DNA strand methylation and sister chromatid exchanges in mammalian cells in vitro

Among other targets, DNA demethylating agents are known to affect the sister chromatid exchange (SCE) frequency in mammalian cells in vitro. The SCE increase appears to be maintained for many (10-16) cell cycles after the end of the pulse in a given cell population, unlike SCEs induced by DNA damaging agents. Yet, epigenetic changes (such as demethylation) would not be expected to affect SCE at all. In the present report we challenge the working hypothesis of a relation between SCEs and demethylation by comparing SCE induction during different rounds of replication when the parental strands were normally methylated or demethylated. Azacytidine (AZA), ethionine (ETH), mitomycin-C (MMC), UV-irradiation (UV) and hydrogen peroxide (H(2)O(2)) were tested for SCE induction in a Chinese hamster ovary cell line after a single pulse, one or two cell cycles before fixation. Whereas MMC, UV and H(2)O(2) induce SCE in both protocols, AZA and ETH show an effect on SCEs only if administered two cycles before fixation. Because two cell cycles are needed in order to achieve demethylation of the parental DNA strand, the data reported here support our working hypothesis that demethylation in the parental DNA strand, at the level of the replication fork (i.e., the region where SCEs are formed), is responsible for an increase in mistaken ligations of processed damage, eventually yielding an increase in SCEs.