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-sta te inhibitor of L-aspartate transcarbamylase, which catalyzes the bios ynthesis of carbamyl-L-aspartate in the de novo pyrimidine biosyntheti c pathway. 5-Bromodeoxyuridine (BrdUrd) is known to be a potent radios ensitizer of proliferating cells when it is incorporated into DNA. The experiments described herein were performed to test the hypothesis th at depletion of cellular pyrimidine precursors by PALA may increase bo th the incorporation of BrdUrd into DNA and the sensitivity of these c ells 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 carci noma cells was determined. BG-1 cells exposed to the most effective PA LA + BrdUrd treatment schedule were then irradiated to determine if PA LA could enhance the radiosensitization already achieved by pretreatme nt with BrdUrd alone. Results: A 72-h exposure to PALA (greater than o r 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. Us ing a clonogenic assay, the IC50 for a 72-h PALA exposure was approxim ately 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 urid ine-5'-triphosphate (UTP) and cytidine-5'-triphosphate (CTP) levels, b ut had no effect on the intracellular adenosine-5'-triphosphate (ATP) level. This decrease in the UTP and CTP pools promoted a fivefold incr ease in the incorporation of [H-3]BrdUrd into the DNA of BG-1 cells. T he most effective treatment schedule involved a 72-h time course, cons isting of a 48-h pretreatment with PALA alone, followed by an addition al 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 t he interaction between the two agents was independent and additive. Th e most effective three-agent treatment schedule consisting of PALA, Br dUrd, and radiation resulted in a greater than 30-fold increase in cyt otoxicity over that expected if the interactions and the three agents were additive (p < 0.05). Conclusions: These data indicate that PALA a lone enhances radiation cytotoxicity and further enhances the radiosen sitization already achieved with the halogenated pyrimidines. These ef fects could be clinically beneficial.