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Gemcitabine and ara-C have multiple mechanisms of action: DNA incorporation and for gemcitabine also ribonucleotide reductase (RNR) inhibition. Since dCTP competes with their incorporation into DNA, dCTP depletion can potentiate their cytotoxicity. We investigated whether additional RNR inhibition by Triapine (3-AP), a new potent RNR inhibitor, enhanced cytotoxicity of gemcitabine and ara-C in four non-small-cell-lung-cancer (NSCLC) cell lines, using the multiple-drug-effect analysis.Simultaneous and sequential exposure (preexposure to 3-AP for 24 h) in a constant molar ratio of 3-AP and gemcitabine was antagonistic/additive in all cell lines. Preexposure to 3-AP at an IC25 concentration for 24 h before variable concentrations of gemcitabine was synergistic. RNR inhibition by 3-AP resulted in a more synergistic interaction in combination with ara-C, which does not inhibit RNR.Two cell lines with pronounced synergism (SW1573) or antagonism (H460) for gemcitabine/3-AP, were evaluated for accumulation of the active metabolites, dFdCTP and ara-CTP. Simultaneous exposure induced no or a small increase, but ara-CTP increased after pretreatment with 3-AP, 4-fold in SW1573 cells, but not in H460 (<1.5 fold). Ara-C and gemcitabine incorporation into DNA were more pronounced (about 2-fold increased) for sequential treatment in SW1573 compared to H460 cells (<1.5 fold). This was not related to the activity and expression of deoxycytidine kinase and the M2 subunit of RNR.In conclusion, RNR inhibition by 3-AP prior to gemcitabine or ara-C exposure stimulates accumulation of the active metabolites and incorporation into DNA. The combination 3-AP/Ara-C is more synergistic than 3-AP/gemcitabine possibly because gemcitabine already inhibits RNR, but ara-C does not.