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.
Jinno
Hideto
H
Division of Environmental Chemistry, National Institute of Health Sciences, Setagaya-ku, Tokyo 158-8501, Japan.
Saeki
Mayumi
M
Saito
Yoshiro
Y
Tanaka-Kagawa
Toshiko
T
Hanioka
Nobumitsu
N
Sai
Kimie
K
Kaniwa
Nahoko
N
Ando
Masanori
M
Shirao
Kuniaki
K
Minami
Hironobu
H
Ohtsu
Atsushi
A
Yoshida
Teruhiko
T
Saijo
Nagahiro
N
Ozawa
Shogo
S
Sawada
Jun-ichi
J
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