Mutated ras p21 as a target for cancer therapy in mouse transitional cell carcinoma

Purpose: To establish an experimental mouse model for bladder cancer immunotherapy using mutated ras as a target. Materials and Methods: A tumorigenic mouse bladder transitional cell carcinoma (TCC) line MB49 (C57BL/6 origin) was analyzed for its c-ras gene status by DNA cloning and sequencing. Aberrant expression of the ras gene was measured with Western blotting. 13-mer peptides corresponding to residues 5 to 17 of the ras protein were synthesized and tested for immunogenicity in syngeneic C57BL/6 mice. Induction of specific immune responses was evaluated by analyzing splenocyte activity in vitro and tumor suppression in vivo. Results: MB49 cells were found to contain a single amino acid substitution of serine for glycine at codon 12 in K-ras loci and an abundant amount of cellular mutated ras p21 protein. C57BL/6 mice immunized with the 13-mer serine-containing ras peptide exhibited mutation-specific immune responses in splenocyte proliferation, cytokine production and cytotoxicity. Specific antitumor immunity in the form of tumor growth delay in vivo was observed in mice immunized with the same mutant peptide followed by subcutaneous MB49 tumor challenge and was enhanced by the addition of low dose interleukin-12. Conclusions: The mouse bladder TCC line MB49 contains a serine mutation at codon 12 of its K-ras gene that is sufficient to induce mutation-specific immune responses in vitro and specific protective immunity to MB49 tumor in vivo. Mutated oncoproteins may be ideal targets for the development of specific immunotherapy-regimens for bladder cancer immunotherapy.