DNA methylation in 5-aza-2'-deoxycytidine-resistant variants of C3H 10T1/2 C18 cells.

Abstract

A cell line (T17) was derived from C3H 10T1/2 C18 cells after 17 treatments with increasing concentrations of 5-aza-2'-deoxycytidine. The T17 cell line was very resistant to the cytotoxic effects of 5-aza-2'-deoxycytidine, and the 50% lethal dose for 5-aza-2'-deoxycytidine was ca. 3 ,uM, which was 30-fold greater than that of the parental C3H 10T1/2 C18 cells. Increased drug resistance was not due to a failure of the T17 cell line to incorporate 5-aza-2'-deoxycytidine into DNA. The cells were also slightly cross-resistant to 5-azacytidine. The percentage of cytosines modified to 5-methylcytosine in T17 cells was 0.7%, a 78% decrease from the level of 3.22% in C3H 1OT1/2 C18 cells. The DNA cytosine methylation levels in several clones isolated from the treated lines were on the order of 0.7%, and clones with methylation levels lower than 0.45% were not obtained even after further drug treatments. These highly decreased methylation levels appeared to be unstable, and DNA modification increased as the cells divided in the absence of further drug treatment. The results suggest that it may not be possible to derive mouse cells with vanishingly low levels of 5-methylcytosine and that considerable de novo methylation can occur in cultured lines.

DNA hypomethylation can activate Xist expression and silence X-linked genes.

“…Reactivation of HPRT correlated with demethylation of the CpG island associated with the HPRT gene (Yen et al 1984;Wolf et al 1984b). It is important to point out here that treatment of cells with 5-aza-dC achieves a moderate level of genomic DNA hypomethylation (Flatau et al 1984;Taylor et al 1984;Michalowsky and Jones 1989) that may be insufficient for the activation of Xist. This can be inferred from our previous observation that the Xist gene, under conditions of limited Dnmt, is more resistant to demethylation than other genes (Beard et al 1995).…”

Polymerase chain reaction-aided genomic sequencing of an X chromosome-linked CpG island: methylation patterns suggest clonal inheritance, CpG site autonomy, and an explanation of activity state stability.

“…DNA methyltransferase is processive (32) and is inactivated when it encounters a site having recently incorporated 5-azadeoxycytidine in place of cytosine (33,34). This may lead to clusters of hemimethylated sites, many of which, however, will be repaired during the 2-day recovery period, in which much remethylation is known to occur (35,36). This "recovery" methylation could be, in large part, random.…”

Comments on “Epigenetic inheritance in evolution”

“…The process can be seen in vivo under certain circumstances. Treatment of cell over several weeks with an inhibitor of DNA methylation reduced genomic methylation drastically, but following removal of the drug, methylation recovers over the succeeding weeks to its original level (Flatau et al, 1984). Slavish copying of the existing methylation pattern would not predict this result.…”