PALA enhancement of bromodeoxyuridine incorporation into DNA increases radiation cytotoxicity to human ovarian adenocarcinoma cells

Purpose: N-(phosphonacetyl)-L-aspartic acid (PALA) is a transition-state inhibitor of L-aspartate transcarbamylase, which catalyzes the biosynthesis of carbamyl-L-aspartate in the de novo pyrimidine biosynthetic pathway. 5-Bromodeoxyuridine (BrdUrd) is known to be a potent radiosensitizer of proliferating cells when it is incorporated into DNA. The experiments described herein were performed to test the hypothesis that depletion of cellular pyrimidine precursors by PALA may increase both the incorporation of BrdUrd into DNA and the sensitivity of these cells to the cytotoxic effect of radiation. Methods and Materials: The effect of PALA concentration and exposure time on the incorporation of BrdUrd into the DNA of exponentially growing BG-1 human ovarian carcinoma cells was determined. BG-1 cells exposed to the most effective PALA + BrdUrd treatment schedule were then irradiated to determine if PALA could enhance the radiosensitization already achieved by pretreatment with BrdUrd alone. Results: A 72-h exposure to PALA (greater than or equal to 25 mu M) delayed the growth of human ovarian adenocarcinoma BG-1 cells by 40% compared to that of the untreated control cells. Using a clonogenic assay, the IC50 for a 72-h PALA exposure was approximately 25 mu M, and the cell killing efficacy was dependant on both the concentration and duration of the exposure. A 72-h exposure to 25 mu M PALA produced approximately a 90% decrease in the intracellular uridine-5'-triphosphate (UTP) and cytidine-5'-triphosphate (CTP) levels, but had no effect on the intracellular adenosine-5'-triphosphate (ATP) level. This decrease in the UTP and CTP pools promoted a fivefold increase in the incorporation of [H-3]BrdUrd into the DNA of BG-1 cells. The most effective treatment schedule involved a 72-h time course, consisting of a 48-h pretreatment with PALA alone, followed by an additional 24-h treatment with both PALA and BrdUrd. The two agent treatments, PALA (25 mu M) + BrdUrd (16 mu M), PALA (25 mu M) + radiation (6 Gy), and BrdUrd (16 mu M); radiation (6 Gy) produced a 2.1-, 7.4-, and 13.2-fold increase in cytotoxicity, respectively, over that expected if the interaction between the two agents was independent and additive. The most effective three-agent treatment schedule consisting of PALA, BrdUrd, and radiation resulted in a greater than 30-fold increase in cytotoxicity over that expected if the interactions and the three agents were additive (p < 0.05). Conclusions: These data indicate that PALA alone enhances radiation cytotoxicity and further enhances the radiosensitization already achieved with the halogenated pyrimidines. These effects could be clinically beneficial.