Cellular localization domains of a rabbit and a human carboxylesterase: influence on irinotecan (CPT-11) metabolism by the rabbit enzyme.
Enzyme activation of prodrugs to improve the therapeutic index of specific anticancer agents is an attractive alternative to current chemotherapy regimens. This study addresses the potential for activating irinotecan (CPT-11) with recombinant carboxylesterases (CEs). CEs are a ubiquitous class of enzymes thought to be involved in the detoxification of xenobiotics. Their primary amino acid sequence indicates that these proteins should be localized to the endoplasmic reticulum. By PCR-mediated mutagenesis of a rabbit liver and a human alveolar macrophage CE cDNA, expression in Cos7 cells, and subsequent immunohistochemical localization, we have determined that an 18-amino acid NH2-terminal hydrophobic signal peptide is responsible for the localization of these proteins to the endoplasmic reticulum. By similar approaches, we have demonstrated that the COOH-terminal amino acids HIEL prevent secretion of the proteins from the cell. Enzymatic activity was lost by removing the NH2-terminal domain; however, active enzyme could be detected in the culture media of cells expressing the COOH-terminally truncated proteins. Secretion of CEs lacking the six COOH-terminal amino acids could be prevented with brefeldin A, confirming that these truncated enzymes were processed and released from cells by endoplasmic reticulum-mediated exocytosis. Double-truncation mutant enzymes lacking both NH2- and COOH-terminal sequences demonstrated immunostaining patterns similar to those of the NH2-terminally truncated proteins and also lacked CE activity. In all cases, metabolism of the classic esterase substrate o-nitrophenyl acetate predicted the sensitivity of cells expressing the rabbit CE to the anticancer agent CPT-11. In addition, the secreted enzyme sensitized Cos7 cells to this drug, indicating that protein association with a lipid bilayer is not required for substrate metabolism.
Department of Molecular Pharmacology, St. Jude Children's Research Hospital, Memphis, Tennessee 38105, USA.
P M
PM
Potter
J S
JS
Wolverton
C L
CL
Morton
M
M
Wierdl
M K
MK
Danks
D000818:Animals; D000972:Antineoplastic Agents, Phytogenic / Q000378:metabolism*; D019556:COS Cells; D002166:Camptothecin / Q000031:analogs & derivatives* / Q000378:metabolism; D002265:Carboxylic Ester Hydrolases / Q000032:analysis* / Q000378:metabolism; D016895:Culture Media, Serum-Free; D004721:Endoplasmic Reticulum / Q000201:enzymology*; D005822:Genetic Vectors; D006801:Humans; D000077146:Irinotecan; D016297:Mutagenesis, Site-Directed; D016133:Polymerase Chain Reaction / Q000379:methods; D011817:Rabbits; D013045:Species Specificity; D014162:Transfection
D000972:Antineoplastic Agents, Phytogenic; D016895:Culture Media, Serum-Free; D000077146:Irinotecan; D002265:Carboxylic Ester Hydrolases; D002166:Camptothecin