Article Figures & Data
Figures
- Figure 1.
Antiestrogen treatment and the induction of macroautophagy in viable MCF-7 cells. A, MCF-7 cells (detached versus adherent) were analyzed for autophagosome levels following treatment with E2 or E2 plus 4-OHT for 72 h. Detached and adherent cells were collected and independently processed for electron microscopic examination. B to D, nuclear morphology was examined for chromatin compaction in detached, 4-OHT–treated (B), adherent, 4-OHT–treated (C), and adherent, E2-treated cells (D). Irregularly clumped chromatin as seen in B is indicative of ACD II. The percentage of nonviable/viable nuclei in detached, 4-OHT–treated (B) adherent, 4-OHT–treated (C), and adherent, E2-treated (D) cell populations is provided at the bottom of each panel. For these experiments, 20 randomly chosen, detached, or adherent cells per treatment were analyzed in three independent experiments in a blind fashion. Statistically significant differences (P < 0.05) determined by the Student's t test are indicated as follows: *, statistically different compared with E2 treatment group; #, statistically different between the E2 plus 4-OHT treatment groups. Magnifications, ×7,700 (B), ×6,000 (C), and ×10,000 (D). Arrows, autophagosome structures; brackets, cytosolic area with increased vacuolation; N, nucleus; asterisk, condensed chromatin.
- Figure 2.
Macroautophagy: a caspase-independent prosurvival response in antiestrogen-sensitive breast cancer cells. A, treatment of MCF-7 cells with the pan-caspase inhibitor z-VAD-fmk blocked 4-OHT–induced cleavage of PARP and lamin A but did not inhibit 4-OHT–induced macroautophagy. For these experiments, MCF-7 cells were pretreated with z-VAD-fmk or vehicle (< 0.5% DMSO) for 1 h and then treated for an additional 72 and 96 h with 50 μmol/L z-VAD-fmk with or without E2 or E2 plus 4-OHT. At 72 h of treatment, the MDC sequestration assay was done as detailed in Materials and Methods. At 96 h of hormonal therapy, cells were harvested for protein and analyzed by immunoblotting. B, 3-MA treatment induced cell death and blocked autophagosome formation. Cells were treated with or without E2 and 10 mmol/L 3-MA for 120 h; detached (nonviable) and adherent (viable) cells were harvested independently for cell counts in the presence of trypan blue. C, Beclin 1 RNAi treatment induced cell death in MCF-7 cells. MCF-7 cells were treated with Beclin 1 RNAi for 48 h followed by treatment with E2 or E2 plus 4-OHT for 96 h. Protein lysates were harvested from the cells and analyzed for levels of Beclin 1, cleaved PARP, and cleaved lamin A. D, 4-OHT treatment induced caspase-6 activation. Caspase-6 activity profiles were determined using a commercially available kit according to the manufacturer's instructions (Calbiochem). Cleared lysates were corrected for equal protein concentration using the EZQ Protein Quantitation kit (Molecular Probes, Inc.) and incubated with fluorogenic tetrapeptide substrate Ac-LEHD-AFC for 2 h at 37°C to measure caspase-6 activity levels, respectively. Liberated AFC fluorescence was measured (400 nm excitation, 505 nm emission) using a TECAN microplate fluorometer and software. All experiments were done in triplicate and statistical significance (P < 0.05) was determined using the Student's t test: *, significance compared with the E2 treatment group; #, significant increase in the number of detached (nonviable) cells following 3-MA treatment.
- Figure 3.
Stepwise 4-OHT selection of MCF-7 cells and the establishment of 4-OHT–resistant sublines with increased autophagosome levels. A, stepwise 4-OHT selection used to establish the TR5 subline. Cell populations resistant to cell death induced by 1.5, 2, 3, 4, and 5 μmol/L were designated TR1.5, TR2, TR3, TR4, and TR5, respectively. B, a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay was done as previously described (32) and compared the dose response of TR5 with MCF-7 parent cells at 72 h of treatment with E2 plus varying concentrations of 4-OHT. C, high-level MDC sequestration and increased LC3 II (16 kDa) expression were seen in 4-OHT–treated TR5 cells compared with TR5 cells growing in E2 for 72 h or to 4-OHT–treated MCF-7 cells. *, statistically significant increase in MDC sequestration for 4-OHT–treated MCF-7, 4-OHT–treated TR5, and E2-treated TR5 cells compared with the E2-treated MCF-7 cells; #, statistically significant increase in MDC sequestration for 4-OHT–treated TR5 cells compared with 4-OHT–treated MCF-7 cells. D, a clonogenic assay showed reduced cloning efficiency of TR5 compared with MCF-7 cells. Cells (300, 500, 1,000, and 1,500) were seeded per 60-mm dish in DMEM/F12 medium with 5% DCC FBS plus E2 in quadruplicate. After 21 d, cells were stained with methylene blue (0.5% in 50% ethanol) and counted. All experiments were done three independent times and statistical significance (P < 0.05) was determined using the Student's t test.
