Document Detail

Inhibition of post-translational modification and surface expression of a melanoma-associated chondroitin sulfate proteoglycan by diethylcarbamazine or ammonium chloride.
MedLine Citation:
PMID:  3514609     Owner:  NLM     Status:  MEDLINE    
Cultured human melanoma M21 cells were treated with diethylcarbamazine (DEC), an inhibitor of proteoglycan biosynthesis in rat chondrosarcoma cells, to examine the assembly and transport of a chondroitin sulfate proteoglycan to the plasma membrane. Pretreatment of melanoma cells at 37 degrees C for 15 min with increasing doses of DEC followed by a 60-min pulse with [35S]sulfate in the presence of DEC resulted in a dose-related inhibition of incorporation of [35S]sulfate into macromolecules. In cells incubated for 75 min with both 1 mM beta-D-xyloside and 15 mM DEC, synthesis and secretion of beta-D-xyloside-bound 35S-glycosaminoglycans were inhibited by more than 80% as compared to cells treated with beta-D-xyloside alone; this inhibition was reversible. As assessed by [3H]serine incorporation into protein, overall protein synthesis was not substantially inhibited by DEC treatment. Detergent lysates from [35S]methionine-labeled melanoma cells were incubated with a monoclonal antibody (9.2.27) that specifically recognizes the peptide core of the melanoma proteoglycan. As assessed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the immunoprecipitate, a 240,000 Mr endoglycosidase H (Endo-H)-sensitive intermediate was the only form of the proteoglycan present inside the cells when the cultures were treated for 60-120 min with 10-15 mM DEC. When the melanoma cells were incubated for 10 min with 15 mM DEC and 100 mu Ci/ml of [35S]methionine, washed, and then chased for 15 min to 4 h in radioactive-free medium, the 240,000 Mr Endo-H-sensitive intermediate was slowly converted to a 250,000 Endo-H-resistant intermediate but not to a mature proteoglycan molecule that possessed chondroitin sulfate glycosaminoglycans. SDS-PAGE analysis of cell surface immunoprecipitates revealed that only a small amount of the 250,000 Mr intermediate was transported to the plasma membrane within 5 h of incubation in the presence of DEC. Proteoglycan synthesis was also inhibited when the melanoma cells were incubated for 60-120 min with ammonium chloride, but unlike DEC-treated cells the majority of the synthesized peptide core was converted to a 245,000 Mr Endo-H-resistant intermediate that was detected on the cell surface. Light and electron microscopic analysis of DEC-treated melanoma cells revealed large vacuoles and a distended Golgi and endoplasmic reticulum. Ammonium chloride-treated cells contained fewer vacuoles than DEC-treated cells but more vacuoles than normal cells.(ABSTRACT TRUNCATED AT 400 WORDS)
R C Spiro; W G Parsons; S K Perry; J P Caulfield; A Hein; R A Reisfeld; J R Harper; K F Austen; R L Stevens
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Publication Detail:
Type:  Journal Article; Research Support, U.S. Gov't, P.H.S.    
Journal Detail:
Title:  The Journal of biological chemistry     Volume:  261     ISSN:  0021-9258     ISO Abbreviation:  J. Biol. Chem.     Publication Date:  1986 Apr 
Date Detail:
Created Date:  1986-05-14     Completed Date:  1986-05-14     Revised Date:  2007-11-14    
Medline Journal Info:
Nlm Unique ID:  2985121R     Medline TA:  J Biol Chem     Country:  UNITED STATES    
Other Details:
Languages:  eng     Pagination:  5121-9     Citation Subset:  IM    
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MeSH Terms
Ammonium Chloride / pharmacology*
Cell Line
Diethylcarbamazine / pharmacology*
Disaccharides / analysis
Exocytosis / drug effects
Glycosaminoglycans / metabolism
Glycosides / pharmacology
Immunosorbent Techniques
Melanoma / metabolism*,  ultrastructure
Microscopy, Electron
Molecular Weight
Protein Processing, Post-Translational / drug effects*
Proteochondroitin Sulfates / metabolism*
Proteoglycans / metabolism*
Sulfates / metabolism
Sulfur Radioisotopes
Grant Support
Reg. No./Substance:
0/Disaccharides; 0/Glycosaminoglycans; 0/Glycosides; 0/Proteochondroitin Sulfates; 0/Proteoglycans; 0/Sulfates; 0/Sulfur Radioisotopes; 0/xylosides; 12125-02-9/Ammonium Chloride; 90-89-1/Diethylcarbamazine

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