Overexpression of the HER2/neu oncogene is associated with tumorigenicity and drug resistance in many types of cancer. Three different human Ewing's sarcoma cell lines (TC71, RD, and A4573) were found to express high levels of the HER2/neu protein. Transduction of TC71 cells with the E1A gene using an adenoviral vector (Ad-E1A) down-regulated HER2/neu overexpression in those cells and increased cytostasis. E1A-induced apoptosis was demonstrated by both flow cytometric analysis and Western blot analysis using a poly(ADP-ribose) polymerase antibody. After transduction of the E1A gene into these cells, the sensitivity of these cells to VP-16 (etoposide) was enhanced 18-fold and to Adriamycin 5-fold. However, no change was seen in cisplatin sensitivity. E1A also significantly increased topoisomerase IIalpha protein expression, indicating that the up-regulation of topoisomerase IIalpha may be one of the mechanisms by which E1A enhanced the sensitivity to topoisomerase II-targeting anticancer drugs, such as VP-16 and Adriamycin, but not cisplatin. In summary, these studies demonstrated that Ad-E1A can down-regulate HER2/neu overexpression and up-regulate topoisomerase IIalpha expression in human Ewing's sarcoma cells, increasing their apoptosis rate and enhancing their sensitivity to VP-16 and ADRIAMYCIN: