Transfection assays with allele-specific constructs: functional analysis of UDP-glucuronosyltransferase variants.
Adverse drug reactions (ADRs) are a major clinical problem. A rapidly growing body of evidence suggests that genetic factors, at least in part, determine individual susceptibility to ADRs. A large number of pharmacogenetic studies have identified a number of polymorphisms as predictors of drug efficacy and/or adverse events. These candidate markers should be investigated further to ascertain the underlying mechanism of action, for example, changes in the kinetic parameters of an enzyme, or transcriptional activity of a promoter region. In this chapter, we describe a transient transfection assay for the functional characterization of naturally occurring variants of UDP-glucuronosyltransferase (UGT) 1A1. This phase II drug metabolizing enzyme is involved in the glucuronidation of SN-38, an active metabolite of the anti-cancer drug irinotecan. Single-nucleotide polymorphisms of the UGT1A1 gene have been correlated to irinotecan-induced ADRs. Variant UGT1A1s are heterologously expressed in COS-1 cells and characterized in terms of the level of protein expression and enzyme kinetics.
Jinno
Hideto
H
Division of Environmental Chemistry, National Institute of Health Sciences, Tokyo, Japan.
Hanioka
Nobumitsu
N
Tanaka-Kagawa
Toshiko
T
Saito
Yoshiro
Y
Ozawa
Shogo
S
Sawada
Jun-ichi
J
D000328:Adult; D000483:Alleles; D000818:Animals; D001681:Biological Assay; D015153:Blotting, Western; D019556:COS Cells; D002166:Camptothecin / Q000031:analogs & derivatives / Q000378:metabolism; D002522:Chlorocebus aethiops; D004795:Enzyme Stability; D020719:Glucuronides / Q000378:metabolism; D014453:Glucuronosyltransferase / Q000032:analysis* / Q000235:genetics; D006801:Humans; D000077146:Irinotecan; D008099:Liver / Q000187:drug effects / Q000378:metabolism; D016297:Mutagenesis, Site-Directed; D010957:Plasmids; D020641:Polymorphism, Single Nucleotide; D014162:Transfection / Q000379:methods*; D000099148:UGT1A1 Enzyme
D002166:Camptothecin; D020719:Glucuronides; D014453:Glucuronosyltransferase; D000077146:Irinotecan; D000099148:UGT1A1 Enzyme