Pizzorno G, Moroson B A, Cashmore A R, Russello O, Mayer J R, Galivan J, Bunni M A, Priest D G, Beardsley G P
Department of Pediatrics, Yale University School of Medicine, New Haven, Connecticut 06510.
Cancer Res. 1995 Feb 1;55(3):566-73.
5,10-dideaza-5,6,7,8-terrahydrofolic acid (DDATHF) is a potent antiproliferative agent in cell culture systems and in vivo in a number of murine and human xenograft tumors. In contrast to classical antifolates, which are dihydrofolate reductase inhibitors, DDATHF primarily inhibits GAR transformylase, the first folate-dependent enzyme along the pathway of de novo purine biosynthesis. The (6R) diastereomer of DDATHF (Lometrexol), currently undergoing clinical investigation, was used to develop CCRF-CEM human leukemia sublines resistant to increasing concentrations of the drug. Three cell lines were selected for ability to grow in medium containing 0.1 microM, 1.0 microM, and 10 microM of (6R)DDATHF, respectively. Impaired polyglutamylation was identified as a common mechanism of resistance in all three cell lines. A progressive decrease in the level of polyglutamylation was associated with diminished folylpolyglutamate synthetase activity and paralleled increasing levels of resistance to the drug. However, the expression of folylpolyglutamate synthetase RNA was not altered in the resistant cell lines compared to the parent cells. The most resistant cell subline also displayed an increased activity of gamma-glutamyl hydrolase. The sublines were scrutinized for other possible mechanisms of resistance. No alterations in drug transport or in purine economy were found. Modest increases were found in the activity of methylene tetrahydrofolate dehydrogenase but no alterations of other folate-dependent enzymes were observed. Increases in accumulation and conversion of folic acid to reduced forms, particularly 10-formyltetrahydrofolate, was also seen. The resistant cell lines were sensitive to dihydrofolate reductase inhibitors, methotrexate and trimetrexate, for a 72-h exposure period but showed cross-resistance to methotrexate for 4 and 24 h exposures. Cross-resistance was also shown toward other deazafolate analogues for both short- and long-term exposures.
5,10-二去氮-5,6,7,8-四氢叶酸（DDATHF）在细胞培养系统以及多种小鼠和人异种移植肿瘤的体内实验中是一种有效的抗增殖剂。与作为二氢叶酸还原酶抑制剂的经典抗叶酸剂不同，DDATHF主要抑制甘氨酰胺核糖核苷酸转甲酰酶，这是从头嘌呤生物合成途径中第一种依赖叶酸的酶。DDATHF的（6R）非对映异构体（洛美曲索）目前正在进行临床研究，用于培育对该药物浓度不断增加具有抗性的CCRF-CEM人白血病亚系。分别选择了三种细胞系，它们能够在含有0.1微摩尔、1.0微摩尔和10微摩尔（6R）DDATHF的培养基中生长。多聚谷氨酸化受损被确定为所有这三种细胞系抗性的共同机制。多聚谷氨酸化水平的逐渐降低与叶酰聚谷氨酸合成酶活性的降低相关，并且与对该药物抗性水平的增加平行。然而，与亲本细胞相比，抗性细胞系中叶酰聚谷氨酸合成酶RNA的表达没有改变。抗性最强的细胞亚系还表现出γ-谷氨酰水解酶活性增加。对这些亚系进行了其他可能抗性机制的检查。未发现药物转运或嘌呤代谢方面的改变。发现亚甲基四氢叶酸脱氢酶活性有适度增加，但未观察到其他依赖叶酸的酶有改变。还观察到叶酸积累增加以及向还原形式，特别是10-甲酰四氢叶酸的转化增加。这些抗性细胞系在72小时的暴露期内对二氢叶酸还原酶抑制剂甲氨蝶呤和三甲曲沙敏感，但在4小时和24小时暴露时对甲氨蝶呤表现出交叉抗性。对于短期和长期暴露，对其他去氮叶酸类似物也表现出交叉抗性。
