Long-term tumor growth suppression in mice immunized with naked DNA of the human tumor antigen mucin (MUC1).

Naked DNA is an attractive tool for vaccination studies. We have studied naked DNA vaccination against the human tumor antigen mucin, encoded by the gene MUC1. C57/BL6 mice were immunized twice, on day 1 and day 10. with plasmid pCI-MUC1, intramuscularly. Five days after the last immunization tumor challenge experiments were performed using the tumor cell line MC38, expressing human MUC1. In 85% of mice immunized with the mucin plasmid tumor growth inhibition was observed, whereas control mice developed tumors. Re-tumor challenge after three months revealed no tumor growth in mice immunized with the mucin plasmid. These encouraging results, showing long-term protection against tumor growth, indicate the potential usefulness of naked DNA vaccination for clinical immunotherapy.

The second messenger binding site of inositol 1,4,5-trisphosphate 3-kinase is centered in the catalytic domain and related to the inositol trisphosphate receptor site.

A segment of inositol 1,4,5-trisphosphate 3-kinase responsible for inositol 1,4,5-trisphosphate (InsP(3)) binding was characterized and confirmed by three different approaches employing the fully active expressed catalytic domain of the enzyme. Part of this moiety was protected from limited tryptic proteolysis by InsP(3). Sequencing of two fragments of 16 and 21 kDa, generated in the absence or presence of InsP(3), respectively, identified segment Glu-271 to Arg-305 as being protected. 15 monoclonal antibodies, all binding to epitopes within this region, inhibited enzyme activity and interfered with inositol phosphate binding. Detailed enzyme kinetic parameters of 32 site-directed mutants revealed residues Arg-276 and Lys-303 in this segment and Arg-322, located nearby, as directly involved in and five other closely neighbored residues, all located within a segment of 73 amino acids, as also influencing InsP(3) binding. Part of this region is similar in sequence to an InsP(3) binding segment in InsP(3) receptors. Combined with the finding that mutants influencing only ATP binding all lie outside this region, these data indicate that an InsP(3) binding core domain is inserted between two segments acting together in ATP binding and phosphate transfer.