Levels and stability of DNA methylation in random surviving cell clones derived from a Chinese hamster cell line after prolonged treatment with 5-aza-2'-deoxycytidine.

SCC30 cells (derived from a single cell from the Chinese hamster ovary CHO-K1 cell line, selected on the basis of a stable chromosome complement) were used to select cell variants with hypomethylated DNA. Cells were treated with 5-aza-2'-deoxycytidine (5azadCyd) at 0.1, 1, or 5 microM for two weeks with the medium and drug renewed twice weekly. From the few surviving cells, 25 random single cell-derived clones were grown for freezing cell stocks, and for DNA isolation for 5-methyldeoxycytidine (5medCyd) estimations. After a minimum of one month's recovery from the drug, these cells showed a continuum of 5medCyd levels ranging from ones with the same as the parental clone (2.93%) to ones having lost almost 50% of their DNA methylation. The modal value corresponded to a loss of one third to one quarter of methylated sites. Five subclones with hypomethylated DNA were grown from the frozen stocks. These cells were shown not to be 5azaCyd-resistant cell variants. By the time sufficient cells had been grown to determine DNA methylation levels, the average percentage of 5medCyd had increased to 76% of the SCC30 value compared to 67% at the time of freezing cell stocks. However, this level of DNA hypomethylation remained constant over two months of continuous culture. Cells of one of these hypomethylated subclones were subjected to a second cycle of 5azaCyd treatment. Six random clones from the survivors showed a further decrease averaging 11% in the level of DNA methylation but, by two months in continuous culture, 5medCyd levels had returned to that present before the second cycle of selection. Hence, cell variants can be readily obtained which have lost some 8-10 million methylated sites (pairs of methylated deoxycytidines), and this loss does not compromise cell viability in in vitro culture. This is consistent with mammalian genomes containing a high level of background methylation in non-essential sites. The usefulness of such single cell-derived clones with stably hypomethylated genomes is discussed in relation to understanding the functions of deoxycytidine methylation in mammalian DNA.