MCF7 breast tumor cells overexpressing human c-Jun exhibit a transformed phenotype characterized not only by increased tumorigenicity but also by enhanced motility and invasion. The cellular phenotypic response to c-Jun overexpression is likely due, at least in part, to altered patterns of gene expression. In order to begin to understand the complexities by which elevated production of c-Jun alters the state of the cell, we have profiled the expression of 588 different genes by comparative hybridization. By using this approach, we have identified a total of 21 upregulated or downregulated gene targets responsive to c-Jun overexpression. Interestingly, 8 of these genes have been previously found associated with c-Jun or AP-I activity and therefore provide internal validation for this approach to target gene discovery. The remaining 13 genes represent potential new c-Jun regulated target genes. Genomic sequence information was available for 15 of the 21 genes identified in this screen. Analysis of these genomic sequences revealed the presence of AP-I or AP-I-like sequences in 12 of the 15 genes examined. Consistent with a direct mechanism of target regulation by c-Jun, gel shift analysis of selected AP-I-containing promoter regions revealed elevated and specific binding by proteins present in nuclear extracts of c-Jun expressing MCF7 cells. (C) 2000 Wiley-Liss, Inc.