Tamoxifen induction of CCAAT enhancer-binding protein α is required for tamoxifen-induced apoptosis

Low concentrations of tamoxifen or its active metabolite 4-hydroxytamoxifen (OHT) induce estrogen receptor alpha(ER alpha)-dependent apoptosis. To analyze the pathway of OHT-ER alpha-induced apoptosis, we developed stably transfected lines of HeLa cells expressing wild-type ER and an inactive mutant ER alpha unable to bind estrogen response elements. HeLa cells expressing the mutant ER alpha and HeLa cells expressing wild-type ER alpha in which the ER was knocked down with an ER-specific small interfering RNA were not killed by Tam or OHT, suggesting that estrogen response element-mediated transcription is required for Tam- and OHT-induced apoptosis. Microarray analysis to identify a gene(s) whose expression is important in OHT-ER-mediated apoptosis identified 19 mRNAs that OHT up-regulated by > 1.6-fold and 15 down-regulated mRNAs. Gene function and the time course of induction by OHT-ER alpha led us to further investigate CCAAT enhancer-binding protein alpha (C/EBP alpha), which has roles in cell cycle progression and apoptosis, and p21. Quantitative reverse transcription-PCR, Western blot analysis, and RNA interference knockdown suggest that cell cycle arrest resulting from OHT-ER alpha induction of p21 may facilitate apoptosis. OHT-ER alpha, but not E-2-ER alpha, induced C/EBP alpha mRNA and protein. RNA interference knockdown of C/EBP alpha nearly abolished OHT-ER alpha-induced apoptosis. We isolated stable cell lines that were resistant to OHT-induced apoptosis, contain full-length functional ER alpha, and undergo apoptosis in response to etoposide. In these OHT-resistant cell lines both before and after OHT treatment, C/EBP alpha levels are much lower than in OHT-sensitive cells. These studies establish a novel molecular site responsible for Tam- and OHT-ER alpha-induced apoptosis of cancer cells.