Nucleoside Transporters: Pharmacology of hENT3 and hCNT3

描述(由申请人提供)：质膜核苷转运体在将核苷药物运送到靶细胞中是重要的，在那里它们被代谢并成为细胞毒药。核苷类药物在临床上用于癌症的治疗(例如。阿糖胞苷(白血病)和病毒性疾病(如艾滋病的AZT)。多个核苷转运体已被克隆，分为钠依赖的浓缩型核苷转运体(CNTs)和钠非依赖性的平衡型核苷转运体(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ENT3和CNT基因家族的新成员人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成纤维细胞中的表达，使细胞对核苷类药物的细胞毒性产生耐药，但对核碱基药物的细胞毒作用无明显影响。因此，我们假设hENT3的过度表达是耐药的原因，而质膜核苷转运蛋白(hENT1、hENT2或hCNT1-3)的过度表达则是药物敏感性增加的原因。在这一应用中，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体抗体的产生)、生物化学(膜分离和重建)、分子生物学(靶向标签和定点突变)、生理学(内源性核苷转运体系统的特征)和药理学(耐药性和核苷药物转运体)。我们研究的一个长期目标是利用分子水平上的核苷转运知识来促进核苷类似物的设计，用于治疗各种病毒、寄生虫和肿瘤疾病，并用作心血管保护剂。HENT3作为高尔基核苷转运蛋白的发现为研究耐药的分子机制开辟了新的平台。

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.