[Identification of a specific protein in flat revertant cell lines derived from ras oncogene-transformed cells].
Total proteins from a mouse embryo fibroblast cell line NIH/3T3, NIH/3T3 cells transformed by human activated c-Ha-ras (EJ-ras) oncogene (EJ-NIH/3T3), and the two flat revertant cell lines, R1 and R2 were analyzed by two-dimensional gel electrophoresis (IEF and NEPHGE). Several hundred polypeptides were resolved as seen by silver staining. Common alterations in four polypeptide spots were observed in the revertants when compared with NIH/3T3 and EJ-NIH/3T3 cells. In these alterations, a new polypeptide spot p92-5.7 (designated by molecular weight X 10(-3) and pI) was detected only in the revertants, but not in NIH/3T3 and EJ-NIH/3T3 cells. Furthermore, the expression level of p92-5.7 seemed to be associated with the flat morphology and the reduced tumorigenicity of the revertants. The polypeptide p92-5.7 was also not detected in the total proteins extracted from BALB/3T3 cells, NIH Swiss mouse primary embryo fibroblasts. Subcellular fractionation of total protein from R1 cells revealed that the p92-5.7 was present in the cytosol. The p92-5.7 was not phosphorylated in the steady state of R1 cells. Western blot analysis using an anti-gelsolin antibody demonstrated that the p92-5.7 might be a variant form of gelsolin which is thought to be an actin regulatory protein or a gelsolin-like polypeptide. The expressions of gelsolin mRNA in the revertants were higher than in the EJ-NIH/3T3 cells. These results may suggest that the expression of p92-5.7 detected only in the revertants is associated, at least in part, with the reversion. This may be the first demonstration of the specific protein expression in the flat revertants.
Laboratory of Molecular Genetics, Hokkaido University, School of Medicine, Sapporo, Japan.
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Fujita
D000469:Alkaline Phosphatase / Q000302:isolation & purification; D000818:Animals; D002461:Cell Line, Transformed; D015342:DNA Probes; D015180:Electrophoresis, Gel, Two-Dimensional; D005347:Fibroblasts / Q000032:analysis*; D011905:Genes, ras; D051379:Mice; D010455:Peptides / Q000235:genetics / Q000302:isolation & purification*; D013347:Subcellular Fractions / Q000032:analysis
D015342:DNA Probes; D010455:Peptides; D000469:Alkaline Phosphatase