Histone deacetylase inhibition modulates deoxyribonucleotide pools and enhances the antitumor effects of the ribonucleotide reductase inhibitor 3'-C-methyladenosine in leukaemia cells.

Histone deacetylase (HDACAC) inhibitors are a new class of epigenetic agents that were reported to enhance the cytotoxic effects of classical anticancer drugs through multiple mechanisms. However, which of the possible drug combi-nations would be the most effective and clinically useful are to be determined. We treated the HL60 and NB4 promyelocytic leukaemia cells with a combination of the ribonucleotide reductase (RRRR) inhibitor 3'-C-methyladenosine (3'-Me-Ado) and several hydroxamic acid-derived HDACAC inhibitors, including two recently synthesized molecules, MC1864 and MC1879, and the reference compound trichostatin A (TSA). The results showed significant growth inhibitory and apoptotic synergistic effects with the combinations. Hence, we evaluated the effects of the combinations on cell cycle distribution and on the level of several proteins involved in the apoptotic process (p21, caspase-3, Bcl-2, Bax, AIFAIFAIF). Since HDACAC inhibitors increased the G1-S transition block induced by 3'-Me-Ado, an effect on RRRR activity was hypothesized. Indeed, the HPLCLC evaluation of intracellular deoxyribonucleotide (dNTNTP) pools showed that both TSA and MC1864 induced a decrease in dNTNTPs, even if with a somewhat different pattern, suggesting that RRRR inhibition contributes to the observed synergism. Furthermore, while TSA was shown to activate the intrinsic apoptotic pathway, MC1864 induced a dose-dependent increase in ROROS and AIFAIFAIF levels. Moreover, the treatment with the radical scavenger N-acetylcysteine determined a significant inhibition of MC1864- but not TSA-mediated synergistic effects. Hence, our findings are consistent with a possible role of HDACAC inhibitor mediated-ROROS induction in RRRR inhibition and in the potentiation of RRRR inhibitor-mediated apoptosis.