- Figure 4.
4-OHT induces macroautophagy in 4-OHT–resistant TR5 cells. A and B, representative electron micrographs of 4-OHT–treated TR5 cells at ×6,000 and ×12,500, respectively. Examination by electron microscopy determined that the number of autophagosomes per 4-OHT–treated TR5 cell varied, with some cells having high numbers (75 per cell) and some cells having ∼10 per cell. The inset is a magnified (×21,500), 4-OHT–induced autophagosome in TR5 cells with lamellated structure indicative of active protein synthesis. N, nucleus; arrows, autophagosomes. Abundant mitochondria (designated by <) also are evident in the cells. C and D, LC3-GFP localization studies identified increased autophagosome formation in 4-OHT–treated TR5 cells compared with E2-treated TR5 cells. TR5 cells were transfected (transiently) with LC3-GFP followed by treatment with E2 or E2 plus 4-OHT (5.0 μmol/L). High-level punctate fluorescence of LC3-GFP in D is representative of 4-OHT–treated TR5 cells in comparison with E2-treated TR5 cells (C) and is consistent with the electron microscopic examination of these antiestrogen-resistant cells.
- Figure 5.
Blockade of autophagosome formation/function in 4-OHT–resistant TR5 cells and the induction of caspase-9 and cell death. A and B, E2-treated and E2 plus 4-OHT–treated TR5 cells were treated with 0, 5.0, or 10 mmol/L of 3-MA for 168 h to block autophagosome formation/function. Cell counts using trypan blue (TB) uptake as a measure of cell death were determined (A) and protein isolated from cells was subjected to immunoblotting (B). Increased levels of cleaved PARP, cleaved lamin A, and cleaved caspase-9 confirmed 3-MA induction of cell death. C, 3-MA treatment reduced autophagosome number in TR5 cells. TR5 cells were transfected with LC3-GFP followed by treatment with E2 or E2 plus 4-OHT for 36 h. Punctate fluorescence of LC3-GFP in TR5 cells was quantified by manual counting of 20 randomly selected cells in two independent experiments. The total number of autophagosomes per cell was decreased in 3-MA–treated TR5 cells. D, 3-MA reduced LC3 II (16 kDa) protein in 4-OHT–treated TR5 cells. Protein was isolated from cells treated with E2 plus 4-OHT with or without 3-MA for 48 h and analyzed by immunoblotting.
- Figure 6.
A prosurvival role for autophagosome function in 4-OHT–resistant TR5 cells. A, Beclin 1 RNAi targeting in antiestrogen-resistant TR5 cells reduced cell number and induced cell death. At 96 and 144 h of antiestrogen treatment, total cell number was determined and protein lysates were harvested from cells, respectively. Immunoblotting determined that PARP and lamin A cleavage were increased in 4-OHT–treated TR5 cells following Beclin 1 RNAi-mediated knockdown. B, TR5 cells were treated with either Beclin 1 RNAi or nontargeting RNAi for 72 h at which time E2 was added to the medium. Cells were harvested at 144 or 240 h for cell counts with trypan blue to determine the ratio of viable versus nonviable cells. Beclin 1 levels were also determined by immunoblotting at 240 h. C, caspase-9 inhibition by z-LEHD-fmk enhances MDC sequestration (macroautophagy) and blocks lamin A cleavage. MCF-7 cells were pretreated with vehicle (<0.5% DMSO) or z-LEHD-fmk for 1 h before a 72-h hormonal therapy with or without z-LEHD-fmk (50 μmol/L). Cleaved lamin A levels were reduced by 5-fold when z-LEHD-fmk was present during 4-OHT treatment of cells; signal intensity of 0.2 compared with an arbitrary value of 1.0 in 4-OHT–treated cells. MDC sequestration levels were determined as outlined in Materials and Methods. Columns, mean of three independent experiments; bars, SE. Statistical significance (P < 0.05) was determined using the Student's t test: *, statistical significance when compared with E2 treatment; #, significance when compared with E2 plus 4-OHT treatment. D, a schematic model in which the blockade of autophagosome function can induce death in the surviving ER+ breast cancer cell population due to the activation of caspase-9 and caspase-6, which then cleaves lamin A to facilitate nuclear destruction.
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Supplementary Material, Samaddar et al
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Volume 7, Issue 9
