Stable cloned sublines of the Lewis lung carcinoma have been isolated that are either highly metastatic or non-metastatic when injected into syngeneic mice. An antigen (called B1) that is present specifically on the metastatic variants has been identified and a monoclonal antibody was raised against it. Studies using the antibody demonstrated a direct correspondence between presence of the epitope and metastatic potential of clones. Alteration of the metastatic potential of subclones by treatment of the cells with 5-azacytidine was accompanied by a corresponding change in the detection of the B1 antigen. In order to study the expression and function of this cell surface protein in normal and tumor cells, the gene encoding the antigen will be isolated. Protein structural information will be obtained by microsequence analyses of immunoaffinity purified antigen. Protein sequence information will be used to design synthetic oligonucleotide probes for screening cDNA libraries constructed from the metastatic cell lines. Alternate cloning approaches, including direct screening of expression libraries using the monoclonal antibody, differential screening, or mRNA isolation by polysome immunoabsorption, will be employed as necessary. Full length cDNA clones and genomic clones will be isolated for studies on the structure and organization of the gene. Using these clones as probes, investigation of the molecular differences between the metastatic and non-metastatic cell lines are proposed. Recombinant DNA techniques will be used to alter the expression of the B1 antigen in cultured cells; the effect of these alterations on the metastatic activity of the derived cell lines will be tested by injecting the cells into mice.
