Tamoxifen induction of CCAAT enhancer-binding protein alpha is required for tamoxifen-induced apoptosis.
Low concentrations of tamoxifen or its active metabolite 4-hydroxytamoxifen (OHT) induce estrogen receptor alpha (ERalpha)-dependent apoptosis. To analyze the pathway of OHT-ERalpha-induced apoptosis, we developed stably transfected lines of HeLa cells expressing wild-type ER and an inactive mutant ERalpha unable to bind estrogen response elements. HeLa cells expressing the mutant ERalpha and HeLa cells expressing wild-type ERalpha 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-ERalpha led us to further investigate CCAAT enhancer-binding protein alpha (C/EBPalpha), 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-ERalpha induction of p21 may facilitate apoptosis. OHT-ERalpha, but not E2-ERalpha, induced C/EBPalpha mRNA and protein. RNA interference knockdown of C/EBPalpha nearly abolished OHT-ERalpha-induced apoptosis. We isolated stable cell lines that were resistant to OHT-induced apoptosis, contain full-length functional ERalpha, and undergo apoptosis in response to etoposide. In these OHT-resistant cell lines both before and after OHT treatment, C/EBPalpha levels are much lower than in OHT-sensitive cells. These studies establish a novel molecular site responsible for Tam- and OHT-ERalpha-induced apoptosis of cancer cells.
Cheng
Jingwei
J
Department of Biochemistry, University of Illinois, Urbana, Illinois 61801, USA.
Yu
David V
DV
Zhou
Jian-Hua
JH
Shapiro
David J
DJ
D000972:Antineoplastic Agents, Phytogenic / Q000494:pharmacology; D017209:Apoptosis / Q000187:drug effects* / Q000235:genetics; D022763:CCAAT-Enhancer-Binding Protein-alpha / Q000235:genetics / Q000378:metabolism*; D004965:Estrogen Antagonists / Q000494:pharmacology*; D047628:Estrogen Receptor alpha / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D005047:Etoposide / Q000494:pharmacology; D020869:Gene Expression Profiling; D005786:Gene Expression Regulation / Q000187:drug effects / Q000235:genetics; D006367:HeLa Cells; D006801:Humans; D009154:Mutation; D020411:Oligonucleotide Array Sequence Analysis; D034741:RNA, Small Interfering / Q000494:pharmacology; D013629:Tamoxifen / Q000031:analogs & derivatives* / Q000494:pharmacology
D000972:Antineoplastic Agents, Phytogenic; D022763:CCAAT-Enhancer-Binding Protein-alpha; D004965:Estrogen Antagonists; D047628:Estrogen Receptor alpha; D034741:RNA, Small Interfering; D013629:Tamoxifen; C016601:afimoxifene; D005047:Etoposide