Since current treatments for almost all patients with non-small-cell lung cancer are not satisfactory, and since prognosis is adversely affected by the overexpression of EGF receptors, we are attempting to identify new genes whose expression is modulated by the EGF signaling pathway in A549 lung carcinoma cells. The cells were treated with either EGF or C225 (a neutralizing antibody that blocks the EGF receptor). We have shown that these treatments affect the epithelial-to-mesenchymal transdifferentiation of these cells. We prepared complementary DNA libraries from EGF-treated, C225Ab-treated or control cells and subjected them to suppression subtractive hybridization to identify both up- and downregulated genes. These enriched cDNAs were fluorescently labeled and used to screen 9,600 human genes on a microarray. Thirty genes were upregulated and eighty downregulated by fourfold or more over background. To confirm subtraction efficiency, we picked approximately 700 clones from the C225-upregulated subtraction library; spotted their inserts, amplified by the polymerase chain reaction, on glass slides and hybridized them with the subtracted cDNAs. The majority of the clones exhibit high intensity when hybridized with the upregulated cDNAs and low intensity when hybridized with the downregulated cDNAs. The differential expression of these clones is now being confirmed by quantitative polymerase chain reaction with revserse transcription or northern blot analysis, and their function will be evaluated using cell differentiation and motility assays.