METHOTREXATE UPTAKE AND METABOLISM IN HEPATIC CELLS

这些研究的基础是理解谷氨酰化的作用 抗叶酸的细胞毒活性及其与细胞毒性的关系 细胞叶基多谷氨酸盐。我们将主要利用一种啮齿动物肝癌 培养中的细胞系。我们将评估这种酶的活性 在生长的细胞中催化谷氨酰化，叶基多谷氨酸合成酶 在许多已知条件下改变谷氨酸添加速率 在完好无损的细胞中。甲氨蝶呤多聚谷氨酸酯类的周转 叶基多谷氨酸盐以及这些池中的每一个对 其他人将被审查。 一系列新的叶酸和反叶酸，用氟取代 将评估谷氨酸部分的4个位置上的氢。这个 氟取代可阻止甲氨蝶呤的谷氨酰化，因此 可以用来评估谷氨酰化在活性中所起的作用 药物的作用。使用同样的原理，减少叶酸辅酶 将合成(二氢叶酸、叶酸、5-甲基四氢叶酸) 作为4氟衍生物。通过这些探头，我们将检查 含氟类似物的运输和代谢，两者之间的关系 叶酸的谷氨酰化与生长依赖，敏感性 谷氨酰化和非谷氨酰化甲氨蝶呤的毒性预防 嘌呤和嘧啶，以及谷氨酰化在救援方案中的作用 使用叶酸。 其他需要调查的领域是以交通为基础的特征 甲氨蝶呤耐药性和更详细的细胞杀伤力分析 甲氨蝶呤的活性。这些研究旨在产生更大的 了解抗叶酸，以便它们可以更有选择性地 在肿瘤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.