Evaluation of division-arrested cells for cell-based high-throughput screening and profiling.
Just-in-time cell supply for cell-based high-throughput screening (HTS) is frequently problematic. In addition to scheduling and logistical issues, quality issues and variability due to passage effect, cell cycle, or confluency contribute to day-to-day signal variability in the course of cell-based HTS campaigns. Cell division-arrest and cryopreservation technologies permit the use of cells as assay-ready reagents for HTS and other cell-based profiling and structure-activity studies. In this report, the authors compare division-arrested and dividing cells in 2 assay types that are dependent on movement of proteins within or through cell membranes: a receptor tyrosine kinase assay involving A431 cells responsive to epidermal growth factor, and a secretion reporter assay, which measures secretion of a reporter gene, secreted alkaline phosphatase. In both assays, dividing and division-arrested cells yielded similar basal and maximal signals at a given cell density. Similar IC50s were obtained for reference inhibitors in each assay, type in both dividing and division-arrested cells. In addition, for the secretion reporter assay, when comparing IC50s obtained from 44 compounds randomly chosen from a primary screening hit list, the rank order of potency obtained from dividing cells and division-arrested cells was essentially identical. Furthermore, the results show that, under certain assay conditions, data generated using division-arrested cells are less variable than those generated using dividing cells. In summary, the results suggest that, in many cases, division-arrested cells can substitute for dividing cells and offer certain advantages for cell-based assays.
Digan
Mary Ellen
ME
Lead Discovery Center/Discovery Technologies, Novartis Institute for Biomedical Research, Inc., Cambridge, MA 02139, USA. mary.digan@novartis.com
Pou
Chantevy
C
Niu
Honglin
H
Zhang
Ji-Hu
JH
D000469:Alkaline Phosphatase / Q000378:metabolism; D002455:Cell Division; D002460:Cell Line; D045744:Cell Line, Tumor; D002462:Cell Membrane / Q000378:metabolism; D002626:Chemistry, Pharmaceutical / Q000379:methods; D004121:Dimethyl Sulfoxide / Q000737:chemistry; D004305:Dose-Response Relationship, Drug; D004353:Drug Evaluation, Preclinical / Q000379:methods*; D004345:Drug Industry / Q000379:methods*; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004797:Enzyme-Linked Immunosorbent Assay; D004815:Epidermal Growth Factor / Q000378:metabolism; D066246:ErbB Receptors / Q000378:metabolism; D017930:Genes, Reporter; D006801:Humans; D020128:Inhibitory Concentration 50; D010766:Phosphorylation; D020794:Receptor Protein-Tyrosine Kinases / Q000378:metabolism; D015203:Reproducibility of Results
D004791:Enzyme Inhibitors; D004815:Epidermal Growth Factor; D066246:ErbB Receptors; D020794:Receptor Protein-Tyrosine Kinases; D000469:Alkaline Phosphatase; D004121:Dimethyl Sulfoxide