Isolation of an endocytic compartment from A431 cells using a density modification procedure employing a receptor-specific monoclonal antibody complexed with colloidal gold

1987 ◽  
Vol 87 (4) ◽  
pp. 495-506
Author(s):  
J. Beardmore ◽  
K.E. Howell ◽  
K. Miller ◽  
C.R. Hopkins
Keyword(s):  
Transferrin Receptor ◽  
Colloidal Gold ◽  
A431 Cells ◽  
Receptor Complexes ◽  
Fold Enrichment ◽  
Versus Protein ◽  
Continuous Sucrose Gradient ◽  
Modification Procedure ◽  
Enzyme Markers ◽  
Human Epidermoid Carcinoma

Our objective was to isolate a prelysosomal compartment involved in receptor-mediated endocytosis in human epidermoid carcinoma (A431) cells. The isolation protocol involves density modification of endosome elements in A431 cells, caused by the receptor-dependent binding and internalization at 20 degrees C of colloidal gold-transferrin receptor antibody (B3/25) particles. The use of 125I-labelled gold-B3/25 provides a radioactive marker for the endosome compartment, the major peak being recovered at the bottom of a continuous sucrose gradient at a density of 1.23g ml-1. Enzyme markers characteristic of other cytoplasmic compartments are present only in negligible amounts in this fraction and L-[35S]methionine-labelling of the cells indicates approximately a 200-fold enrichment of 125I-labelled gold-B3/25 versus protein. Electron microscopy of the endosome-rich fraction reveals that we have isolated a highly purified population of small gold-containing vesicles and tubules from which the transferrin receptor can be immunoprecipitated using the B3/25 antibody. Gel electrophoresis and fluorography of L-[35S]-methionine-labelled cells suggests that these elements contain a characteristic profile of approximately 10 major proteins of which three appear to be specifically enriched. In cells incubated with [125I]transferrin, 12% of the ligand sediments with the gold-labelled elements. We conclude, therefore, that the components we have isolated play a role in the intracellular processing of the transferrin-transferrin receptor complexes.

scholarly journals Internalization and processing of transferrin and the transferrin receptor in human carcinoma A431 cells.

1983 ◽  
Vol 97 (2) ◽  
pp. 508-521 ◽  
Author(s):  
C R Hopkins ◽  
I S Trowbridge
Keyword(s):  
Transferrin Receptor ◽  
Transferrin Receptors ◽  
Gold Colloid ◽  
A431 Cells ◽  
Coated Pits ◽  
Gold Complexes ◽  
Thin Sections ◽  
Receptor Complexes ◽  
Intracellular Processing ◽  
Peripheral Cytoplasm

The binding and subsequent intracellular processing of transferrin and transferrin receptors was studied in A431 cells using 125I-transferrin and a monoclonal antibody to the receptor (ATR) labeled with 125I and gold colloid. Using 125I-transferrin we have shown that, whereas at 37 degrees C uptake proceeded linearly for up to 60 min, most of the ligand that was bound was internalized and then rapidly returned to the incubation medium undegraded. At 37 degrees C, the intracellular half-life of the most rapidly recycled transferrin was 7.5 min. 125I-ATR displayed the same kinetics of uptake but following its internalization at 37 degrees C, it was partially degraded. At 22 degrees C and below, the intracellular degradation of 125I-ATR was selectively inhibited and as a result it accumulated intracellularly. Electron microscopy of conventional thin sections and of whole-cell mounts was used to follow the uptake and processing of transferrin receptors labeled with ATR-gold colloid complexes. Using a pulse-chase protocol, the intracellular pathway followed by internalized ATR gold-receptor complexes was outlined in detail. Within 5 min at 22 degrees C the internalized complexes were transferred from coated pits on the cell surface to a system of narrow, branching cisternae within the peripheral cytoplasm. By 15 min they reached larger, more dilated elements that, in thin section, appeared as irregular profiles containing small (30-50-nm diam) vesicles. By 30 min, the gold complexes were located predominantly within typical spherical multivesicular bodies lying in the peripheral cytoplasm, and by 40-60 min, they reached a system of cisternal and multivesicular body elements in the juxtanuclear area. At 22 degrees C, no other compartments became labeled but if they were warmed to 37 degrees C the gold complexes were transferred to lysosome-like elements. Extracting ATR-gold complexes with Triton X after a 30-min chase at 22 degrees C and purifying them on Sepharose-transferrin indicated that the internalized complexes remained bound to the transferrin receptor during their intracellular processing.

