High-frequency generation of altered Mr 70,000 env glycoproteins in N-methyl-N'-nitro-N-nitrosoguanidine-treated murine tumor cells

Abstract

In previous studies we have characterized variant clones established following treatment of mouse Eb lymphoma cells with N-methyl-N'-nitro-N-nitrosoguanidine (MNNG). Some of these clones were impaired in tumorigenicity due to an increased immunogenicity (tum- phenotype). In this paper we investigated the mutagenic effect of MNNG on cell surface molecules. The results show that retroviral Mr 70,000 glycoprotein (gp70) antigens undergo extensive alterations following MNNG treatment. In five of five mutant clones analyzed, the two-dimensional gel electrophoretic patterns of gp70 antigens were altered in comparison to the parental Eb cells. Peptide mapping analysis of immunoprecipitated gp70 molecules using three different enzymes revealed detectable changes in amino acid sequence in three of five mutant clones. In contrast, no alterations were detected in H-2Kd and H-2Dd antigens of the same clones. The gp70 antigens expressed by mutant clones could be resolved in three distinct clusters. Only one cluster induced antibodies in the syngeneic host. When genomic DNAs of MNNG clones were investigated by Southern blot analysis using a gp70-specific probe, an additional 4.5-kilobase hybridizing band could be detected that was not present in parental Eb cells and 5'-azacytidine-treated Eb clones. Collectively, our results show that gp70 antigens and genes are affected by MNNG treatment with high frequency. The possible role of structurally altered gp70 molecules in the immunogenicity of mutagenized tumor cells is discussed.