A novel fluorometric assay for aldo-keto reductase 1C3 predicts metabolic activation of the nitrogen mustard prodrug PR-104A in human leukaemia cells - PubMed (original) (raw)

. 2014 Mar 1;88(1):36-45.

doi: 10.1016/j.bcp.2013.12.019. Epub 2014 Jan 13.

Yongchuan Gu 2, Donya Moradi Manesh 3, Jad El-Hoss 3, Duohui Jing 3, Karen L Mackenzie 3, Christopher P Guise 1, Annika Foehrenbacher 2, Susan M Pullen 2, Juliana Benito 4, Jeffrey B Smaill 1, Adam V Patterson 1, Medhanie A Mulaw 5, Marina Konopleva 4, Stefan K Bohlander 6, Richard B Lock 3, William R Wilson 7

Affiliations

A novel fluorometric assay for aldo-keto reductase 1C3 predicts metabolic activation of the nitrogen mustard prodrug PR-104A in human leukaemia cells

Stephen M F Jamieson et al. Biochem Pharmacol. 2014.

Abstract

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.

Keywords: Aldo-keto reductase 1C3; Fluorogenic assays; Leukaemia; PR-104; SN34037.

Copyright © 2014 Elsevier Inc. All rights reserved.

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