METHOTREXATE UPTAKE AND METABOLISM IN HEPATIC CELLS

这些研究的基础是了解谷氨酰化的作用 在抗叶酸剂的细胞毒活性中起作用， 细胞的叶酸聚谷氨酸盐。 我们将主要利用啮齿动物肝癌 细胞系培养。 我们将评估酶的活性， 在生长的细胞中催化谷氨酰化，叶酰聚谷氨酸合成酶 在许多已知改变谷氨酸盐添加速率的条件下 在完整的细胞中。 甲氨蝶呤聚谷氨酸盐和 以及这些池中的每一个对 其他将被检查。 一系列新的叶酸和叶酸拮抗剂， 将评估谷氨酸部分4位的氢。 的 氟取代防止甲氨蝶呤的谷氨酰化， 可用于评估谷氨酰化在活性中的作用 药物。 使用相同的原理，减少叶酸辅酶 （二氢叶酸，亚叶酸，5-甲基四氢叶酸）将被合成 作为4氟衍生物。 通过这些探针，我们将检查 氟类似物的转运和代谢， 叶酸的谷氨酰化和生长依赖性， 谷氨酰化和非谷氨酰化甲氨蝶呤的毒性预防 嘌呤和嘧啶，以及谷氨酰化在拯救方案中的作用 使用亚叶酸。 其他有待调查的领域是交通基础设施的特点 甲氨蝶呤耐药性和更详细的分析， 甲氨蝶呤的活性。 这些研究旨在产生更大的 了解抗叶酸剂，以便它们可以更有选择性地 在瘤形成的治疗中用作抗增殖剂。

The basis for these studies is understanding the role that glutamylation plays in the cytotoxic activity of antifolates and its relationship to cellular folylpolyglutamates. We will utilize primarily a rodent hepatoma cell line in culture. We will evaluate the activity of the enzyme that catalyzes glutamylation, folylpolyglutamate synthetase, in cells grown under a number of conditions known to alter the rate of glutamate addition in intact cells. The turnover of methotrexate polyglutamates and folylpolyglutamates and the effect that each of these pools has on the other will be examined. A new series of folates and antifolates with a fluorine in place of hydrogen in the 4 position of the glutamate moiety will be evaluated. The fluoro substitution prevents the glutamylation of methotrexate and as such can be used to evaluate the role that glutamylation plays in the activity of the drug. Using the same rationale, reduced folate coenzymes (dihydrofolate, folinic acid, 5-methyltetrahydrofolate) will be synthesized as the 4 fluoro derivative. With these probes we will examine the transport and metabolism of the fluoroanalogs, the relationship between glutamylation of folates and growth dependence, the sensitivity of glutamylated and non-glutamylated methotrexate to toxicity prevention by purines and pyrimidines, and the role of glutamylation in rescue protocols employing folinic acid. Other areas to be investigated are the characteristics of transport-based methotrexate resistance and a more detailed analysis of the cytocidal activity of methotrexate. These studies are intended to yield greater understanding about antifolates so that they may be more selectively employed as antiproliferative agents in the treatment of neoplasia.