N-Phosphonacetyl-l-aspartic acid (PALA) resistance may be due to the ability of tumor cells to utilize preformed circulating pyrimidine nucleosides, thereby overcoming the block of de novo pyrimidine biosynthesis which PALA causes. To test this hypothesis we examined the effects of PALA and nitrobenzylthioinosine (NBMPR) alone and in combination on B16 melanoma cells in vitro using a clonogenic assay and in vivo using growth delay. In medium containing purine and pyrimidine nucleosides at a final concentration of 28 μM, exposure to PALA (100 μM) alone or to NBMPR (10 μM) alone for periods up to 72 hr did not result in any cytotoxicity. However, exposures to PALA (100 μM) plus NBMPR (10 μM) resulted in a decrease in clonogenic survival to 0.011 at 72 hr. In medium without nucleosides, PALA (100 μM) exposure for 72 hr caused a similar decrease in survival to 0.015. whereas NBMPR (10 μM) had no effect on survival. The addition of uridine resulted in a concentration-dependent reversal of the cytotoxic effects of PALA. C57 Bl female mice bearing B16 melanoma were treated intraperitoneally daily for 4 days with PALA, the phosphate of NBMPR (NBMPR-P), or PALA plus NBMPR-P. PALA, 300 mg/kg daily × 4, resulted in a 6-day tumor growth delay but NBMPR-P, 100 mg/kg daily × 4, had no effect. PALA, 150 mg/kg daily × 4, plus NBMPR, 50 or 100 mg/kg daily × 4, resulted in a 6-day tumor growth delay also. These studies demonstrate that: (1) circulating pyrimidine nucleosides are determinants of the cytotoxic effects of PALA; (2) in vitro PALA and NBMPR combine to cause significant cytotoxicity whereas either agent alone has no effect; (3) in vivo the combination of PALA and NBMPR-P results in the same antitumor affect as PALA alone at twice the dose; and (4) due to an increase in animal toxicity, no therapeutic advantage could be demonstrated for the combination over PALA alone in vivo. We conclude that the cytotoxic effect of PALA is modulated by the levels of the preformed circulating nucleosides and that combining PALA with an inhibitor of salvage pyrimidine uptake would not increase the therapeutic efficacy of PALA because of an increase in toxicity.
ASJC Scopus subject areas