Carrier Mediated Antifolate Transport and Resistance

描述（由申请人提供）：上一个资助期的研究旨在阐明小鼠白血病细胞中转运相关获得性抗叶酸剂耐药的分子基础，并深入了解小鼠RFC（RFC）的结构-功能。目前的建议代表了这项工作的发展，基于该实验室最近的研究，其特征在于在人类实体瘤细胞系中普遍表达的，突出的，具有低pH最适的叶酸转运活性，有助于抗叶酸剂的活性，并在叶酸的肠道吸收中发挥作用。数据表明，存在至少两种类型的低pH叶酸转运活性，其代表为（i）大鼠肠上皮细胞（IEC-6），其中低pH活性的主要部分（但不是全部）以某种方式与RFC表达相关，和（ii）Hela细胞，其中低pH活性完全不依赖RFC。拟议研究的目的是探索人类实体瘤细胞系中与转运相关的抗叶酸剂耐药性的潜在机制，并更好地定义低pH叶酸转运途径的基础和药理学重要性及其与RFC的关系。新一代抗叶酸剂培美曲塞在这些研究中受到重视，因为越来越多的证据表明其临床实用性、对低pH转运蛋白的明显高亲和力以及其他新特性。这些研究建议：（ii）开发RFC缺陷的人实体瘤细胞系，以区分RFC相关的和RFC非依赖的低pH转运活性，以提供对人RFC的结构-功能的进一步了解，以及这如何影响抗叶酸活性;（iii）选择人肿瘤细胞系的原发性抗性， 培美曲塞，以评估RFC和/或低pH转运活性的作用，评估交叉耐药模式，并确定假定的培美曲塞酶靶点在药物活性中的作用;（iv）阐明RFC功能与小肠来源细胞和可能鉴定的肿瘤中的低pH转运活性相关的机制;（v）实施在Hela细胞中克隆低pH、不依赖RFC的转运蛋白的方法。这些研究得到了初步数据和各种细胞系的支持，这些细胞系已经开发出RFC功能受损，改变了低pH转运活性或其他独特的特性，这些特性将促进这项工作。

DESCRIPTION (provided by applicant): Studies over the last funding period were directed to elucidating the molecular basis for transport-associated acquired resistance to antifolates in murine leukemia cells and gaining insight into the structure-function of the murine RFC (RFC). The current proposal represents an evolution of this work, based on recent studies from this laboratory, that have characterized ubiquitously expressed, prominent, folate transport activities with low-pH optima in human solid tumor cell lines that contribute to the activity of antifolates and play a role in intestinal absorption of folates. The data indicate that there are at least two types of low-pH folate transport activities typified by (i) rat intestinal epithelial cells, (IEC-6) in which the major portion, but not all, of the low pH activity is linked in some way to RFC expression and (ii) Hela cells in which the low-pH activity is entirely RFC- independent. The objectives of the proposed studies are to explore mechanisms underlying transport-related antifolate resistance in human solid tumor cell lines and to better define the basis for, and pharmacological importance of, low-pH folate transport routes and their relationship to RFC. The new generation antifolate, pemetrexed, is emphasized in these studies because of increasing evidence of its clinical utility, its apparent high affinity for low pH transporter(s), and its other novel properties. The studies proposed: (i) assess the prevalence, and characterize the properties of, folate transport activities with low-pH optima; (ii) develop RFC-deficient human solid tumor cell lines in order to distinguish between RFC-associated, and RFC-independent, low pH transport activities, to provide further insight into the structure-function of human RFC, and how this impacts on antifolate activities; (iii) select human tumor cell lines for primary resistance to pemetrexed in order to assess the roles of RFC and/or low-pH transport activities, assess cross-resistance patterns, and determine the role of putative pemetrexed enzyme targets in drug activity; (iv) clarify mechanisms by which RFC function is related to low-pH transport activity in cells of small intestinal origin, and possibly tumors that may be identified; (v) implement methodologies for cloning the low pH. RFC-independent, transporter in Hela cells. The proposed studies are supported by preliminary data and variety of cell lines that have been developed with impaired RFC function, altered low pH-transport activity, or other unique properties that will facilitate this work.