Hydroxyurea with Azacitidine or Decitabine in Combination Is Antagonistic on DNA Methylation Inhibition.

5-azacytidine (azacitidine, Vidaza) and 5-aza-2′deoxycytidine (decitabine, Dacogen) are cytidine analogs currently used to treat myelodysplastic syndrome and as an investigational agent to treat acute myelogenous leukemia, sickle cell disease and other cancers. Decitabine is phosphorylated to a triphosphate form and incorporated into DNA where it can covalently trap DNA methyltransferase. Azacitidine is also phosphorylated to a triphosphate form, but requires reduction from a ribonucleotide to a deoxyribonucleotide by ribonucleotide reductase (RR) before incorporation into DNA. Hydroxyurea (Hydrea) is a drug routinely used to treat patients with leukemia and sickle cell disease. Hydroxyurea inhibits cell proliferation by inhibiting RR. Inhibition of RR leads to a depletion of nucleotide pools and cell cycle arrest in S-phase. Therefore, we hypothesized that the combination of hydroxyurea with azacitidine would be antagonistic to the ability of azacitidine to inhibit DNA methylation, and synergistic with decitabine in inhibiting DNA methylation. Hydroxyurea inhibits RR, which could block the conversion of azacitidine from a ribonucleotide to a deoxyribonucleotide, and therefore prevent its incorporation into DNA. In contrast, decitabine could increase its incorporation into DNA by taking advantage of a depletion of other deoxynucleotides induced RR inhibition by hydroxyurea. In order to test this hypothesis, we treated HL-60 (leukemia) and T24 (bladder cancer) cell lines with hydroxyurea in combination with either decitabine or azacitidine. DNA methylation was quantitated using Bisulfite-PCR Pyrosequencing of LINE-1 DNA repetitive elements, the MAGE-A1 gene and the p16 gene promoter region. We clearly showed that hydroxyurea at concentrations above 0.5 mM completely blocked the ability of azacitidine to inhibit DNA methylation (IC50=0.36 ± 0.04 mM). Surprisingly, we found that hydroxyurea at concentrations above 0.5mM also completely inhibited the effect of decitabine on DNA methylation (IC50=0.24 ± 0.06 mM). Further investigation showed that inhibition of the ability to block DNA methylation correlated with inhibition of the cell cycle. Using flow cytometry we clearly show that the antagonistic effect of hydroxyurea on DNA methylation inhibitors correlates with the arresting of cell division in S-phase. Aphidicolin, which inhibits DNA polymerase, also blocked the cell cycle and the ability of azacitidine and decitabine to inhibit DNA methylation. Thus, azacitidine and decitabine are S-phase specific drugs and are antagonized when used in combination with drugs that arrest the cell cycle. However, we also show that sequential treatment of hydroxyurea followed by azacitidine or decitabine could be used to avoid this antagonism. Therefore, due to the antagonistic effect of hydroxyurea with the mechanistic inhibitors of DNA methylation, azacitidine and decitabine, we recommend sequential combination treatment strategy.