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

Among other targets, DNA demethylating agents are known to affect the siste r chromatid exchange (SCE) frequency in mammalian cells in vitro. The SCE i ncrease 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 damagi ng agents. Yet, epigenetic changes (such as demethylation) would not be exp ected to affect SCE at all. In the present report we challenge the working hypothesis of a relation between SCEs and demethylation by comparing SCE in duction during different rounds of replication when the parental strands we re normally methylated or demethylated. Azacytidine (AZA), ethionine (ETH), mitomycin-C (MMC), UV-irradiation (UV) and hydrogen peroxide (H2O2) were t ested 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 H2O2 in duce SCE in both protocols, AZA and ETH show an effect on SCEs only if admi nistered two cycles before fixation. Because two cell cycles are needed in order to achieve demethylation of the parental DNA strand, the data reporte d here support our working hypothesis that demethylation in the parental DN A 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 proce ssed damage, eventually yielding an increase in SCEs. (C) 1999 Elsevier Sci ence B.V. All rights reserved.