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/O 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/O 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/O but reduces it by only 75 and 50% in LL/
PALA-C and LL/
PALA-J, respectively. Similarly,
PALA treatment (20 microM) of
tumor lines in culture provokes a dramatic decrease in the incorporation of NaH14CO3 into
pyrimidine intermediates and
nucleotides in the LL/O 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.