Kensler T W, Mutter G, Hankerson J G, Reck L J, Harley C, Han N, Ardalan B, Cysyk R L, Johnson R K, Jayaram H N, Cooney D A
Cancer Res. 1981 Mar;41(3):894-904.
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.
通过用重复的亚治愈剂量的N-（膦酰乙酰基）-L-天冬氨酸（PALA）处理荷瘤小鼠，筛选出对PALA具有抗性的Lewis肺癌变体。在产生的四个变体中测量了靶酶L-天冬氨酸转氨甲酰酶（ATCase）的比活性。其中三个变体的ATCase活性显著升高；然而，第四系LL/PALA-C的ATCase活性与亲本PALA敏感系（LL/O）相同。在随后关于对PALA抗性机制的生化研究中，将一个高ATCase变体LL/PALA-J和LL/PALA-C与LL/O进行了比较。发现所有三个系中磷酸化嘧啶核苷和脱氧核苷的补救途径中的酶活性相似。这些系中的ATCase与其天然底物以及PALA表现出密切可比的动力学。单次治疗剂量的PALA后ATCase恢复的时间进程表明，两个抗性变体都比亲本更快地恢复全部活性。此外，在抗性系中，分级剂量的PALA处理24小时后对ATCase的抑制作用较低。体外将[14C]PALA摄取到源自三种Lewis肺癌的细胞系中显然是通过被动扩散发生的，并且在敏感和抗性细胞中的摄取速率相当。对肿瘤核苷酸含量的分析揭示了LL/O和LL/PALA-C系中嘌呤和嘧啶核苷酸水平的可比光谱，而LL/PALA-J系具有增加的核苷酸库。在所有三个系中。用PALA（400mg/kg）处理24小时后，尿苷和胞苷核苷酸水平大幅降低（70%至80%），而三磷酸腺苷和三磷酸鸟苷水平升高（50%至100%）。通过测量用吡唑呋林（一种乳清苷-5'-单磷酸脱羧酶抑制剂）和0.9%氯化钠溶液或PALA处理的小鼠肿瘤中的乳清酸和乳清苷水平来估计从头嘧啶途径的前体通量，结果表明PALA处理消除了LL/O中乳清酸和乳清苷的积累，但在LL/PALA-C和LL/PALA-J中分别仅将其减少了75%和50%。同样，仅在LL/O细胞系中，用20μM的PALA处理培养的肿瘤系会导致NaH14CO3掺入嘧啶中间体和核苷酸的量急剧减少。对该途径中其他酶的比活性的测定表明，限速步骤氨甲酰磷酸合成酶II的活性在两个抗性系中均升高了2至3倍。由于氨甲酰磷酸合成酶II与ATCase形成复合物，因此有人提出氨甲酰磷酸合成酶II的水平是PALA活性的重要协同决定因素。抗性变体中增加的氨甲酰磷酸池可能用于竞争性地将PALA从ATCase中置换出来，减少酶抑制，并使嘧啶生物合成尽管有治疗仍能继续进行。
