1. Volume 24, Issue 1, Pages 120-129 (July 2013)
Targeting BCL-2 with the BH3 Mimetic ABT-199 in Estrogen Receptor-Positive Breast Cancer 
François Vaillant, Delphine Merino, Lily Lee, Kelsey Breslin, Bhupinder Pal, Matthew E. Ritchie, Gordon K. Smyth, Michael Christie, Louisa J. Phillipson, Christopher J. Burns, G. Bruce Mann, Jane E. Visvader, Geoffrey J. Lindeman 
Cancer Cell 
Volume 24, Issue 1, Pages (July 2013)
DOI: /j.ccr
Copyright © 2013 Elsevier Inc. Terms and Conditions

2. Figure 1
BCL-2 and BCL-XL Expression in Breast Cancer and Primary Xenograft Tumors
(A) Box-plots of BCL2 and BCL2L1 expression in breast tumor subtypes.
(B) Immunostaining for ER, PR, BCL-2, and Ki-67 expression in passage 2 breast tumor xenografts 23T, 315T, 50T, and 838T. Percent Ki-67 scores were 33% ± 3%, 37% ± 3%, 63% ± 3%, and 47% ± 9%, respectively (mean ± SEM of three tumors). Inset: BCL-2 control (no primary antibody). Scale bar, 50 μm.
(C) Western blot analysis of ER, BCL-2, MCL-1, BCL-XL, BIM, NOXA, PUMA, p53, AKT, pAKT, and PI3K expression in tumor xenografts. Actin was used as a loading control.
See also Figure S1 and Table S1.
Cancer Cell  , DOI: ( /j.ccr )
Copyright © 2013 Elsevier Inc. Terms and Conditions

3. Figure 2
The BH3 Mimetic ABT-737 Improves Tumor Response to Tamoxifen in BCL-2-Expressing ER-Positive Primary Xenograft Tumors
(A) Kaplan-Meier survival curves of mice bearing 23T (n = 6–8 per arm), 315T (n = 5–8 per arm), 50T (n = 5–7 per arm), and 838T (n = 6 or 7 per arm) tumors treated with vehicle alone, ABT-737, tamoxifen (administered by s.c. injection for 23T and slow release pellet for 315T, 50T, and 838T models), and combined tamoxifen and ABT-737. Log rank (Mantel-Cox) p values are shown for combination therapy versus tamoxifen alone.
(B) Cleaved caspase-3 immunostaining following 48 hr treatment. The histogram shows the number of cleaved caspase-3-positive cells per 1.5 mm2 field. Values represent the mean ± SEM (n = 3; two or three fields per tumor).
(C) Immunostaining of bromodeoxyuridine (BrdU) uptake in 23T tumors 48 hr after the treatment. Histogram shows number of BrdU-positive cells per 1.5 mm2 field. Values represent the mean ± SEM (n = 3; two or three fields per tumor). Scale bars, 50 μm. One asterisk denotes p < 0.05; two asterisks denote p < 0.005; three asterisks denote p < 0.001; and four asterisks denote p < NS, not significant (values compared to vehicle control).
See also Figure S2.
Cancer Cell  , DOI: ( /j.ccr )
Copyright © 2013 Elsevier Inc. Terms and Conditions

4. Figure 3
Tamoxifen Treatment Increases BCL-2 and Sensitizes Cells to ABT-199
(A) Western blot analysis showing BCL-2, BCL-XL, MCL-1, and BIM levels in 23T tumors from mice treated for 48 hr with vehicle, tamoxifen (Tam, 0.5 mg in oil, s.c., daily), and/or ABT-737 (50 mg/kg, i.p., daily). Actin was used as a loading control.
(B) Western blot of BCL-2 and BIM following immunoprecipitation of BCL-2. Lysates were from 23T tumor cells treated in vitro for 24 hr in the presence or absence of tamoxifen (10 μM) and 5 hr in the presence or absence of ABT-737 (5 μM). Lane 1, no antibody control.
(C) 23T, 315T, and 50T tumor cells were cultured for 24 hr in mammosphere media in the presence of ABT-737 (1 μM) or ABT-199 (1 μM) with or without tamoxifen (10 μM) and viability determined as compared to no treatment (NT). Shown are the mean ± SEM of triplicates from three independent experiments. One asterisk denotes p < 0.05 and two asterisks denote p <
(D) Mice bearing 23T, 315T, and 50T tumors (n = 6–8 per arm) were treated with vehicle, ABT-199, tamoxifen, combined tamoxifen, and ABT-737 or combined tamoxifen and ABT-199 and tumor volume measured. Values represent the mean ± SEM.
See also Figure S3 and Table S2.
Cancer Cell  , DOI: ( /j.ccr )
Copyright © 2013 Elsevier Inc. Terms and Conditions

5. Figure 4
Combination Therapy with ABT-737 or ABT-199 Counteracts Tamoxifen-Associated Endometrial Hyperplasia
(A) Images of uterine horns (with ovaries at superior poles) in NOD-SCID-IL2Rγc−/− mice following 10 day treatment with vehicle, ABT-737 (50 mg/kg, i.p., daily), tamoxifen (0.5 mg in oil, s.c., daily), or ABT-737 and tamoxifen. Tissue from ABT-737-treated animals shows a slight yellow tinge. Scale bar, 1 cm.
(B) Hematoxylin and eosin staining of transverse sections of the miduterine horn following 10 days of treatment. Scale bar, 500 μm.
(C) Histogram showing mean endometrial diameter (±SEM) measured perpendicular to the uterine ligament. (n = 6 mice per group; three or four sections per animal). The size and morphology of the myometrium and serosal layer were not altered. The asterisk denotes p <
(D) Cleaved caspase-3 immunostaining of uterine sections. Arrows show apoptotic cells shed into the endometrial lumina following combined treatment. Scale bar, 100 μm.
Cancer Cell  , DOI: ( /j.ccr )
Copyright © 2013 Elsevier Inc. Terms and Conditions

6. Figure 5
ABT-737 or ABT-199 Synergizes with mTOR Inhibitors to Induce Apoptosis
(A) MCF-7 cells were treated with tamoxifen (10 μM), ABT-737 (1 μM), and/or AZD8055 (1 μM) for 24 hr and then subjected to toxicity assays using phosphatidylinositol (PI) staining.
(B) MCF-7 cells were treated with increasing concentrations of AZD8055 and ABT-737 for 24 hr and then subjected to toxicity assays using Cell Titer Glo followed by BLISS score analysis. BLISS synergy values are >0 on the vertical z axis.
(C) Western blot analysis of lysates from MCF-7 cells treated with 1 μM of ABT-737 or AZD8055. Actin served as a loading control.
(D) MCF-7 cells were treated with ABT-737 or ABT-199 (1 μM) and/or AZD8055 (1 μM) for 24 hr prior to performing toxicity assays using PI staining.
(E) 315T cells were treated for 24 hr with the drug combinations as indicated (n = 3). BH3 mimetics (ABT-737 and ABT-199) and PI3K/mTOR inhibitors (PKI-587, AZD8055, and BEZ235) were applied at 1 μM. For (A), (D), and (E), the mean ± SEM is shown for three independent experiments.
(F) Kaplan-Meier survival curve of mice bearing 315T tumors (n = 7 or 8 per arm) treated with vehicle, PKI-587, tamoxifen + ABT-737, and tamoxifen + ABT-737 + PKI-587. Log rank (Mantel-Cox) p value is shown for triple therapy versus tamoxifen + ABT-737.
See also Figure S4.
Cancer Cell  , DOI: ( /j.ccr )
Copyright © 2013 Elsevier Inc. Terms and Conditions