ISOLATION AND PARTIAL CHARACTERIZATION OF A CDNA-ENCODING A RABBIT LIVER CARBOXYLESTERASE THAT ACTIVATES THE PRODRUG IRINOTECAN (CPT-11)

We have isolated a cDNA encoding a rabbit carboxylesterase (CE; EC 3.1 .1.1) that converts the camptothecin-derived prodrug irinotecan (CPT-1 1) to the potent topoisomerase I inhibitor 7-ethyl-10-hydroxycamptothe cin. NH2-terminal amino acid sequencing of a purified rabbit CE allowe d the design of redundant oligonucleotides to perform PCR from rabbit liver cDNA, DNA sequencing of the PCR product contirmed the identity o f the clone, and after both 5' and 3' rapid amplification of cDNA ends , oligonucleotide primers were designed to amplify the entire cDNA. Th e 1698-bp open reading frame encoded a 565-amino acid protein containi ng the characteristic CE B-1 and B-2 motifs, a hydrophobic NH2-termina l leader sequence, and the COOH-terminal residues HIEL that are though t to be responsible for protein localization in the endoplasmic reticu lum, Transient expression of the cDNA in COS-7 cells resulted in CE ac tivity in fell 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 mac rophage cDNA encoding a highly homologous CE produced no change in dru g sensitivity.