Zhang H, Cooney D A, Zhang M H, Ahluwalia G, Ford H, Johns D G
Laboratory of Medicinal Chemistry, National Cancer Institute, NIH, Bethesda, Maryland 20892.
Cancer Res. 1993 Dec 1;53(23):5714-20.
Cyclopentenyl cytosine (CPEC) exhibits oncological activity in murine and human tumor cells and has now entered Phase I clinical trials. Its mode of action as an antitumor agent appears to be inhibition by its triphosphate (CPEC-TP) of CTP synthase, the enzyme which converts UTP to CTP. In an attempt to elucidate the mechanism of resistance to CPEC, a murine leukemia cell line resistant to CPEC (L1210/CPEC) was developed by N-methyl-N-nitro-N-nitrosoguanidine-induced mutagenesis and subsequent selection by cultivation of the L1210 cells in the presence of 2 microM CPEC. Resistant clones were maintained in CPEC-free medium for 6 generations before biochemical studies were performed. The resistant clone selected for further studies was approximately 13,000-fold less sensitive to growth inhibition by CPEC than the parental cells, and the concentration of CPEC required to deplete CTP in the resistant cells was 50-fold higher than in the sensitive cells. A comparison of the kinetic properties of CTP synthase from sensitive and resistant cells indicated alteration in the properties of the enzyme from the latter; the median inhibitory concentration for CPEC-TP increased from 2 to 14 microM, Km for UTP decreased from 126 to 50 microM, and Vmax increased 12-fold from 0.2 to 2.3 nmol/mg/min. Northern blot analyses of polyadenylated RNA from the resistant and sensitive cells indicated a 3-fold increase in transcripts of the CTP synthase gene in the resistant line. Consistent with these alterations in the properties of the enzyme, the resistant cells exhibited significantly expanded CTP and dCTP pools (4- 5-fold) when compared with the sensitive cells. No change was observed, however, in the properties of uridine-cytidine kinase, the enzyme responsible for the initial phosphorylation of CPEC; despite this, however, cellular uptake of CPEC was greatly decreased, and phosphorylation of CPEC and its incorporation into RNA were 10-fold less than in the parental cells. These latter observations are most readily explained by feedback inhibition by the increased CTP levels of the resistant cells of uridine-cytidine kinase and/or of the membrane transport process used for initial entry of CPEC.
环戊烯基胞嘧啶（CPEC）在小鼠和人类肿瘤细胞中表现出肿瘤活性，目前已进入I期临床试验。它作为一种抗肿瘤药物的作用模式似乎是其三磷酸酯（CPEC-TP）抑制CTP合酶，该酶将UTP转化为CTP。为了阐明对CPEC的耐药机制，通过N-甲基-N-硝基-N-亚硝基胍诱导诱变，随后在2 microM CPEC存在下培养L1210细胞，建立了对CPEC耐药的小鼠白血病细胞系（L1210/CPEC）。在进行生化研究之前，将耐药克隆在无CPEC的培养基中传代6代。选择用于进一步研究的耐药克隆对CPEC生长抑制的敏感性比亲本细胞低约13000倍，耐药细胞中耗尽CTP所需的CPEC浓度比敏感细胞高50倍。对敏感和耐药细胞中CTP合酶的动力学特性进行比较表明，后者的酶特性发生了改变；CPEC-TP的半数抑制浓度从2 microM增加到14 microM，UTP的Km从126 microM降低到50 microM，Vmax从0.2 nmol/mg/min增加了12倍。对耐药和敏感细胞的聚腺苷酸化RNA进行Northern印迹分析表明，耐药细胞系中CTP合酶基因的转录本增加了3倍。与酶特性的这些改变一致，与敏感细胞相比，耐药细胞的CTP和dCTP池显著扩大（4-5倍）。然而，负责CPEC初始磷酸化的尿苷-胞苷激酶的特性没有变化；尽管如此，CPEC的细胞摄取量大大降低，CPEC的磷酸化及其掺入RNA的量比亲本细胞少10倍。耐药细胞中CTP水平升高对尿苷-胞苷激酶和/或用于CPEC初始进入的膜转运过程的反馈抑制最容易解释这些后一种观察结果。
