Lack of correlation between DNA-methylating activity and appearance of the immunogenic phenotype in clones of a murine lymphoma treated with mutagens

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Abstract
The relationship between induction of novel immunogenicity by xenogenizing chemicals and DNA-methylating activity in murine tumors was investigated at the clonal level in L1210Ha cells treated with 5-azacytidine, N-methyl-N′-nitro-N-nitrosoguanidine (MNNG) or 1-(p-chlorophenyl)-3,3-dimethyltriazene (DM-Cl). Cells were exposed to the drugs in vitro, cloned by limiting dilution, and assayed for transplantation immunogenicity and 5-methylcytosine content. The results showed that 0% (0/29, 5-azacytidine), 6.8% (2/29, MNNG) and 87.5% (28/32, DM-Cl) of the resulting clones were highly immunogenic, as judged by their tumorigenicity in intact compared to immunodepressed hosts. Frequency distribution analysis of the 5-methylcytosine content of drug-treated and parental clones showed that the methylation pattern was not significantly modified by tumor exposure to either 5-azacytidine or MNNG, and the two immunogenic clones induced by MNNG had methylcytosine levels very close to the 50th percentile value. In contrast, the extent of DNA methylation was increased in the cells treated with DM-Cl, but no obvious association was found between methylation status and immunogenicity of the drug-treated clones. In four 5-azacytidine-treated clones that displayed little or no immunogenicity, additional rounds of drug exposure led to progressive DNA demethylation, but failed, as a rule, to enhance tumor cell immunogenicity. Taken together, the present data indicate that, at least for the examined tumor, immunogenic variants are generated by mutagen treatment at high (MNNG) or very high (DM-Cl) frequencies under conditions in which hypomethylation-induced antigen amplification is unlikely.