The biological effects of N-(phosphonacetyl)-l-aspartate (PALA) and 5-fluorouracil (5-FU) were examined singly, and in combination, on the growth of a human mammary carcinoma (MDA) cell line in culture. All combinations of 5-FU (2.5 × 10-7 to 1.5 × 10-5M) and PALA (6.0 × 10-5 to 3.6 × 10-3 M) resulted in synergistic inhibition of cell growth as revealed by a 50 per cent isobologram.To examine the biochemical basis for the synergism, measurements of the incorporation of [3H]-5-FU into total non-poly(A)- and poly(A)-RNA, and of the simultaneous incorporation of [14C]deoxyguanosine and [3H]deoxyuridine into DNA, were determined. The combination of 3.7 × 10-5M PALA and 1 × 10-6 M 5-FU produced 65-85 per cent inhibition of cell growth after continuous treatment for 13 days. Treatment of the cells for 3 or 24 hr with the same drug regimen produced approximately a 170 per cent increase in the incorporation of 1 × 10-6M [3H]-5-FU into poly(A)RNA in comparison to [3H]-5-FU treatment alone; exposure for 24 hr to 3.7 × 10-5 M PALA and 1 × 10-6 M [3H]-5-FU resulted in a 285 per cent increase in the incorporation of [3H]-5-FU into non-poly(A)RNA. The incorporation of either [14C]deoxyguanosine or [3H]deoxyuridine into DNA was not inhibited by this drug regimen; however, the incorporation of [3H]deoxyuridine into DNA was elevated significantly upon 12 or 24 hr of exposure to PALA alone. PALA and 5-FU treatment resulted in a 75 per cent reduction in the concentration of UTP and no change in the concentration of 5-fluorouridine-5′triphosphate 5-FUTP) versus 5-FU treatment alone. Thus, the proportion of 5-FUTP in the total 5FUTP + UTP pool was enhanced more than 3-fold by the combination regimen. These results indicate that the synergistic effect of the combination of PALA and 5-FU on the growth of MDA cells correlates with an increased proportion of 5-FUTP in the pyrimidine nucleotide pool and, consequently, with an enhanced incorporation of 5-FU into RNA, but not with inhibition of DNA synthesis.
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