TY - JOUR
T1 - A novel fluorometric assay for aldo-keto reductase 1C3 predicts metabolic activation of the nitrogen mustard prodrug PR-104A in human leukaemia cells
AU - Jamieson, Stephen M.F.
AU - Gu, Yongchuan
AU - Manesh, Donya Moradi
AU - El-Hoss, Jad
AU - Jing, Duohui
AU - Mackenzie, Karen L.
AU - Guise, Christopher P.
AU - Foehrenbacher, Annika
AU - Pullen, Susan M.
AU - Benito, Juliana
AU - Smaill, Jeffrey B.
AU - Patterson, Adam V.
AU - Mulaw, Medhanie A.
AU - Konopleva, Marina
AU - Bohlander, Stefan K.
AU - Lock, Richard B.
AU - Wilson, William R.
N1 - Funding Information:
We thank Dianne Ferry for assistance with the coumberone assays, Sree Sreebhavan for assistance with LC-MS/MS and Stephen Edgar for flow cytometry. This research was supported by a Programme Grant from the Health Research Council of New Zealand ( 11/1103 ), a grant from the German Ministry for Education and Research (BMBF, NGFN-Plus: 01GS0876 ), and by a Project Grant and Fellowship from the National Health and Medical Research Council of Australia . Children's Cancer Institute Australia for Medical Research is affiliated with the University of New South Wales and the Sydney Children's Hospitals Network.
PY - 2014/3/1
Y1 - 2014/3/1
N2 - Aldo-keto reductase 1C3 (AKR1C3, EC 1.1.1.188) metabolises steroid hormones, prostaglandins and xenobiotics, and activates the dinitrobenzamide mustard prodrug PR-104A by reducing it to hydroxylamine PR-104H. Here, we describe a functional assay for AKR1C3 in cells using the fluorogenic probe coumberone (a substrate for all AKR1C isoforms) in conjunction with a specific inhibitor of AKR1C3, the morpholylurea SN34037. We use this assay to evaluate AKR1C3 activity and PR-104A sensitivity in human leukaemia cells. SN34037-sensitive reduction of coumberone to fluorescent coumberol correlated with AKR1C3 protein expression by immunoblotting in a panel of seven diverse human leukaemia cell lines, and with SN34037-sensitive reduction of PR-104A to PR-104H. SN34037 inhibited aerobic cytotoxicity of PR-104A in high-AKR1C3 TF1 erythroleukaemia cells, but not in low-AKR1C3 Nalm6 pre-B cell acute lymphocytic leukaemia (B-ALL) cells, although variation in PR-104H sensitivity confounded the relationship between AKR1C3 activity and PR-104A sensitivity across the cell line panel. AKR1C3 mRNA expression showed wide variation between leukaemia patients, with consistently higher levels in T-ALL than B-ALL. In short term cultures from patient-derived paediatric ALL xenografts, PR-104A was more potent in T-ALL than B-ALL lines, and PR-104A cytotoxicity was significantly inhibited by SN34037 in T-ALL but not B-ALL. Overall, the results demonstrate that SN34037-sensitive coumberone reduction provides a rapid and specific assay for AKR1C3 activity in cells, with potential utility for identifying PR-104A-responsive leukaemias. However, variations in PR-104H sensitivity indicate the need for additional biomarkers for patient stratification.
AB - Aldo-keto reductase 1C3 (AKR1C3, EC 1.1.1.188) metabolises steroid hormones, prostaglandins and xenobiotics, and activates the dinitrobenzamide mustard prodrug PR-104A by reducing it to hydroxylamine PR-104H. Here, we describe a functional assay for AKR1C3 in cells using the fluorogenic probe coumberone (a substrate for all AKR1C isoforms) in conjunction with a specific inhibitor of AKR1C3, the morpholylurea SN34037. We use this assay to evaluate AKR1C3 activity and PR-104A sensitivity in human leukaemia cells. SN34037-sensitive reduction of coumberone to fluorescent coumberol correlated with AKR1C3 protein expression by immunoblotting in a panel of seven diverse human leukaemia cell lines, and with SN34037-sensitive reduction of PR-104A to PR-104H. SN34037 inhibited aerobic cytotoxicity of PR-104A in high-AKR1C3 TF1 erythroleukaemia cells, but not in low-AKR1C3 Nalm6 pre-B cell acute lymphocytic leukaemia (B-ALL) cells, although variation in PR-104H sensitivity confounded the relationship between AKR1C3 activity and PR-104A sensitivity across the cell line panel. AKR1C3 mRNA expression showed wide variation between leukaemia patients, with consistently higher levels in T-ALL than B-ALL. In short term cultures from patient-derived paediatric ALL xenografts, PR-104A was more potent in T-ALL than B-ALL lines, and PR-104A cytotoxicity was significantly inhibited by SN34037 in T-ALL but not B-ALL. Overall, the results demonstrate that SN34037-sensitive coumberone reduction provides a rapid and specific assay for AKR1C3 activity in cells, with potential utility for identifying PR-104A-responsive leukaemias. However, variations in PR-104H sensitivity indicate the need for additional biomarkers for patient stratification.
KW - Aldo-keto reductase 1C3
KW - Fluorogenic assays
KW - Leukaemia
KW - PR-104
KW - SN34037
UR - https://www.scopus.com/pages/publications/84896695644
UR - https://www.scopus.com/pages/publications/84896695644#tab=citedBy
U2 - 10.1016/j.bcp.2013.12.019
DO - 10.1016/j.bcp.2013.12.019
M3 - Article
C2 - 24434189
AN - SCOPUS:84896695644
SN - 0006-2952
VL - 88
SP - 36
EP - 45
JO - Biochemical Pharmacology
JF - Biochemical Pharmacology
IS - 1
ER -