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

  • Biochem Pharmacol. 2014 Mar 1;88(1):36-45. doi: 10.1016/j.bcp.2013.12.019.
Stephen M F Jamieson  1 ,  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
  • 1. Auckland Cancer Society Research Centre, the University of Auckland, Auckland, New Zealand; Maurice Wilkins Centre for Molecular Biodiscovery, the University of Auckland, Auckland, New Zealand.
  • 2. Auckland Cancer Society Research Centre, the University of Auckland, Auckland, New Zealand.
  • 3. Children's Cancer Institute Australia for Medical Research, University of New South Wales, Sydney, Australia.
  • 4. Division of Cancer Medicine, The University of Texas MD Anderson Cancer Center, Houston, TX, United States.
  • 5. Comprehensive Cancer Centre Ulm, Institute for Experimental Tumor Research, University of Ulm, Germany.
  • 6. Department of Molecular Medicine and Pathology, The University of Auckland, Auckland, New Zealand.
  • 7. Auckland Cancer Society Research Centre, the University of Auckland, Auckland, New Zealand; Maurice Wilkins Centre for Molecular Biodiscovery, the University of Auckland, Auckland, New Zealand. Electronic address: [email protected].
PMID: 24434189 DOI: 10.1016/j.bcp.2013.12.019
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.
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