Nucleoside Transporters: Pharmacology of hENT3 and hCNT3

核苷转运蛋白：hENT3 和 hCNT3 的药理学

• 批准号: 6730547
• 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/6730547
• 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）：质膜核苷转运蛋白在将核苷类药物转运到靶细胞中代谢并产生细胞毒性方面很重要。核苷类药物在临床上用于治疗癌症。白血病中的Ara C)和病毒性疾病（如艾滋病中的AZT）。已经克隆了多个核苷转运体，并将其分为na依赖性浓缩核苷转运体（CNTs）和na非依赖性平衡核苷转运体（ENTs）。CNTs和ENTs之间没有同源性。本实验室克隆了ENT基因家族的新成员人类ENT3和CNT基因家族的新成员人类CNT3。hCNT3在Cos7L细胞中的瞬时表达证实了hCNT3是一种具有广泛选择性的核苷转运蛋白。对于hENT3，免疫定位研究表明hENT3是一种高尔基核苷转运蛋白。hENT3在含有功能性质膜核苷转运体的PS120成纤维细胞中表达，使细胞对核苷类药物具有细胞毒性，但对核碱基类药物没有细胞毒性。因此，我们假设hENT3的过表达是耐药的原因，而质膜核苷转运蛋白（hENT1、hENT2或hCNT1-3）的过表达是药物敏感性增加的原因。在这个应用中，我们提出表征hENT3 （Aim1）和hCNT3 （Aim2）的动力学和药理学性质。策略方法包括细胞生物学（免疫定位和核苷转运体抗体的产生）、生物化学（膜分离和重组）、分子生物学（靶向标签和定点诱变）、生理学（内源性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，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，并作为心血管保护剂。hENT3作为高尔基核苷转运体的鉴定为研究耐药分子机制开辟了新的途径。