Isolation and partial characterization of a cDNA encoding a rabbit liver carboxylesterase that activates the prodrug irinotecan (CPT-11).
Isolation and partial characterization of a cDNA encoding a rabbit liver carboxylesterase that activates the prodrug irinotecan (CPT-11).
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发表时间：
1998-06
期刊：
影响因子：
11.2
通讯作者：
P. Potter;C. A. Pawlik;C. Morton;C. Naeve;M. Danks
中科院分区：
文献类型：
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作者：
P. Potter;C. A. Pawlik;C. Morton;C. Naeve;M. Danks
我们已经分离出一种编码兔羧酸酯酶（CE; EC 3.1.1.1）的cDNA，该酶能将喜树碱衍生的前药伊立替康（CPT-11）转化为有效的拓扑异构酶I抑制剂7-乙基-10-羟基喜树碱。纯化兔CE的nh2末端氨基酸测序允许设计冗余寡核苷酸从兔肝cDNA进行PCR。PCR产物的DNA测序证实了克隆的身份，在cDNA末端进行5′和3′快速扩增后，设计寡核苷酸引物扩增整个cDNA。这个1698 bp的开放阅读框编码了一个565个氨基酸的蛋白质，其中包含特征CE B-1和B-2基序，疏水nh2末端先导序列和cooh末端残基HIEL，这些被认为是内质网中蛋白质定位的原因。在COS-7细胞中短暂表达cDNA，可提高细胞提取物中CE活性，提高细胞对CPT-11的敏感性。此外，兔肝CE cDNA在人胶质瘤U-373 MG细胞系中的稳定表达导致CPT-11的IC50值降低56倍，而编码高度同源CE的人肺泡巨噬细胞cDNA的表达对药物敏感性没有变化。
We have isolated a cDNA encoding a rabbit carboxylesterase (CE; EC 3.1.1.1) that converts the camptothecin-derived prodrug irinotecan (CPT-11) to the potent topoisomerase I inhibitor 7-ethyl-10-hydroxycamptothecin. NH2-terminal amino acid sequencing of a purified rabbit CE allowed the design of redundant oligonucleotides to perform PCR from rabbit liver cDNA. DNA sequencing of the PCR product confirmed the identity of the clone, and after both 5' and 3' rapid amplification of cDNA ends, oligonucleotide primers were designed to amplify the entire cDNA. The 1698-bp open reading frame encoded a 565-amino acid protein containing the characteristic CE B-1 and B-2 motifs, a hydrophobic NH2-terminal leader sequence, and the COOH-terminal residues HIEL that are thought to be responsible for protein localization in the endoplasmic reticulum. Transient expression of the cDNA in COS-7 cells resulted in CE activity in cell extracts and increased the sensitivity of cells to CPT-11. Additionally, stable expression of the rabbit liver CE cDNA in the human glioma U-373 MG cell line resulted in a 56-fold decrease in the IC50 value for CPT-11, whereas the expression of a human alveolar macrophage cDNA encoding a highly homologous CE produced no change in drug sensitivity.