Selection of a mutant cell line based on differential expression of glycosphingolipid, utilizing anti-lactosylceramide antibody and complement.

Abstract

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.