Time and sequence dependence of hydroxyurea in combination with gemcitabine in human KB cells

Gemcitabine (Gem) is a deoxycytidine analogue whose active metabolite, dFdCTP, blocks DNA elongation and has a cytotoxic effect. Hydroxyurea (HU) is an S-phase specific inhibitor of ribonucleotide reductase (RR) with a broad spectrum of antitumor effects. We report here that low-dose HU enhanced the activity of Gem in a time- and sequence-dependent manner. Exposure of human oropharyngeal carcinoma KB cells to HU followed by the addition of Gem at various times significantly enhanced cytotoxicity when compared to controls. The greatest enhancement of cytotoxicity occurred when Gem was added 8 hours after HU. By treating KB cells with radiolabeled-Gem following HU treatment, we further confirmed that the incorporation of dFdCTP into DNA increased 6-fold over control reactions under these conditions. The mechanism of the time- and sequence-dependent enhancement is associated with a decrease in hRRM2 RNA, protein, and activity between 4 and 8 hours. The subsequent depletion of dNTP pools allows for increased incorporation of dFdCTP into cells arrested in S-phase, resulting in higher levels of cytotoxicity than either treatment alone.