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Mechanism of Resistance of Variants of the Lewis Lung Carcinoma to N-(Phosphonacetyl)-L-aspartic Acid¹

Laboratories of Toxicology [T. W. K., G. M., J. G. H., L. J. R., C. H., N. H., B. A., H. N. J., D. A. C.] and Chemical Pharmacology [R. L. C.], Developmental Therapeutics Program, Division of Cancer Treatment, National Cancer Institute, Bethesda, Maryland 20205, and Arthur D. Little, Inc., Cambridge, Massachusetts 012140 [R. K. J.]

Variants of the Lewis lung carcinoma were selected for resistance to N-(phosphonacetyl)-L-aspartic acid (PALA) by treatment of tumor-bearing mice with repetitive subcurative doses of PALA. The specific activity of the target enzyme, L-aspartic acid transcarbamylase (ATCase), was measured in the four variants developed. Three had markedly elevated ATCase activities; however, the fourth line, LL/PALA-C, had an ATCase activity identical to that of the parent, PALA-sensitive line (LL/O). One high-ATCase variant, LL/PALA-J, and LL/PALA-C were compared with LL/0 in subsequent biochemical studies on the mechanism of resistance to PALA. Enzyme activities in the salvage pathways which phosphorylate pyrimidine nucleosides and deoxynucleosides were found to be similar in all three lines. ATCase in these lines exhibits closely comparable kinetics with its natural substrates as well as with PALA. The time courses of restitution of ATCase after a single therapeutic dose of PALA show that both resistant variants recover full activity more rapidly than the parent. Additionally, inhibition of ATCase 24 hr following graded doses of PALA is lower in the resistant lines. The uptake of [¹⁴C]PALA in vitro into cell lines derived from the three Lewis lung carcinomas apparently occurs by passive diffusion and at comparable rates in both sensitive and resistant cells. Analysis of the nucleotide content of tumors reveals comparable spectrums of purine and pyrimidine nucleotide levels in the LL/0 and LL/PALA-C lines, whereas the LL/PALA-J line has augmented nucleotide pools. In all three lines, 24 hr after treatment with PALA (400 mg/kg), uridine and cytidine nucleotide levels were substantially diminished (70 to 80%) while adenosine 5'-triphosphate and guanosine 5'-triphosphate levels were elevated (50 to 100%). Estimations of precursor flux through the de novo pyrimidine pathway by measuring orotate and orotidine levels in tumors of mice treated with pyrazofurin (an inhibitor of orotidine-5-'-monophosphate decarboxylase) and either 0.9% NaCl solution or PALA shows that PALA treatment eliminates orotate and orotidine accumulation in LL/0 but reduces it by only 75 and 50% in LL/PALA-C and LL/PALA-J, respectively. Similarly, PALA treatment (20 µM) of tumor lines in culture provokes a dramatic decrease in the incorporation of NaH¹⁴CO₃ into pyrimidine intermediates and nucleotides in the LL/0 cell line only. Determinations of specific activities of the other enzymes in this pathway reveal that the activity of carbamyl phosphate synthetase II, the rate-limiting step, is elevated 2- to 3-fold in both resistant lines. Since carbamyl phosphate synthetase II exists as a complex with ATCase, the suggestion is made that levels of carbamyl phosphate synthetase II are collaterally important determinants of PALA activity. An augmented pool of carbamyl phosphate in the resistant variants may serve to competitively displace PALA from ATCase, diminish enzyme inhibition, and allow pyrimidine biosynthesis to proceed despite therapy.

¹ A preliminary account of this work was presented at the 71st Annual Meeting of the American Association for Cancer Research in San Diego, Calif. (18).

² To whom requests for reprints should be addressed, at Laboratory of Medicinal Chemistry and Biology, Bldg. 37/6D-18, National Cancer Institute, Bethesda, Md. 20205.

Received 8/ 5/80. Accepted 11/21/80.