Generation of Monoclonal Antibodies Specific For ras p21 Glu-12 Oncoproteins: Detection in Carcinogen-Induced Mammary Carcinomas

Abstract

ras genes have been shown to become oncogenes by single point mutations which result in amino acid substitutions that affect either their GTPase activity (positions 12,13,59,61) or their affinity for GTP and GDP. Ras oncogenes and their corresponding proteins have been described in a variety of human cancers as well as in animal tumors induced by physical and chemical carcinogens. One of these animal tumor systems involves the induction of mammary carcinomas in rats by a single dose of N-nitroso-N-methylurea (NMU), a methylating carcinogen. These NMU-induced mammary carcinomas contain transforming H-ras genes activated by G --> A transitions in the second nucleotide of their 12th codon, presumably a consequence of the pre-mutagenic lesions induced by NMU. These G --> A mutations result in the replacement of the normal glycine in the 12th position of the ras p21 protein by a glutamic acid residue. In this study, we report the generation of monoclonal antibodies (Mab) reactive with oncogenic ras p21 proteins containing glutamic acid at position 12 (p21 Glu-12). Mab designated E184 specifically recognized activated ras p21 Glu-12 proteins but not normal p21 (Gly-12) or p21 proteins activated by other position 12 substitutions including arginine, aspartic acid, cysteine, valine or serine residues. Western blot analysis of NMU-induced mammary carcinomas demonstrated that Mab E184 recognized p21 proteins expressed in these rat tumors but not p21 present in normal tissues nor in other carcinogen-induced tumors known to carry H-ras oncogenes activated by mutations at position 61. Mab E184 should be a valuable reagent to determine the presence of activated ras p21 (Glu-12) proteins in animal tumor systems and to correlate their expression with the initiation and/or progression of neoplastic development.