DKFZ DNA replication assay Bromodeoxyuridine incorporation assay Cancer transcription microarray studies commonly deliver long lists of "candidate" genes that are putatively associated with the respective disease. For many of these genes, no functional information, even less their relevance in pathologic conditions, is established as they were identified in large-scale genomics approaches. Strategies and tools are thus needed to distinguish genes and proteins with mere tumor association from those causally related to cancer. Here, we describe a functional profiling approach, where we analyzed 103 previously uncharacterized genes in cancer relevant assays that probed their effects on DNA replication (cell proliferation). The genes had previously been identified as differentially expressed in genome-wide microarray studies of tumors. Using an automated high-throughput assay with single-cell resolution, we discovered seven activators and nine repressors of DNA replication. These were further characterized for effects on extracellular signal-regulated kinase 1/2 (ERK1/2) signaling (G(1)-S transition) and anchorage-independent growth (tumorigenicity). One activator and one inhibitor protein of ERK1/2 activation and three repressors of anchorage-independent growth were identified. Data from tumor and functional profiling make these proteins novel prime candidates for further in-depth study of their roles in cancer development and progression. We have established a novel functional profiling strategy that links genomics to cell biology and showed its potential for discerning cancer relevant modulators of the cell cycle in the candidate lists from microarray studies. flask Nunc 24 Well Plate Nunc 24 Well Plate Nunc DMEM high glucose Invitrogen 31053-028 1235371 fbs1 PenStrep1 NEAH1 lglutamine1 l-glutamine Invitrogen 25030-024 FBS Calf Bovine Serum ATCC 30-2030 Pen/Strep Invitrogen 15070-063 NEAH Invitrogen 11140-035 NIH3T3 ATCC mouse Mus musculus embryo fibroblast adherent cDNA full length cDNA clone cDNA full length cDNA clone Olympus BX high-content screening microscope Olympus flask DMEM high glucose Transfection cDNA CellCulture1 24WellSeedAction Perturbation WashPBSAction1 24WellSeedAction DetachCellsAction StainCellsAction DEMhigh1 Array1 p1 p2