The cytidine analog, 5-azacytidine (5AC) alters gene expression. We examined the mutagenic activity of a series of cytidine analogs, including 5AC, in L5178Y mouse cells and found that their mutagenic potency correlated with their reported ability to induce differentiation C3H10T1/2 cells. We are analyzing 5AC induced mutants in AS52 cells. 5AC was an effective mutagen from 0.5 to 5.0 mug/ml. 6TGr-AS52 clones were induced for molecular analyses with 3.0 mug/ml 5AC. 6TGr clones were isolated and DNA was prepared for polymerase chain reaction (PCR) and sequence analysis. The frequency of deletions and putative point mutations was determined by PCR analysis. Of 149 6TGr clones analyzed, 134 (90%) were putative point mutants (wild-type PCR product observed) and 15 (10%) were deletion mutants (alteration or loss of the gpt PCR product). Of the 134 putative point mutants, 53 (36% of the total mutants examined) showed base mutations in the structural region of the gene. 5AC induced micronuclei in L5178Y cells at concentrations at which it is mutagenic. The induced micronuclei contained chromosomal fragments as evidenced by our inability to observe CREST staining of kinetochores. 5AC did not induce micronuclei via interference with chromosome morphology during prophase or metaphase and it affected mitosis only after the cells entered anaphase. Chromatid bridges were observed in anaphase that extended into telophase and even into interphase. the compound induced micronuclei if added while the cells were in S phase. 5AC had no effect on the spindle formation and morphology and that the metaphase pate looked normal. Some of the chromosomes in cells treated with 5AC were not able to separate during mitosis, resulting in tetraploidy. This occurred during the first mitosis after hypomethylation where one of the sister chromatids would be fully methylated and the second hemimethylated. It appears that maximal endoreduplication occurred during replication of hemimethylated DNA.
