Upon division and exit from mitosis they reattach and reflatten (Fig 1A). therapeutic window in which the checkpoint would be eliminated in cancer cells but sufficiently preserved in normal cells. We developed an assay to identify lead compounds that inhibit the spindle checkpoint. Most cells respond to microtubule drugs by activating the spindle checkpoint and arresting in mitosis with a rounded morphology. Our assay depended on the ability of checkpoint inhibitor compounds to drive mitotic exit and cause cells to flatten onto the substrate in the continuous presence of microtubule drugs. In this study we characterize one of the compounds, OM137, as an inhibitor of Aurora kinases. We find that this compound is growth inhibitory to cultured cells when applied at high concentration and potentiates the growth inhibitory effects Anisomycin of subnanomolar concentrations of paclitaxel. and isolated on GSH Sepharose Fast Flow (GE Healthcare). GST-tagged TAO1 immobilized on GSH Sepharose beads was direclty used in kinase assay in 40 mM HEPES pH 7.5, 10 mM MgCl2, 1 mM EDTA and myelin basic protein like a substrate. CDK1:cyclin B was assayed under the same conditions previously explained for CDK5:p25 (15C17). Video Microscopy S3, Ptk1, or Hela cells were cultivated on 25 mm round coverslips. The coverslips were sealed into Sykes Moore Chambers (Bellco) and medium containing test compounds were added using a syringe. Cells were cultured at 37C within the stage of a Zeiss Axiovert 200 microscope or a Nikon Eclipse TE2000-E microscope. Images were collected at intervals using phase contrast or Nomarski DIC optics with Roper Coolsnap-HQ2 or Hamamtsu Orca-ERG cams using Metamorph software (Molecular Products) or NIS-Elements software (Nikon). Cell Proliferation Assay Hela cells at 80 cells/well were seeded in 96 well plates and permitted to adhere to the substratum for 6 hours while incubating at 37C under 5% CO2. Test compounds were then added; paclitaxel at 0.25 nM and OM137 ranging from 6.25 uM to 100 uM. Settings received equivalent levels of DMSO (the diluent for both compounds). All conditions were assayed in quadruplicate. Cells were incubated for 4 days under these conditions. At the end of the 4th day time, the press was exchanged with new media comprising OM137 at the same concentrations, but paclitaxel was increased to 0.75 nM. Cells were YAP1 incubated for an additional 4 days. The amount of cell proliferation was measured using the CellTiter 96?AQueous 1 Solution Cell Proliferation Assay (Promega Corporation, G3580). Absorbance measurements were obtained using a Tecan Genios plate reader. Data from cells treated solely with OM137 were normalized to untreated cell ideals. Values from cells exposed to taxol and OM137 were normalized to data from cells treated with taxol only. Results Large Throughput Screening Identifies Chemical Inhibitors of the Mitotic Spindle Checkpoint Many cultured cells that are well attached during interphase become rounded during mitosis and maintain only weak attachment to the substratum. Upon division and exit from mitosis they reattach and reflatten (Fig 1A). Cells treated with microtubule medicines such as nocodozole arrest in mitosis through the action of the spindle checkpoint and remain arrested with this rounded state for a number of hours. They can be dislodged very easily with mild agitation of the medium. However, if the spindle checkpoint is definitely inactivated these cells will flatten and reattach without division (Fig Anisomycin 1A). We transferred nocodazole-arrested mitotic cells to wells of 384 well dishes and tested a library of small molecules for their ability to Anisomycin induce mitotic exit in the caught cells. Compounds that inactivate the checkpoint caused cells to exit mitosis, flatten, and reattach securely to the substratum. The cells in wells comprising inactive compounds remained rounded and were very easily washed from the dishes (Fig 1B). After fixation in a solution comprising a fluorescent DNA label, we used a fluorescence plate reader to rapidly assess which test compounds could induce mitotic exit and cell reattachment. Because the assay requires cells to actively flatten onto the substrate it selects against compounds that are merely cytotoxic. Open in a separate window Number 1 A high throughput, whole cell assay for small molecule inhibitors of the mitotic spindle checkpoint. A, In normal cell division cells become rounded during mitosis and flatten onto the substrate following cytokinesis. In response to microtubule medicines the spindle checkpoint is definitely activated and cells remain arrested as rounded cells while the checkpoint is active. If the checkpoint fails, cells flatten.