Characterization of monoclonal antibody R256, specific for activated ras p21 with arginine at 12, and analysis of breast carcinoma of v-Harvey-ras transgenic mouse.
The ras family of proto-oncogenes can be activated into transforming genes by single point mutations resulting in p21 products with amino acid substitutions at positions 12, 13 and 61. Here we describe a monoclonal antibody designated R256 which reacts specifically with a synthetic peptide corresponding to amino acids 5 to 16 of ras p21 activated by the substitution of arginine for glycine at position 12. It does not react with peptides representing other activating substitutions at position 12. Western blot studies showed that R256 reacts specifically with recombinant ras p21 containing an arginine-12 substitution but is unreactive with the normal glycine-12 recombinant p21. R256 binds the activated Kirsten p21 in human lung tumor cell line A2182, which contains an activated arginine-12 p21. R256 is also specific for arginine-12 p21 extracted from NIH3T3 cells transformed by the viral Harvey-ras p21, containing arginine at 12, and is not reactive with transformants containing activated p21s with valine, serine, aspartic acid, glutamic acid, cysteine or normal glycine at position 12. When R256 was used to test for expression of arginine-12 p21 in tissues of transgenic mice containing an MMTV/v-Harvey-ras transgene, the monoclonal antibody was able to determine that the arginine-12 p21 transgene product was present in breast tumors but not in normal tissues. The ability of R256 to react specifically with arginine-12 p21 may prove to be valuable in the study of ras oncogenesis in model systems and in determining the presence of arginine-12 p21 in human tumors.
Pullano
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E.I. Du Pont, Medical Products Department, North Billerica, Massachusetts 01862.
Sinn
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Carney
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D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology*; D000918:Antibody Specificity; D001120:Arginine; D015153:Blotting, Western; D004797:Enzyme-Linked Immunosorbent Assay; D000939:Epitopes; D011905:Genes, ras; D006801:Humans; D008325:Mammary Neoplasms, Experimental / Q000503:physiopathology*; D051379:Mice; D008822:Mice, Transgenic; D011518:Proto-Oncogene Proteins / Q000276:immunology*; D016283:Proto-Oncogene Proteins p21(ras); D011994:Recombinant Proteins / Q000276:immunology; D014162:Transfection
D000911:Antibodies, Monoclonal; D000939:Epitopes; D011518:Proto-Oncogene Proteins; D011994:Recombinant Proteins; D001120:Arginine; C501009:HRAS protein, human; D016283:Proto-Oncogene Proteins p21(ras)