Received: 2 June 2008, Revised: 13 November 2008, Accepted: 14 November 2008, Published online in Wiley InterScience: 20 January 2009 Modulation of choline kinase activity in human cancer cells observed by dynamic 31 P NMR Cristina Gabellieri a , Mounia Beloueche-Babari a , Yann Jamin a , Geoffrey S. Payne a , Martin O. Leach a and Thomas R. Eykyn a * Choline metabolites are widely studied in cancer research as biomarkers of malignancy and as indicators of therapeutic response. However, endogenous phosphocholine levels are determined by a number of processes that confound the interpretation of these measurements, including membrane transport rates and a series of enzyme catalysed reactions in the Kennedy pathway. Employing a dynamic 31 P NMR assay that is specific to choline kinase (ChoK) we have measured the rates of this enzyme reaction in cell lysates of MDA-MB-231 breast, PC-3 prostate and HeLa cervical cancer cells and in solutions of purified human ChoK. The rates are sensitive to inhibition by hemicholinium-3 (HC-3), a competitive ChoK inhibitor, and to N-[2-bromocinnamyl(amino)ethyl]-5-isoquinoline- sulphonamide (H-89), an agent commercialized as a specific cyclic-AMP-dependent protein kinase A (PKA) inhibitor. Copyright ß 2009 John Wiley & Sons, Ltd. Keywords: 31 P NMR; enzymatic assay; choline kinase; hemicholinium-3; H-89 INTRODUCTION Choline metabolites are widely studied in cancer research as diagnostic markers of malignancy and also as indicators of therapeutic response (1,2). Choline is incorporated into mem- brane phospholipids through the three-step Kennedy pathway. Following choline transport into the cells, the first step of this process, the ATP-dependent phosphorylation of choline (Cho) to phosphocholine (PCho), is catalysed by choline kinase (ChoK) (ATP: choline phosphotransferase, EC 2.7.1.32) in the presence of Mg 2þ . The final product, phosphatidylcholine (PtdCho), is an integral component of the cell membrane. Malignant and oncogenic transformations are often associated with altered membrane phospholipid metabolism; phosphorus NMR spec- troscopy ( 31 P NMR) has revealed increased levels of PCho in almost all forms of cancer, both as a precursor and as a breakdown product of the synthesis of membrane phospholipids, while choline metabolites are present at a lower level in normal tissue (3). Choline kinase is overexpressed in several human cancer cell lines (4) and a strong correlation between increased ChoK activity in ex vivo breast tumour tissues and high histological grade has been found (5). Furthermore, previous studies reported that chemical carcinogens (6) and oncogenes (7,8) lead to enhanced activity of ChoK in mammalian cells and highlight the central role of ChoK in tumour transformation. Choline kinase is now an attractive target for mechanism- based anti-cancer treatment with agents aimed at blocking its activity now in pre-clinical development (9). Rational drug development relies on the discovery of pharmacodynamic biomarkers, particularly those that are non-invasive, to aid tumour diagnosis and staging, treatment schedule planning and subsequent assessment of response to therapy (10,11). Previous work has shown that inhibition of ChoK either with the small molecule inhibitor MN58b (9) or RNA interference (12) correlates with decreased cellular PCho levels. 31 P NMR has been widely used to study the kinetics of phosphoryl transferring enzymes both in vitro and in vivo (13–15). In this study, we have applied a dynamic real-time 31 P NMR method to measure ChoK activity in solutions of purified ChoK and in lysates of human MDA-MB-231 breast, PC-3 prostate and HeLa cervical cancer cells. We have measured the modulation of ChoK activity following exposure to the ChoK inhibitor hemi- cholinium-3 (HC-3) (16) and to N-[2-bromocinnamyl(amino) ethyl]-5-isoquinolinesulphonamide (H-89). Hemicholinium is a known inhibitor of ChoK and also of choline transport (17) whereas H-89 is an agent commercialized as a specific cyclic-AMP-dependent protein kinase A (PKA) inhibitor (18). H-89 has also been shown to inhibit ChoK although it was unclear whether this effect is due to direct inhibition of ChoK or due to (www.interscience.wiley.com) DOI:10.1002/nbm.1361 Research Article * Correspondence to: T. R. Eykyn, Cancer Research UK Clinical Magnetic Reson- ance Research Group, The Institute of Cancer Research, Royal Marsden NHS Foundation Trust, Downs Road, Sutton, Surrey, SM2 5PT, UK. E-mail: Thomas.Eykyn@icr.ac.uk a C. Gabellieri, M. Beloueche-Babari, Y. Jamin, G. S. Payne, M. O. Leach, T. R. Eykyn Cancer Research UK Clinical Magnetic Resonance Research Group, The Institute of Cancer Research, Royal Marsden NHS Foundation Trust, Downs Road, Sutton, Surrey, SM2 5PT, UK Contract/grant sponsor: Cancer Research UK [CUK]; contract/grant numbers: C1060/A5117; C1060/A6916. Abbreviations used: Cho, choline; ChoK, choline kinase; D-MEM, Dulbecco’s modified Eagle’s medium; DMSO, dimethyl sulfoxide; EDTA, ethylene diamine tetraacetic acid; HC-3, hemicholinium; PBS, phosphate buffered saline; PCho, phosphocholine; Pi, inorganic phosphate; PKA, protein kinase A; PLC, phospholipase C; PLD, phospholipase D; PtdCho, phosphatidylcholine. NMR Biomed. 2009; 22: 456–461 Copyright ß 2009 John Wiley & Sons, Ltd. 456