Nucleoside Transporters: Pharmacology of hENT3 and hCNT3

核苷转运蛋白：hENT3 和 hCNT3 的药理学

• 批准号: 6858592
• 负责人: CHUNG-MING TSE
• 金额: $ 27.18万
• 依托单位: JOHNS HOPKINS UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2003
• 资助国家: 美国
• 起止时间: 2003-04-01 至 2008-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6858592
• 关键词: Golgi apparatus; RNase protection assay; cell line; cell membrane; confocal scanning microscopy; cytotoxicity; drug design /synthesis /production; drug hypersensitivity; drug resistance; enzyme linked immunosorbent assay; gene expression; glycoproteins; glycosylation; hybrid antibody; intracellular transport; membrane transport proteins; microorganism disease chemotherapy; molecular cloning; neoplasm /cancer chemotherapy; nucleosides; pharmacokinetics; polymerase chain reaction; protein structure function; site directed mutagenesis; tissue /cell culture; western blottings

DESCRIPTION (provided by applicant): Plasma membrane nucleoside transporters are important in transporting nucleoside drugs into their target cells where they are metabolized and become cytotoxic. Nucleoside drugs are clinically used in the treatment of cancer (eg. Ara C in leukemia) and viral diseases (eg AZT in AIDS). Multiple nucleoside transporters have been cloned and are divided into Na-dependent Concentrative Nucleoside Transporters (CNTs) and Na-independent Equilibrative Nucleoside Transporters (ENTs). There is no homology between CNTs and ENTs. Our laboratory has cloned a new member of ENT gene family, human ENT3, and a new member of CNT gene family, human CNT3. Transient expression of hCNT3 in Cos7L cells confirms that hCNT3 is a broadly selective nucleoside transporter. For hENT3, immunolocalization studies show that hENT3 is a Golgi nucleoside transporter. Expression of hENT3 in PS120 fibroblasts containing functional plasma membrane nucleoside transporters, confers the cells resistant to the cytotoxicity of nucleoside drugs but not to the cytotoxicity of nucleobase drugs. Thus, we hypothesize that overexpression of hENT3 accounts for drug resistance, while overexpression of plasma membrane nucleoside transporter (hENT1, hENT2 or hCNT1-3) accounts for increase in drug sensitivity. In this application, we propose to characterize the kinetic and pharmacological properties of hENT3 (Aim1) and hCNT3 (Aim2). Strategic approaches include cell biology (immunolocalization and production of nucleoside transporter antibodies), biochemistry (membrane fractionation and reconstitution), molecular biology (targeting tag and site-directed mutagenesis), physiology (characterization of endogenous nucleoside transport systems) and pharmacology (drug resistance and nucleoside drug transport). A long-term goal of our studies is to use the knowledge of nucleoside transport at the molecular level to facilitate the design of nucleoside analogue drugs for the treatment of various viral, parasitic, and neoplastic diseases and as cardiovascular protective agents. The identification of hENT3 as a Golgi nucleoside transporter opens a new venue for studying molecular mechanisms of drug resistance.

描述（由申请人提供）：质膜核苷转运蛋白对于将核苷药物转运到其靶细胞中很重要，在靶细胞中它们被代谢并变得具有细胞毒性。核苷类药物临床上用于治疗癌症（如白血病中的Ara C）和病毒性疾病（如艾滋病中的AZT）。多种核苷转运蛋白已被克隆，分为钠依赖型集中核苷转运蛋白（CNT）和钠非依赖型平衡核苷转运蛋白（ENT）。 CNT 和 ENT 之间没有同源性。我们实验室克隆了ENT基因家族的新成员——人ENT3，以及CNT基因家族的新成员——人CNT3。 hCNT3 在 Cos7L 细胞中的瞬时表达证实 hCNT3 是一种广泛选择性的核苷转运蛋白。对于 hENT3，免疫定位研究表明 hENT3 是高尔基体核苷转运蛋白。 hENT3 在含有功能性质膜核苷转运蛋白的 PS120 成纤维细胞中表达，赋予细胞对核苷药物的细胞毒性的抵抗力，但对核碱基药物的细胞毒性没有抵抗力。因此，我们假设 hENT3 的过度表达导致耐药性，而质膜核苷转运蛋白（hENT1、hENT2 或 hCNT1-3）的过度表达导致药物敏感性增加。在此应用中，我们建议表征 hENT3 (Aim1) 和 hCNT3 (Aim2) 的动力学和药理学特性。战略方法包括细胞生物学（免疫定位和核苷转运蛋白抗体的产生）、生物化学（膜分级分离和重构）、分子生物学（靶向标签和定点突变）、生理学（内源核苷转运系统的表征）和药理学（耐药性和核苷药物转运）。我们研究的长期目标是利用分子水平上核苷转运的知识来促进核苷类似物药物的设计，用于治疗各种病毒、寄生虫和肿瘤疾病以及作为心血管保护剂。 hENT3 作为高尔基体核苷转运蛋白的鉴定为研究耐药性分子机制开辟了新的途径。