An automated image capture and quantitation approach to identify proteins affecting tumor cell proliferation.
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.
Bhawe
Kaumudi M
KM
Target Discovery Department, Sugen, Inc. (A Subsidiary of Pfizer), South San Francisco, CA, USA. kbhawe@exelixis.com
Blake
Robert A
RA
Clary
Douglas O
DO
Flanagan
Peter M
PM
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
D009363:Neoplasm Proteins; D001973:Bromodeoxyuridine