Cellular metabolism of 5,6-dihydro-5-azacytidine and its incorporation into DNA and RNA of human lymphoid cells CEM/O and CEM/dCk(-).
5,6-Dihydro-5-azacytidine (DHAC) is a hydrolytically stable analog of 5-azacytidine (5-aza-C) that has antileukemic activity against experimental leukemias and, like 5-aza-C, causes DNA hypomethylation. We report the cellular metabolism of DHAC and its incorporation into nucleic acids in the CCRF/CEM/O and deoxycytidine kinase mutant CCRF/CEM/dCk(-) human lymphoid cell lines. The cells were incubated with their respective IC50 concentrations for 24 h, then aliquot samples were removed at predetermined intervals and extracted for nucleotides. The acid-soluble extracts of the cells were assayed on HPLC for nucleotides of DHAC. The major anabolite of [3H]DHAC, [3H]DHACTP, peaked at 110.3 +/- 30.7 microM in CEM/O and at 96.3 +/- 41.9 microM in CEM/dCk(-) cells at 9 and 12 h, respectively. The intracellular concentrations of the deoxyribonucleoside triphosphate, [3H]DHAdCTP, peaked at 13.5 +/- 7.7 microM at 4 h in CEM/O and at 80.8 +/- 13.8 microM at 12 h, a 6-fold greater cellular concentration, in the dCk mutant cell line. The amount of DHAC anabolites incorporated into CEM/O nucleic acids reached a plateau in RNA at 552.6 +/- 7.8 pmol/10(7) cells and in DNA at 64.55 +/- 10.0 pmol/10(7) cells. In CEM/dCk(-) cells, DHAC anabolites reached a plateau in RNA and DNA at 4,256.3 +/- 631.0 and 395.5 +/- 145.4 pmol/10(7) cells, respectively. Thus, with equitoxic treatments of DHAC, the incorporation of its analog anabolites into RNA and DNA was 8- and 6-fold greater in CEM/dCk(-) cells. DNA methylation levels were depressed equally despite a 6-fold greater incorporation of the analog in DNA in the CEM/dCk(-) cells indicating that hypomethylation may be saturated after DHAC treatment. The DNA methylation levels reached a nadir of 0.19% and 0.20% methyl-C (percentage of methylation) in the two cell lines at 6 and 12 h after the beginning of drug treatment and remained relatively constant for the duration of the 24-h treatment. A curve-linear relationship was obtained between the DNA methylation levels in both cell lines and the amounts of DHAC anabolite incorporated into DNA.
Avramis
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Department of Pediatrics, School of Medicine, University of Southern California, Los Angeles 90027.
Powell
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Mecum
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D000964:Antimetabolites, Antineoplastic / Q000493:pharmacokinetics*; D001374:Azacitidine / Q000031:analogs & derivatives* / Q000493:pharmacokinetics; D002460:Cell Line; D004273:DNA, Neoplasm / Q000378:metabolism*; D006801:Humans; D007938:Leukemia / Q000378:metabolism*; D008214:Lymphocytes / Q000378:metabolism*; D008745:Methylation; D009154:Mutation; D012334:RNA, Neoplasm / Q000378:metabolism*; D013997:Time Factors; D014316:Tritium; D014407:Tumor Cells, Cultured
D000964:Antimetabolites, Antineoplastic; D004273:DNA, Neoplasm; D012334:RNA, Neoplasm; C013938:5,6-dihydro-5-azacytidine; D014316:Tritium; D001374:Azacitidine