Functional characterization of human UDP-glucuronosyltransferase 1A9 variant, D256N, found in Japanese cancer patients.
SN-38 (7-ethyl-10-hydroxycamptothecin), an active metabolite of the antitumor prodrug irinotecan, is conjugated and detoxified to SN-38 10-O-beta-d-glucuronide by hepatic UDP-glucuronosyltransferase (UGT) 1A1. Recent studies have revealed that other UGT1A isoforms, UGT1A7 and UGT1A9, also participate in SN-38 glucuronidation. Although several genetic polymorphisms are reported for UGT1A1 and UGT1A7 that affect the SN-38 glucuronidation activities, no such polymorphisms have been identified for UGT1A9. In the present study, UGT1A9 exon 1 and its flanking regions were sequenced from 61 Japanese cancer patients who were all treated with irinotecan. A novel nonsynonymous single nucleotide polymorphism was identified in UGT1A9 exon 1, heterozygous 766G>A resulting in the amino acid substitution of D256N. The wild-type and D256N UGT1A9s were transiently expressed at similar protein levels in COS-1 cells, and their membrane fractions were characterized in vitro for the glucuronidation activities toward SN-38. The apparent Km values were 19.3 and 44.4 microM, and the Vmax values were 2.94 and 0.24 pmol/min/mg of membrane protein for the wild-type and D256N variant, respectively. The SN-38 glucuronidation efficiency (normalized Vmax/Km) of D256N was less than 5% that of wild-type UGT1A9. These results clearly indicate that the D256N variant is essentially nonfunctional with regard to SN-38 glucuronidation. These findings highlight the importance of further studies into the potential influence of UGT1A9 D256N variant to irinotecan metabolism in vivo.
Division of Environmental Chemistry, National Institute of Health Sciences, Setagaya-ku, Tokyo 158-8501, Japan.
Hideto
H
Jinno
Mayumi
M
Saeki
Yoshiro
Y
Saito
Toshiko
T
Tanaka-Kagawa
Nobumitsu
N
Hanioka
Kimie
K
Sai
Nahoko
N
Kaniwa
Masanori
M
Ando
Kuniaki
K
Shirao
Hironobu
H
Minami
Atsushi
A
Ohtsu
Teruhiko
T
Yoshida
Nagahiro
N
Saijo
Shogo
S
Ozawa
Jun-ichi
J
Sawada
D019943:Amino Acid Substitution; D000818:Animals; D000970:Antineoplastic Agents / Q000009:adverse effects* / Q000627:therapeutic use; D001216:Asparagine / Q000235:genetics; D001224:Aspartic Acid / Q000235:genetics; D019556:COS Cells; D002166:Camptothecin / Q000009:adverse effects* / Q000031:analogs & derivatives* / Q000378:metabolism* / Q000494:pharmacology / Q000627:therapeutic use; D005091:Exons; D014644:Genetic Variation; D005965:Glucuronates / Q000378:metabolism* / Q000494:pharmacology; D020723:Glucuronic Acid / Q000378:metabolism; D014453:Glucuronosyltransferase / Q000187:drug effects / Q000235:genetics* / Q000378:metabolism; D006801:Humans; D000077146:Irinotecan; D007564:Japan; D009154:Mutation / Q000187:drug effects*; D009369:Neoplasms / Q000188:drug therapy / Q000235:genetics / Q000473:pathology; D020641:Polymorphism, Single Nucleotide; D014162:Transfection; D000090264:UDP-Glucuronosyltransferase 1A9
C114685:7-ethyl-10-hydroxycamptothecin beta-glucuronide; D000970:Antineoplastic Agents; D005965:Glucuronates; C000715591:UGT1A9 protein, human; D001224:Aspartic Acid; D001216:Asparagine; D000077146:Irinotecan; D020723:Glucuronic Acid; D014453:Glucuronosyltransferase; D000090264:UDP-Glucuronosyltransferase 1A9; D002166:Camptothecin