Effects of lipophilic thymidylate synthase (TS) inhibitor AG337 on human head and neck squamous cell carcinoma (HNSCC) cell lines and CCRF-CEM human leukemia cells and sublines with acquired methotrexate (MTX) resistance were assayed using continuous or intermittent drug exposure. During 120-h continuous exposure, HNSCC cell lines A253 and FaDu are equally MTX sensitive (EC50 equals approximately 15nM); AG337 is less potent (EC50 approximately equals 1 microM). A253 is intrinsically resistant to 24-h intermittent MTX exposure (EC50 equals approximately 17 microM; FaDu, EC50 equals approximately 0.3 microM); both HNSCC cell lines are resistant to 24-h AG337 exposure (EC50 >100 microM). CCRF-CEM shows MTX (EC50 =14 nM) and AG337 (EC50 equals approximately 0.6 microM) sensitivity in continuous exposure similar to HNSCC; however, AG337 retains potency against CCRF-CEM cells in intermittent exposure (24-h, EC50 equals approximately 2 microM; 6-h, EC50 equals approximately 48 microM). The reduced folate leucovorin (LV) at > or = 0.1 microM fully protects from growth inhibition by continuous MTX exposure, but growth inhibition by AG337 is reversed only slightly by < or = 100 microM LV. Thymidine fully protects A253 and FaDu against growth inhibition by AG337, while hypoxanthine alone is without effect, suggesting inhibition is TS-specific. CCRF-CEM sublines with acquired MTX-resistance resulting from DHFR overexpression, defective MTX transport, or defective MTX polyglutamylation retain full sensitivity to AG337 in continuous exposure (all EC50 =0.4 microM). These data indicate that AG337 may be useful in therapy of tumors that have acquired resistance to MTX by most common mechanisms.

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