Selection of strongly immunogenic "tum-" variants from tumors at high frequency using 5-azacytidine.

Abstract

Highly immunogenic tum- (non-tumorigenic in normal syngeneic hosts) clonal variants can be selected from a variety of poorly immunogenic and highly tumorigenic mouse cell lines at very high frequencies (e.g., > 80%) after treatment in vitro with chemical mutagens such as ethyl methanesulfonate (EMS) or N-methyl-N''-nitro-N-nitrosoguanidine (MNNG). The same result can be obtained with the poorly mutagenic cytidine analog, 5-azacytidine, a strong DNA hypomethylating agent. 5-Azacytidine and EMS were equally and comparably effective, or ineffective, in inducing tum- variants from 3 different highly tumorigenic mouse cell lines. Like mutagen-induced tum- variants, those obtained after 5-azacytidine treatment generated usually strong cytolytic T lymphocyte (CTL) responses in vitro, and could grow in immunosuppressed (nude mouse) host. Pretreatment of the tumor cell lines with 5-azacytidine did not cause significant increases in mutations at several independent drug-resistant gene loci, whereas EMS did. Treatment of cells with 5-azacytidine can induce transcriptional activation of silent genes through a reduction of DNA 5-methylcytosine content, a process that can also be effected by mutagenic DNA alkylating agents such as EMS and MNNG. An epigenetic mechanism (DNA hypomethylation) leading to activation and expression of genes coding for potential tumor antigens is probably involved in the generation at high frequency of tum- variants after mutagen treatment. The implications of these findings to mechanisms of tumor progression and the generation of tumor heterogeneity are discussed.