Gemcitabine and pemetrexed are effective agents in the treatment of non-small-cell lung cancer (NSCLC), and the present study investigates cellular and genetic aspects of their interaction against A549, Calu-1, and Calu-6 cells. Cells were treated with pemetrexed and gemcitabine, and their interaction was assessed using the combination index. The role of drug metabolism in gemcitabine cytotoxicity was examined with inhibitors of deoxycytidine kinase (dCK), 5Ј-nucleotidase, and cytidine deaminase, whereas the role of pemetrexed targets, thymidylate synthase (TS), dihydrofolate reductase (DHFR), and glycinamide ribonucleotide formyltransferase (GARFT) in drug chemosensitivity was analyzed in cytotoxicity rescue studies. The effect of gemcitabine and pemetrexed on Akt phosphorylation was investigated with enzyme-linked immunosorbent assay, whereas quantitative polymerase chain reaction (PCR) was used to study target gene-expression profiles and its modulation by each drug. Synergistic cytotoxicity was demonstrated, and pemetrexed significantly decreased the amount of phosphorylated Akt, enhanced apoptosis, and increased the expression of dCK in A549 and Calu-6 cells, as well as the expression of the human nucleoside equilibrative transporter 1 (hENT1) in all cell lines. PCR demonstrated a correlation between dCK expression and gemcitabine sensitivity, whereas expression of TS, DHFR, and GARFT was predictive of pemetrexed chemosensitivity. These data demonstrated that 1) gemcitabine and pemetrexed synergistically interact against NSCLC cells through the suppression of Akt phosphorylation and induction of apoptosis; 2) the gene expression profile of critical genes may predict for drug chemosensitivity; and 3) pemetrexed enhances dCK and hENT1 expression, thus suggesting the role of gene-expression modulation for rational development of chemotherapy combinations.Despite recent advances in early diagnosis and treatment, non-small-cell lung cancer (NSCLC) is a disease with a grim prognosis. Extensive clinical studies demonstrated that chemotherapy increases survival in the adjuvant setting (Arriagada et al., 2004) and in patients with advanced disease (Reck and Gatzemeier, 2004). Nonetheless, response rates remain lower than 15%, and median survival is less than 6 months, thus emphasizing the need for new effective drugs and combination regimens (Rosell and Crinò, 2002). However, the rationale for chemotherapy combinations has remained mostly empirical, determined from the antitumor activity of each agent and the lack of overlapping toxicities, despite many attempts to discover preclinical models for rational selection of drug interactions.Gemcitabine (2Ј,2Ј-difluorodeoxycytidine) is a deoxycytidine analog with a broad spectrum of anticancer activity against several solid tumors in preclinical models, and it is M.D.T. was supported by unrestricted research grants from Eli Lilly (Florence, Italy). R. Article, publication date, and citation information can be found at http://molpharm.aspetjournals.org.doi:10.1124/...