Fernandes D J, Sur P, Kute T E, Capizzi R L
Department of Biochemistry, Wake Forest University, Winston-Salem, North Carolina 27103.
Cancer Res. 1988 Oct 15;48(20):5638-44.
The basis for the proliferation-dependent cytotoxicity of methotrexate has been investigated in mice bearing the L5178Y ascites leukemia. Methotrexate at 60 mg/kg i.p. reduced the viability of logarithmically growing ascites cells (55% active S phase cells) to 28% of control, whereas the viability of the slowly growing cells (18% active S phase) was decreased to only 59% of control. Log phase tumor cells accumulated 8-fold higher levels of methotrexate polyglutamates compared to cells that had approached the stationary phase. However, no differences between log phase and slowly growing tumor cells were observed in the cellular levels of unmetabolized methotrexate. Intestinal mucosa and bone marrow from non-tumor-bearing mice resembled slowly growing tumor cells and had markedly lower levels of methotrexate polyglutamates than logarithmically growing cells. The greater accumulation of methotrexate polyglutamates in the logarithmically growing tumor cells was consistent with an increased synthesis of methotrexate polyglutamates in these cells. The enhanced methotrexate polyglutamylation in log phase versus slowly growing cells was not related to changes in the rates of either cellular methotrexate transport, transmembrane efflux of methotrexate, or hydrolysis of methotrexate polyglutamates. Thymidylate synthase activity measured in situ and in extracts from log phase cells was 4- and 2-fold higher, respectively, than in the more slowly growing cells. Methotrexate produced a 2.4-fold greater depletion of poly-gamma-glutamyl derivatives of 5,10-methylenetetrahydropteroylglutamate in log phase cells compared to slowly growing cells, and this was a function of both the increased methotrexate polyglutamate accumulation and thymidylate synthase activity in the rapidly proliferating cells. These results provide further evidence that the selectivity of methotrexate for tumors with a high growth fraction is a consequence of the rapid rates of both cellular methotrexate polyglutamate synthesis and oxidation of 5,10-methylenetetrahydropteroyl polyglutamates by thymidylate synthase.
在携带L5178Y腹水白血病的小鼠中，对甲氨蝶呤增殖依赖性细胞毒性的基础进行了研究。腹腔注射60mg/kg的甲氨蝶呤可使对数生长期腹水细胞（55%为活跃S期细胞）的活力降至对照组的28%，而生长缓慢的细胞（18%为活跃S期）的活力仅降至对照组的59%。对数期肿瘤细胞积累的甲氨蝶呤多聚谷氨酸水平比接近静止期的细胞高8倍。然而，在未代谢的甲氨蝶呤细胞水平上，未观察到对数期和生长缓慢的肿瘤细胞之间存在差异。来自无肿瘤小鼠的肠黏膜和骨髓类似于生长缓慢的肿瘤细胞，其甲氨蝶呤多聚谷氨酸水平明显低于对数生长期细胞。对数生长期肿瘤细胞中甲氨蝶呤多聚谷氨酸的大量积累与这些细胞中甲氨蝶呤多聚谷氨酸合成增加一致。与生长缓慢的细胞相比，对数期细胞中甲氨蝶呤多聚谷氨酸化增强与细胞甲氨蝶呤转运速率、甲氨蝶呤跨膜外排或甲氨蝶呤多聚谷氨酸水解的变化无关。原位测量及对数期细胞提取物中测得的胸苷酸合成酶活性分别比生长较慢的细胞高4倍和2倍。与生长缓慢的细胞相比，甲氨蝶呤在对数期细胞中使5,10-亚甲基四氢蝶酰谷氨酸的多聚γ-谷氨酰衍生物消耗增加2.4倍，这是快速增殖细胞中甲氨蝶呤多聚谷氨酸积累增加和胸苷酸合成酶活性增加共同作用的结果。这些结果进一步证明，甲氨蝶呤对高生长分数肿瘤的选择性是细胞中甲氨蝶呤多聚谷氨酸合成以及胸苷酸合成酶对5,10-亚甲基四氢蝶呤多聚谷氨酸氧化的快速速率共同作用的结果。
