title: Inhibition of post-translational modification and surface expression of a melanoma-associated chondroitin sulfate proteoglycan by diethylcarbamazine or ammonium chloride.
abstract: 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)
mesh_terms: D000643:Ammonium Chloride / Q000494:pharmacology*; D002460:Cell Line; D011508:Chondroitin Sulfate Proteoglycans / Q000378:metabolism*; D004049:Diethylcarbamazine / Q000494:pharmacology*; D004187:Disaccharides / Q000032:analysis; D005089:Exocytosis / Q000187:drug effects; D006025:Glycosaminoglycans / Q000378:metabolism; D006027:Glycosides / Q000494:pharmacology; D006801:Humans; D007163:Immunosorbent Techniques; D008545:Melanoma / Q000378:metabolism* / Q000648:ultrastructure; D008854:Microscopy, Electron; D008970:Molecular Weight; D011499:Protein Processing, Post-Translational / Q000187:drug effects*; D011509:Proteoglycans / Q000378:metabolism*; D013431:Sulfates / Q000378:metabolism; D013462:Sulfur Radioisotopes
chemical_list: D011508:Chondroitin Sulfate Proteoglycans; D004187:Disaccharides; D006025:Glycosaminoglycans; D006027:Glycosides; D011509:Proteoglycans; D013431:Sulfates; D013462:Sulfur Radioisotopes; C010807:xylosides; D000643:Ammonium Chloride; D004049:Diethylcarbamazine