methylnitronitrosoguanidine and Mast-Cell-Sarcoma

Appreciable yields of cutaneous mast cell tumors were induced in a two-stage skin carcinogenesis protocol comprising N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) initiation followed by 12-O-tetradecanoylphorbol-13-acetate (TPA) promotion in 4 of 5 strains of mice. Only female mice of each of the 5 strains were studied. The incidences of benign and/or malignant lesions differed considerably between strains; 27% in DBA/2, 22% in BDF1, 11% in BALB/c, 10% in CDF1 and 0% in C57BL/6 mice and no mast cell tumors were detected in any of the strains when treated with the initiator alone. First found in a DBA/2 mouse at week 50, most tumors were observed after 100 weeks of promotion, and were usually small in size (less than 2 mm in diameter) and predominantly located within the corium, although they occasionally extended into the subcutaneous tissue. Histologically, the benign mast cell tumors were composed of non-encapsulated, well circumscribed densely packed sheets of discrete cuboidal or rhomboid cells. Metachromatic granules were clearly visible in the cytoplasm by Toluidine Blue staining. Two of the tumors induced in DBA/2 mice were diagnosed as malignant mast cell tumors on the twin bases of cellular atypia and deep infiltration into the muscular layer. The cutaneous mast cell tumors were constantly accompanied by subepidermal mast cell aggregations which were also commonly observed in tumor-free skin of mice receiving the initiation-promotion procedure.

Topics: Animals; Cell Aggregation; Cocarcinogenesis; Female; Mast Cells; Mast-Cell Sarcoma; Methylnitronitrosoguanidine; Mice; Mice, Inbred Strains; Skin Neoplasms; Species Specificity; Tetradecanoylphorbol Acetate

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., greater than 80%) after treatment in vitro with chemical mutagens such as ethyl methanesulfonate (EMS) or N-methyl-N'-nitro-N-nitrosoguanidine (MNNG). We herein demonstrate that the same result can be obtained with the poorly mutagenic cytidine analogue, 5-azacytidine, a strong DNA hypomethylating agent. 5-Azacytidine and EMS were equally and comparably effective, or ineffective, in inducing tum- variants from three 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) hosts. However, 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. It is known that 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. We therefore hypothesize that an "epigenetic" mechanism (DNA hypomethylation) leading to activation and expression of genes coding for potential tumor antigens is 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.

Topics: 5-Methylcytosine; Animals; Antigens, Neoplasm; Azacitidine; Cell Transformation, Neoplastic; Cytosine; DNA; Ethyl Methanesulfonate; Mammary Neoplasms, Experimental; Mast-Cell Sarcoma; Methylnitronitrosoguanidine; Mice; Mice, Inbred A; Mice, Inbred DBA; Mice, Nude; Mutagens; Neoplasm Transplantation; Phenotype

A previous report from this laboratory demonstrated that treatment of mouse mastocytoma P815 with the mutagen N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) produces tumor cell variants that are unable to form tumors in syngeneic animals. We examined whether repeated mutagen treatment could increase the frequency of tum- variants above that obtained after a single treatment. This was found to occur with frequencies increasing from a few percent after 1 treatment to more than 90% after 8 treatments. Moreover, uncloned survivor populations obtained after 8 or more MNNG cycles that contained such a high proportion of tum- variants had a markedly decreased tumorigenicity for syngeneic mice. As reported for tum- variants obtained after 1 mutagen treatment, several tum- variants obtained after repeated treatments carried new variant-specific antigens that elicited a specific cytolytic T cell response. Some of these tum- antigens were found to consist of multiple determinants that could be lost independently. We observed that the resistance of the mutagenized populations to MNNG increased gradually with the number of mutagen treatments. In addition, some tum- variants obtained after 8 mutagen treatments showed a reduced sensitivity to mitomycin C.

Topics: Animals; Cell Division; Clone Cells; Epitopes; Genetic Variation; Lymphocyte Culture Test, Mixed; Mast-Cell Sarcoma; Methylnitronitrosoguanidine; Mice; Mice, Inbred DBA; Mitomycin; Mitomycins; Mutagens; Neoplasm Transplantation; T-Lymphocytes, Cytotoxic

Topics: Adenocarcinoma; Animals; Ethyl Methanesulfonate; Mammary Neoplasms, Experimental; Mast-Cell Sarcoma; Methylnitronitrosoguanidine; Mice; Mice, Inbred BALB C; Mice, Inbred DBA; Mutagens; Ouabain; Thioguanine