title: An automated image capture and quantitation approach to identify proteins affecting tumor cell proliferation.
abstract: To facilitate the characterization of proteins that negatively regulate tumor cell proliferation in vitro, the authors have implemented a high-throughput functional assay that measures S-phase progression of tumor cell lines. For 2 tumor cell lines-human melanoma A375 and human lung carcinoma A549-conditions were established using the cyclin-dependent kinase inhibitor, p27kip; the tumor suppressor p53, a kinase-inactive allele of the cell cycle-regulated serine/threonine kinase Aurora2; and the G1/S drug block, aphidicolin. For screening purposes, gene libraries were delivered by adenoviral infection. Cells were fixed and labeled by immunocytochemistry, and an automated image acquisition and analysis package on a Cellomics ArrayScanII was used to quantify the effects of these treatments on cell proliferation. The assay can be used to identify novel proteins involved in proliferation and serves as a more robust, reproducible, and sensitive alternative to enzyme-linked immunosorbent assay (ELISA)-based technologies.
authors.affiliation: Target Discovery Department, Sugen, Inc. (A Subsidiary of Pfizer), South San Francisco, CA, USA. kbhawe@exelixis.com
authors.forename: Kaumudi M
authors.initials: KM
authors.lastname: Bhawe
authors.forename: Robert A
authors.initials: RA
authors.lastname: Blake
authors.forename: Douglas O
authors.initials: DO
authors.lastname: Clary
authors.forename: Peter M
authors.initials: PM
authors.lastname: Flanagan
mesh_terms: D001331:Automation; D001973:Bromodeoxyuridine / Q000378:metabolism; D002277:Carcinoma / Q000378:metabolism / Q000473:pathology; D002455:Cell Division / Q000502:physiology; D004797:Enzyme-Linked Immunosorbent Assay; D005453:Fluorescence; D006801:Humans; D007091:Image Processing, Computer-Assisted / Q000379:methods*; D007150:Immunohistochemistry / Q000379:methods*; D008175:Lung Neoplasms / Q000378:metabolism / Q000473:pathology; D008545:Melanoma / Q000378:metabolism / Q000473:pathology; D009363:Neoplasm Proteins / Q000032:analysis*; D009369:Neoplasms / Q000378:metabolism / Q000473:pathology*; D012680:Sensitivity and Specificity; D014407:Tumor Cells, Cultured
chemical_list: D009363:Neoplasm Proteins; D001973:Bromodeoxyuridine