N-(phosphonacetyl)-
L-aspartic acid (
PALA) is a transition- state inhibitor of
L-aspartate transcarbamylase, which catalyses 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 (> or = 25 microM) 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 microM and the cell killing efficiency was dependent on both the concentration and duration of the exposure. A 72-h exposure to 25 microM
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 [3H]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 microM) + BrdUrd (16 microM),
PALA (25 microM) + radiation (6 Gy), and BrdUrd (16 microM) + 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: