ◽  
1998 ◽  
Vol 91 (10) ◽  
pp. 3862-3874 ◽  
Author(s):  
Joseph Albanese ◽  
Sarkis Meterissian ◽  
Maria Kontogiannea ◽  
Catherine Dubreuil ◽  
Arthur Hand ◽  
...  

Exfoliation of plasma membrane components is a directed process that consumes energy and requires active cell metabolism. Proteins involved in regulating the survival and proliferation of eukaryotic cells are released on exfoliated vesicles. We examine here whether the Fas receptor and its cognate ligand (FasL) are present on vesicles shed from high metastatic potential CX-1 cells and low metastatic potential MIP-101 cells and from HuT 78 cells, respectively. Rates of exfoliation at 2 hours and cumulative levels of extracellular vesicles in serum-free medium conditioned by CX-1 cells are increased by 1.8-fold and 1.6-fold, respectively, relative to that in medium conditioned by MIP-101 cells. Although vesicles shed from both cancer cell lines contain Fas antigen, the amount of Fas per vesicle and the percentage of vesicles containing Fas are increased for vesicles isolated from MIP-101 cells, relative to those from CX-1 cells, as determined by immunogold particle labeling and electron microscopy and by immunofluorescence microscopy and flow cytometry. Results of metabolic labeling with 35S-methionine indicate that Fas biosynthesis is reduced by up to 3.3-fold for CX-1 cells, relative to that of MIP-101 cells, consistent with the finding of decreased Fas on vesicles shed from the plasma membrane of CX-1 cells. Although mRNA for soluble Fas receptor is detectable in both cell lines, depletion of shed vesicles from serum-free medium by ultracentrifugation removes all detectable biological activity. FasL is detected on vesicles exfoliated from HuT 78 cells by immunoelectron microscopy and Western blot analysis. FasL-bearing vesicles induce apoptosis of Fas-expressing cancer cells at the same level as observed by treatment with monoclonal anti-Fas antibody. Furthermore, Fas-bearing extracellular vesicles from MIP-101 but not from CX-1 cells protect the CX-1 cell line from FasL-induced and anti-Fas–mediated apoptosis, indicating that Fas present on shed vesicles is biologically active. We conclude that the Fas antigen and its cognate ligand are exfoliated from the cell surface in a bioactive configuration. Exfoliation may provide a mechanism for long-range signal-directed apoptosis while maintaining Fas/FasL on a membrane surface.


2018 ◽  
Vol 118 (03) ◽  
pp. 480-489 ◽  
Author(s):  
Anat Aharon ◽  
Anni Sabbah ◽  
Liron Issman ◽  
Hila Berkovich ◽  
Reem Copty ◽  
...  

Background The involvement of extracellular vesicles (EVs) in cancer-associated thrombosis (CT) is unclear. This study aimed to explore the properties of EVs derived from breast cancer (BC) cells following exposure to high- or low-dose chemotherapeutic agents and evaluate thrombogenic effects of these EVs on endothelial cells (ECs). Methods EVs were isolated from BC cell lines (non-metastatic MCF7, high-metastatic MDA-MB-231), pre-exposed to serum-free medium (control), with or without increasing doses of doxorubicin or paclitaxel. EV structure and size were studied using electron microscopy and Nano-sight. Antigen levels were measured by fluorescence-activated cell sorting (FACS). EV effects on EC thrombogenicity were assessed using FACS, factor Xa chromogenic assay and RT-PCR. Results Serum-free medium BC cell resulted in EV shedding that additionally increased when MDA-MB-231 cells were exposed to high doses of both agents. Tissue factor (TF) levels were similarly low (9–13%) in all EVs compared with the high expression on their parental MDA-MB-231 cells (76–83%). EVs derived from MDA-MB-231 cells stimulated with high-dose doxorubicin demonstrated significantly (fivefold; p < 0.001) elevated levels of negatively charged phospholipids, a 97% decrease in TF pathway inhibitor (TFPI) levels and a sixfold increase (p < 0.001) in procoagulant activity. These EVs also enhanced EC thrombogenicity. Effects of EVs originating from MCF7 cells were less pronounced. Conclusion These findings suggest that thrombogenic properties of BC-derived EVs may depend on the type and dose of the applied chemotherapy agent and may also be affected by the cell metastatic nature.


1996 ◽  
Vol 16 (1) ◽  
pp. 211-218 ◽  
Author(s):  
G A Preston ◽  
T T Lyon ◽  
Y Yin ◽  
J E Lang ◽  
G Solomon ◽  
...  

The role of c-Fos in apoptosis was examined in two Syrian hamster embryo cell lines (sup+I and sup-II) and a human colorectal carcinoma cell line (RKO), using the chimeric Fos-estrogen receptor fusion protein c-FosER. As previously reported, contrasting responses were observed when these two cell lines were placed under growth factor deprivation conditions; sup+I cells were highly susceptible to apoptosis, whereas sup-II cells were resistant. In this report, we show that the activated c-FosER protein induces apoptosis in sup-II preneoplastic cells in serum-free medium, indicating that c-Fos protein can induce apoptotic cell death in these cells. c-Fos-induced apoptosis was not blocked by the protein synthesis inhibitor cycloheximide, suggesting that the c-Fos transcriptional activation activity is not involved. This conclusion was further supported by the observation that overexpression of v-Fos, which is highly proficient in transcriptional activation but deficient in the transcriptional repression activity associated with c-Fos, did not induce apoptosis. Constitutively expressed Bcl-2 delayed the onset of low-serum-induced apoptosis in sup+I cells and enhanced survival in sup-II cells. Further, coexpression of Bcl-2 and c-FosER in sup+I or sup-II cells protected the cells from c-FosER-induced apoptosis. The possibility that c-FosER-induced apoptosis requires a p53 function was examined. Colorectal carcinoma RKOp53+/+ cells, which do not normally undergo apoptosis in serum-free medium, showed apoptotic DNA fragmentation upon expression and activation of c-FosER. Further, when the wild-type p53 protein was diminished in the RKO cells by infection with the papillomavirus E6 gene, subsequent c-FosER-induced apoptosis was blocked. The data suggest that c-Fos protein plays a causal role in the activation of apoptosis in a p53-dependent manner. This activity does not require new protein synthesis and is blocked by overexpression of Bcl-2 protein.


Author(s):  
F. Verhoeye ◽  
Caroline Burteau ◽  
S. Chenu ◽  
Jean Louis Goergen ◽  
Annie Marc ◽  
...  

1988 ◽  
Vol 24 (8) ◽  
pp. 845-854 ◽  
Author(s):  
M. E. Kaighn ◽  
R. F. Camalier ◽  
F. Bertolero ◽  
U. Saffiotti

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