Inhibition of post-translational modification and surface expression of a melanoma-associated chondroitin sulfate proteoglycan by diethylcarbamazine or ammonium chloride.
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)
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
D011508:Chondroitin Sulfate Proteoglycans; D004187:Disaccharides; D006025:Glycosaminoglycans; D006027:Glycosides; D011509:Proteoglycans; D013431:Sulfates; D013462:Sulfur Radioisotopes; C010807:xylosides; D000643:Ammonium Chloride; D004049:Diethylcarbamazine