Mechanism of Cell Death following Thymidylate Synthase Inhibition: 2′-Deoxyuridine-5′-triphosphate Accumulation, DNA Damage, and Growth Inhibition following Exposure to CB3717 and Dipyridamole

Abstract

The thymidylate synthase inhibitor N¹⁰-propargyl-5,8-dideazafolic acid (CB3717) inhibits the growth of human lung carcinoma A549 cells. The cytotoxicity of CB3717 is potentiated by the nucleoside transport inhibitor dipyridamole (DP), which not only inhibits the uptake and therefore salvage of thymidine but also inhibits the efflux of deoxyuridine, thereby enhancing the intracellular accumulation of deoxyuridine nucleotides. Measurement of intracellular deoxyuridine triphosphate (dUTP) pools, by sensitive radioimmunoassay, demonstrated a large increase in response to CB3717, in a dose- and time-related manner, and this accumulation was enhanced by coincubation with DP. In untreated cells and those treated with DP alone, dUTP was close to or below the limit of detection of the assay. In cells treated for 24 h with 3 µm CB3717 (concentration producing 50% growth inhibition) the intracellular dUTP was 46.1 ± 9.6 (SEM) pmol/10⁶ cells and after 24 h exposure to 30 µm CB3717, 337.5 ± 37.9 pmol dUTP/10⁶ cells was detected. There was significant enhancement by DP of the accumulation of dUTP in cells treated with CB3717; coincubation of cells with 1 µm DP + 3 µm CB3717 for 24 h resulted in intracellular dUTP levels of 174.7 ± 57.7 pmol/10⁶ cells. Accumulation of DNA strand breaks, measured by alkaline elution, also increased in response to CB3717 concentration and exposure period. Newly synthesized (nascent) DNA was more sensitive to damage by CB3717 than was mature DNA. As with the accumulation of dUTP, coincubation with DP also enhanced the accumulation of strand breaks, whereas DP alone had little or no effect on DNA fragmentation.

When data for cells treated with CB3717 alone and CB3717 in combination with DP were combined, there was a significant correlation of intracellular dUTP levels with the level of DNA strand breaks. This strongly suggests that growth inhibition following thymidylate synthase inhibition is mediated through an increase in intracellular dUTP, leading to uracil misincorporation into DNA, its subsequent excision, and resultant strand breakage.