METHOTREXATE UPTAKE AND METABOLISM IN HEPATIC CELLS

抗叶酸的多谷氨酸衍生物已被确定为 这类抗肿瘤药物疗效的主要决定因素 探员们。这些衍生品的形成已被表征为 转化细胞系的数量和分离的叶聚谷氨酸 合成酶。相反，这些γ-谷氨酰基的裂解过程 衍生品尚未得到足够深入或详细的评估。这个 这项研究的重点是催化这一过程的酶？？谷氨酰基。 水解酶(GH)。在上一个资助期的研究已经建立了一个 该酶的一些特征：1)它是一种糖蛋白(MR-55 kDa) 具有33 kDa的多肽MR，2)主要是一种分泌酶(在所有 检测的肿瘤细胞)，其主要细胞位置是溶酶体，3)和 细胞内酶活性升高是获得性药物的一种机制 阻力，4)它的上升对减少有更深刻的影响。 细胞抗叶酸多谷氨酸而不是叶基多谷氨酸，以及5)一个cdna 已经克隆并鉴定了大鼠肝癌中的生长激素，并对其进行了鉴定。 推导的氨基酸序列已建立。GH Will的详细评估 以确定其在抗叶酸药理学中的作用 包括1)生长激素基因及其表达改变因素的进一步研究 ， 2)使用定点突变和详细的物理和 生化分析以了解糖蛋白及其蛋白的结构 作用机制，3)酶变化机制的确定 获得性耐药的活性以及对胰岛素等的反应 组织培养条件的扰动，4)评估细胞定位化 N 应用多克隆抗体显微镜观察生长激素的表达 细胞贩运，以及5)使用分子方法来增强或 抑制细胞内生长激素水平，并根据效果评估结果 抗叶酸活性和多谷氨酰化、生长激素功能与叶酸生长 依赖。将进行这些调查，以确定GH是否可以 被用作目标或其功能的修改是否可以利用 边缘 以提高抗叶酸的治疗选择性。最后， 将对生长激素的分泌进行详细的检测，以了解其 生物学和药理学意义，并开发工具来 确定其作为诊断标记物的潜力。

The polyglutamate derivatives of antifolates have been established as being a major determinant in the therapeutic efficiency of this class of antitumo agents. The formation of these derivatives has been characterized in a number of transformed cell lines and with isolated folypolyglutamate synthetase. In contrast, the process of cleavage of these y-glutamyl derivatives has not yet been evaluated in sufficient depth or detail. The focus of this study is the enzyme that catalyzes this process, y-glutamyl hydrolase (GH). Studies in the previous grant period have established a number of features about this enzyme: 1) it is a glycoprotein (Mr-55 kDa) with a peptide Mr of 33kDa, 2) it is primarily a secreted enzyme (in all tumor cells tested) whose primary cellular location is the lysosome, 3) an intracellular elevation in its activity is a mechanism of acquired drug resistance, 4) its elevation has a more profound effect in diminishing cellular antifolate polyglutamates than folyl polyglutamates, and 5) a cDNA for GH from rat hepatoma has been cloned and characterized and the deduced amino acid sequence established. A detailed evaluation of GH will be made in order to establish its role in the pharmacology of the antifolat including 1) further studies on GH gene and factors that alter its expressi , 2) the use of site directed mutagenesis and detailed physical and biochemical analysis to understand the structure of the glycoprotein and it mechanism of action, 3) determining the mechanism of alteration in enzyme activity in acquired drug resistance and in response to insulin and other perturbations in tissue culture conditions, 4) evaluating the cytolocalizat n of GH by microscopy using polyclonal antibodies and investigating its cellular trafficking, and 5) using molecular methodology to enhance or inhibit GH levels in cells and evaluate the outcome in terms of the effect antifolate activity and polyglutamylation, GH function and folate growth dependence. These investigations will be conducted to determine if GH can be exploited as a target or if the modification of its function can be util ed to enhance the therapeutic selectivity of the antifolates. Lastly, the secretion of GH will be examined in detail in order to understand its biological and pharmacological significance and to develop the tools to determine its potential as a diagnostic marker.