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. Author manuscript; available in PMC: 2009 Aug 1.
Published in final edited form as: Cancer Res. 2008 Aug 1;68(15):6387–6395. doi: 10.1158/0008-5472.CAN-08-0538

Figure 3.

Figure 3

Tamoxifen activates the proapoptotic 4ICD BH3-only protein. A, Tamoxifen stimulates mitochondrial accumulation of the 4ICD BH3-only protein. T47D cells were cultured in phenol red-free RPMI containing 5% charcoal-stripped FBS for 48 hrs then were left untreated or treated with 100 pM 17-β-estradiol (Estrogen) alone or in combination with 5 μM 4-hydroxytamoxifen (Tamoxifen) for 1 hr. Mitochondria were isolated and analyzed by western blot for accumulation of 4ICD. Analysis of TOM40 was included as a loading control. B, Inhibitors of γ-secretase and BCL-2 expression suppress tamoxifen induced apoptosis of T47D cells. T47D cells were cultured in phenol red-free RPMI containing 5% charcoal-stripped FBS for 48 hrs and treated with 100 pM 17-β-estradiol (Estrogen) alone or in combination with 5 μM 4-hydroxytamoxifen (Tamoxifen) for 48 hrs. HER4 processing to generate 4ICD was prevented by the addition of 20 nM γ-secretase Inhibitor XXI to the 17-β-estradiol and tamoxifen treatments. Apoptosis was determined visually in a Nikon fluorescent microscope by staining the cells with DAPI and determining the percentage of cells with condensed nuclei (mean +/- SE of at least three experiments). C, Tamoxifen stimulates dimerized activation of mitochondrial BAK. T47D cells were cultured in phenol red-free RPMI containing 5% charcoal-stripped FBS for 48 hrs then were left untreated or treated with 50 ng/ml of heregulin β1 (HRG), or 100 pM 17-β-estradiol (Estrogen) alone or in combination with 5 μM 4-hydroxytamoxifen (Tamoxifen) for 1 hr. Mitochondrial extracts were treated with 1 mM of BMH crosslinker for 30 min. at room temperature and BAK monomers or cross-linked activated BAK dimers were detected by western blot analysis. The HER4 ligand HRG and tamoxifen independently activated dimerization of the mitochondrial dysfunction gateway protein BAK. D, Tamoxifen fails to activate BAX. T47D cells were cultured in phenol red-free RPMI containing 5% charcoal-stripped FBS for 48 hrs then left untreated or treated with 1 μM staurosporine (STS) for 12 hrs, 50 ng/ml of heregulin β1 (HRG), or 100 pM 17-β-estradiol (Estrogen) alone or in combination with 5 μM 4-hydroxytamoxifen (Tamoxifen) for 1 hr. Mitochondrial extracts were analyzed by western blot for BAX dimerized activation.