Use of radiolabelled choline as a pharmacodynamic marker for the signal transduction inhibitor geldanamycin.
There is an urgent need to develop non-invasive pharmacodynamic endpoints for the evaluation of new molecular therapeutics that inhibit signal transduction. We hypothesised that, when labelled appropriately, changes in choline kinetics could be used to assess geldanamycin pharmacodynamics, which involves inhibition of the HSP90 molecular chaperone-->Raf1-->Mitogenic Extracellular Kinase-->Extracellular Signal-Regulated Kinase 1 and 2 signal transduction pathway. Towards identifying a potential pharmacodynamic marker response, we have studied radiolabelled choline metabolism in HT29 human colon carcinoma cells following treatment with geldanamycin. We studied the effects of geldanamycin, on net cellular accumulation of (methyl-(14)C)choline and (methyl-(14)C)phosphocholine production. In parallel experiments, the effects of geldanamycin on extracellular signal-regulated kinase 1 and 2 phosphorylation and cell viability were also assessed. Additional validation studies were carried out with the mitogenic extracellular kinase inhibitor U0126 as a positive control; a cyclin-dependent kinase-2 inhibitor roscovitine and the phosphatidylinositol 3-kinase inhibitor LY294002 as negative controls. Hemicholinium-3, an inhibitor of choline transport and choline kinase activity was included as an additional control. In exponentially growing HT29 cells, geldanamycin inhibited extracellular signal-regulated kinase 1 and 2 phosphorylation in a concentration- and time-dependent manner. These changes were associated with a reduction in (methyl-(14)C)choline uptake, (methyl-(14)C) phosphocholine production and cell viability. Brief exposure to U0126, suppressed phosphocholine production to the same extent as Hemicholinium-3. In contrast to geldanamycin and U0126, which act upstream of extracellular signal-regulated kinase 1 and 2, roscovitine and LY294002 failed to suppress phosphocholine production. Our results suggest that when labelled with carbon-11 isotope, (methyl-(11)C)choline may be a useful pharmacodynamic marker for the non-invasive evaluation of geldanamycin analogues.
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Cancer Research UK PET Oncology group, Department of Cancer Medicine, Imperial College of Science Technology and Medicine, Hammersmith Hospital, MRC Cyclotron Building, Du Cane Road, London W12 0NN, UK.
Hutchinson
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Osman
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Price
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Workman
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Aboagye
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D016227:Benzoquinones; D015153:Blotting, Western; D002250:Carbon Radioisotopes; D002455:Cell Division / Q000187:drug effects; D002470:Cell Survival / Q000187:drug effects; D002794:Choline / Q000031:analogs & derivatives* / Q000378:metabolism* / Q000493:pharmacokinetics; D006801:Humans; D047029:Lactams, Macrocyclic; D020935:MAP Kinase Signaling System / Q000187:drug effects*; D020928:Mitogen-Activated Protein Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism; D010766:Phosphorylation / Q000187:drug effects; D010767:Phosphorylcholine / Q000378:metabolism; D011809:Quinones / Q000494:pharmacology*; D014407:Tumor Cells, Cultured
D016227:Benzoquinones; D002250:Carbon Radioisotopes; D047029:Lactams, Macrocyclic; D011809:Quinones; C017506:methylcholine; D010767:Phosphorylcholine; D020928:Mitogen-Activated Protein Kinases; D002794:Choline; C001277:geldanamycin