Selection of a mutant cell line based on differential expression of glycosphingolipid, utilizing anti-lactosylceramide antibody and complement.
A mouse mammary carcinoma mutant cell line showing a characteristic alteration of glycosphingolipid (GSL) composition, but with unchanged protein glycosylation pattern, was isolated. Parent cell line FM3A/F28-7 is characterized by the predominance of lactosylceramide (LacCer) and virtual absence of more complex GSLs. Mutant cell line FUA169 was isolated after treatment of F28-7 cells with the mutagen N-methyl-N'-nitro-N-nitrosoguanidine and incubation with anti-LacCer monoclonal antibody T5A7 and complement. FUA169 cells were characterized by a dramatic reduction in LacCer and by the presence of a major ganglioside identified as GM3. They also showed high activity of CMP-sialic acid:LacCer 2,3-sialosyltransferase, whereas F28-7 cells had no detectable activity of this enzyme. In contrast to the difference in GSL pattern, F28-7 and FUA169 showed identical protein glycosylation patterns, as evidenced by "Western blotting" with various lectins and surface labeling with galactose oxidase/NaB3H4 and periodate/NaB3H4. Thus, FUA169 is identified as a glycosylation mutant with regard to GM3 expression and will be useful for studies of the functional role of GM3. Growth of FUA169 cells, relative to F28-7 cells, showed greater temperature sensitivity and greater inhibitability based on restriction of growth factors, particularly insulin.
Tsuruoka
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Department of Biochemical Oncology/Membrane Research, Fred Hutchinson Cancer Research Center, Seattle, Washington.
Tsuji
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Nojiri
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Holmes
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EH
Hakomori
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D000818:Animals; D000911:Antibodies, Monoclonal; D015703:Antigens, CD; D002455:Cell Division; D002469:Cell Separation; D000939:Epitopes / Q000276:immunology; D005679:G(M3) Ganglioside / Q000276:immunology; D006028:Glycosphingolipids / Q000032:analysis / Q000276:immunology* / Q000378:metabolism*; D006031:Glycosylation; D007790:Lactosylceramides; D008325:Mammary Neoplasms, Experimental; D051379:Mice; D008809:Mice, Inbred C3H; D016296:Mutagenesis; D012276:Ricin / Q000494:pharmacology; D012799:Sialyltransferases / Q000378:metabolism; D014407:Tumor Cells, Cultured
D000911:Antibodies, Monoclonal; D015703:Antigens, CD; D000939:Epitopes; D005679:G(M3) Ganglioside; D006028:Glycosphingolipids; D007790:Lactosylceramides; C009744:CDw17 antigen; D012276:Ricin; D012799:Sialyltransferases; C021343:haematoside synthetase