Combined Cytotoxic Effect of Low-Dose 5-Fluorouracil and Hydroxyurea on 9L Cells in Vitro¹

Continuous exposure of exponentially growing 9L rat brain tumor cells for 24 hr to a nontoxic dose (0.77 µm) of 5-fluorouracil (5-FUra) produced a progressive increase in S-phase cells from 35% (asynchronous culture) to 70% as shown by DNA histograms based on data obtained by flow cytometry. When 9L cells were treated with the S-phase-specific agent hydroxyurea (1.3 mm) immediately after treatment with 5-FUra, a synergistic cell kill resulted. A centrifugal elutriation study confirmed that enhanced cell kill was caused in part by the S-phase synchrony produced by 5-FUra and to the S-phase specificity of hydroxyurea. A higher dose (7.7 µm) of 5-FUra caused a partial G₁-S block; subsequent treatment with hydroxyurea also enhanced cell kill, but the enhancement was not related to S-phase synchrony. A centrifugal elutriation study suggested that, after 24-hr treatment with 5-FUra, hydroxyurea might kill both cells in S phase, which has the greater number of clonogenic cells, and kill cells that are in other phases of the cell cycle, including cells blocked at the G₁-S border that are vulnerable to the phenomenon of “thymidineless death”; concomitant administration of thymidine along with 5-FUra eliminated enhanced cell kill.