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-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.
Potter
P M
PM
Department of Molecular Pharmacology, St. Jude Children's Research Hospital, Memphis, Tennessee 38105, USA. phil.potter@stjude.org
Pawlik
C A
CA
Morton
C L
CL
Naeve
C W
CW
Danks
M K
MK
D000595:Amino Acid Sequence; D000818:Animals; D000972:Antineoplastic Agents, Phytogenic / Q000737:chemistry / Q000378:metabolism* / Q000494:pharmacology; D001483:Base Sequence; D019556:COS Cells / Q000187:drug effects / Q000201:enzymology; D002166:Camptothecin / Q000031:analogs & derivatives* / Q000737:chemistry / Q000378:metabolism / Q000494:pharmacology; D043182:Carboxylesterase; D002265:Carboxylic Ester Hydrolases / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D002470:Cell Survival / Q000187:drug effects; D018076:DNA, Complementary / Q000737:chemistry / Q000302:isolation & purification*; D005910:Glioma / Q000201:enzymology; D006801:Humans; D000077146:Irinotecan; D008099:Liver / Q000201:enzymology; D008969:Molecular Sequence Data; D011355:Prodrugs / Q000737:chemistry / Q000378:metabolism* / Q000494:pharmacology; D011817:Rabbits; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D014407:Tumor Cells, Cultured / Q000187:drug effects
D000972:Antineoplastic Agents, Phytogenic; D018076:DNA, Complementary; D011355:Prodrugs; D000077146:Irinotecan; D002265:Carboxylic Ester Hydrolases; D043182:Carboxylesterase; D002166:Camptothecin