The effects of cladribine and fludarabine on DNA methylation in K562 cells.
The effects of the antileukemic adenosine analogues, 2-chloro-2'-deoxyadenosine (cladribine) and 9-beta-D-arabinosyl-2-fluoroadenine (fludarabine), on DNA methylation were studied in a cell line K562. It was previously found that both drugs inactivated SAH hydrolase, an enzyme which participates in the "active methyl" cycle. The study examined the effects of these drugs on three aspects of DNA methylation: (i) activity of endogenous C-5 DNA methyltransferase; (ii) capacity of genomic DNA (gDNA) to accept methyl groups, transferred from S-adenosylmethionine by the bacterial methyltransferase, SssI; (iii) estimation of changes of methylated cytosine levels in gDNA, using methylation-dependent restriction analysis. Cladribine and fludarabine inhibited C-5 DNA methyltransferase, with ED(50) values of 3.5 and 47.0 microM, respectively, after 24hr cell growth in the presence of the drugs. After 48 hr growth of cells with cladribine (0.1 microM) or fludarabine (3 microM), the capacity of DNA to accept methyl groups, in the presence of exogenous bacterial SssI methylase, increased by approximately 1.8 and 1.6 times, respectively, compared to control DNA. Digestion of gDNA with endonucleases HpaII and BssHII followed by SssI DNA methylation, indicated that cladribine (0.1 microM) reduced the level of methylated cytosines in both CpG islands and CCGG sequences, sensitive to HpaII restriction enzyme. Inhibition of DNA methylation by fludarabine was observed mainly in CpG dinucleotide located within sequences sensitive to HpaII. The perturbation of DNA methylation was considered as a complex process. Our findings for cladribine and fludarabine should be regarded as an extra element of their antileukemic efficacy.
Wyczechowska
Dorota
D
Department of Medicinal Chemistry, Medical University of Lodz, 6/8 Mazowiecka Street, Lodz 92-215, Poland.
Fabianowska-Majewska
Krystyna
K
D000241:Adenosine / Q000031:analogs & derivatives / Q000494:pharmacology; D000970:Antineoplastic Agents / Q000494:pharmacology*; D002470:Cell Survival / Q000187:drug effects; D017338:Cladribine / Q000494:pharmacology*; D018899:CpG Islands / Q000187:drug effects*; D004248:DNA (Cytosine-5-)-Methyltransferases / Q000378:metabolism; D019175:DNA Methylation / Q000187:drug effects*; D020868:Gene Silencing / Q000187:drug effects; D006801:Humans; D020014:K562 Cells; D014407:Tumor Cells, Cultured; D014740:Vidarabine / Q000031:analogs & derivatives* / Q000494:pharmacology*
D000970:Antineoplastic Agents; D017338:Cladribine; D004248:DNA (Cytosine-5-)-Methyltransferases; D014740:Vidarabine; D000241:Adenosine; C024352:fludarabine