Long term instability and molecular mechanism of 5-azacytidine-induced DNA hypomethylation in normal and neoplastic tissues in vivo.
We have previously shown that treatment of normal and neoplastic cells with the antileukemic drug, 5-azacytidine, led to the rapid synthesis of a low molecular weight RNA containing 5-azacytosine. This fraudulent RNA inhibited tRNA (cytosine-5)-methyltransferase early after drug administration. The absence of tRNA (cytosine-5)-methyltransferase activity resulted in the synthesis of tRNA specifically deficient in 5-methylcytosine. Here, we show that treatment of L1210 cells, grown intraperitoneally in mice, with 5-azacytidine led to a rapid and prolonged inactivation of DNA (cytosine-5)-methyltransferase activity and to the synthesis of undermethylated DNA. DNA isolated from the treated tissue was found to inactivate the DNA methylase (decreased Vmax) in in vitro DNA (cytosine-5)-methyltransferase assays. Kinetic analysis showed noncompetitive inhibition of the substrate by the inhibitor. The persistence of DNA undermethylation after treatment with 5-azadeoxycytidine or 5-azacytidine in animals has not been measured directly; therefore, we have investigated this phenomenon in the intact animal. Prolonged treatment with 5-azacytidine was required to maintain a a fraction of undermethylated sites in DNA of L1210 cells in vivo for up to 4 months or longer after drug withdrawal. Such treatment led to instability of DNA methylation levels in L1210 cells in vivo. At least a partial restoration of DNA 5-methylcytosine levels was observed after acute and chronic 5-azacytidine treatment, respectively. 5-Azacytidine was also found to induce DNA hypomethylation in regenerating, but not in normal adult mouse liver cells. Our results show that: 1) it was extremely difficult to decrease the DNA methylation level to less than 50% of control; and 2) it was also difficult to maintain stable DNA methylation levels in vivo after exposure to the drug.
D000818:Animals; D001374:Azacitidine / Q000494:pharmacology*; D004247:DNA / Q000378:metabolism*; D004248:DNA (Cytosine-5-)-Methyltransferases / Q000378:metabolism*; D004273:DNA, Neoplasm / Q000378:metabolism*; D007700:Kinetics; D007939:Leukemia L1210 / Q000378:metabolism*; D008099:Liver / Q000187:drug effects / Q000378:metabolism*; D008115:Liver Regeneration; D008297:Male; D008745:Methylation; D008780:Methyltransferases / Q000378:metabolism*; D051379:Mice; D008807:Mice, Inbred BALB C; D008811:Mice, Inbred DBA
D004273:DNA, Neoplasm; D004247:DNA; D008780:Methyltransferases; D004248:DNA (Cytosine-5-)-Methyltransferases; D001374:Azacitidine