scholarly journals Mitochondrial Pathway of α-Tocopheryl Succinate-Induced Apoptosis in Human Epidermoid Carcinoma A431 Cells

Acta Naturae ◽  
2012 ◽  
Vol 4 (3) ◽  
pp. 88-94 ◽  
Author(s):  
M. A. Savitskaya ◽  
M. S. Vildanova ◽  
O. P. Kisurina-Evgenieva ◽  
E. A. Smirnova ◽  
G. E. Onischenko
Keyword(s):  
Cell Death ◽  
Vitamin E ◽  
Apoptotic Cell ◽  
Mitochondrial Pathway ◽  
Epidermoid Carcinoma ◽  
A431 Cells ◽  
Human Carcinoma ◽  
Tocopheryl Succinate ◽  
Induced Apoptosis ◽  
Human Epidermoid Carcinoma

Vitamin E derivatives are known to act as agents exhibiting cytotoxity against tumor cells. The effect of vitamin E succinate on human epidermoid carcinoma cell line A431 was investigated in this study using live imaging, immunocytochemistry, and transmission electron microscopy. -Tocopheryl succinate-induced apoptotic cell death in A431 cells was shown to be both dose- and time-dependent. The hyperproduction of reactive oxygen species, changes in size, shape and ultrastructural characteristics of mitochondria followed by the release of cytochrome c from mitochondria to cytosol were observed. These results suggest that -tocopheryl succinate induces apoptosis that occurs via the mitochondrial pathway. Mitochondria are shown to be crucial targets in -tocopheryl succinate-induced caspase-dependent cell death in human carcinoma A431 cells.

scholarly journals Centipede Scolopendra suppresses cell growth in human epidermoid carcinoma cell A431

2017 ◽  
Vol 12 (3) ◽  
pp. 299
Author(s):  
Lei Zheng ◽  
Hao He ◽  
Xuji Shen ◽  
Yanping Sun
Keyword(s):  
Molecular Mechanisms ◽  
Video Clip ◽  
Epidermoid Carcinoma ◽  
Matrix Metalloproteinase 2 ◽  
Migration And Invasion ◽  
A431 Cells ◽  
Matrigel Invasion ◽  
Egfr Expression ◽  
Cancer Intervention ◽  
Human Epidermoid Carcinoma

<p class="Abstract">The aim of this study was to examine the anti-proliferation and anti-migration effect of Centipede Scolopendra extracts (CSE) on human epidermoid carcinoma cells (high-EGFR expression) A431 and elucidate the underlying signaling mechanisms. MTT and colony formation assays were used. Migration and invasion potential of A431 cells were examined by wound-healing assays and matrigel invasion chamber assays. To investigate the underlying molecular mechanisms, we used ELISA to analyze the expression of EGF, Western blotting to analyze the expression of MMP2 (matrix metalloproteinase 2), MMP9 and EGFR, PCR to analyze the mRNA expression of EGFR pretreated with CSE. The results showed that CSE effectively inhibited the proliferation of A431. Furthermore, CSE-mediated cell cycle arrest in S phase. We also observed that CSE treatment led to down-regulation of MMP2 and MMP9 and suppress the migration and invasion in A431. CSE exerted its anti-proliferation and anti-migration by targeting EGFR and related metastasis factors, thus could be a useful therapeutic candidate for high-EGFR expression cancer intervention.</p><p class="Abstract"><strong>Video Clip of Methodology</strong>:</p><p class="Abstract">Cell migration and invasion assay: 2 min 41 sec   <a href="https://www.youtube.com/v/fg27Xn0talE">Full Screen</a>   <a href="https://www.youtube.com/watch?v=fg27Xn0talE">Alternate</a>   </p>

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