The molecular basis for the enhanced growth inhibition of MCF-7 human breast cancer xenografts by a combination of human interferon-beta (IFN-beta) and tamoxifen was investigated. Treatment of MCF-7, MDA-MB-231, and BT-20 cells with the combination of IFN-beta and tamoxifen resulted in enhanced antiproliferative effects in vitro. Treatment with the combination of IFN-beta and tamoxifen enhanced the expression of several IFN-beta-inducible genes in human breast carcinoma cell lines relative to levels induced by IFN-beta alone. Tamoxifen alone did not induce transcription of IFN-stimulated genes (ISGs). Augmentation of ISG expression by the combination of IFN-beta and tamoxifen was noted in breast tumor cell lines irrespective of their functional estrogen receptor (ER) status or their dependence on estradiol for growth, suggesting that upregulation of ISGs was independent of ER status. Enhancement of IFN-stimulated gene expression by tamoxifen occurred at the transcriptional level. Expression of transfected reporter genes under the control of IFN-alpha/beta regulated promoters was also enhanced in IFN-beta and tamoxifen-treated cells. Similarly, transcriptional induction of chimeric reporter plasmids driven by an IFN-gamma inducible promoter (GAS; IFN-gamma activated site) was also enhanced by the combination of IFN-gamma and tamoxifen. In tamoxifen treated cells, IFN-beta and IFN-gamma readily activated transcription factors ISGF-3 and GAF, respectively. Therefore, augmentation of ISG expression by tamoxifen is an early event in the antitumoral activity of this drug combination.