3.1.1 Cell culture of adherent cells
MCF-7 (human mammary epithelial adenocarcinoma), A549 (human alveolar carcinoma) and H1299 (non-small cell lung carcinoma) cells were routinely cultured in phenol red-free high-glucose Dulbecco‟s modified Eagle‟s medium (DMEM) supplemented with 10% fetal bovine serum (FBS), 100 units/ml penicillin, 100 µg/ml streptomycin and 1 mM sodium pyruvate (“normal medium”) at 37 °C under 5% CO2 atmosphere. One or two days prior to hormone treatment, growth medium was changed to high-glucose DMEM containing 5% charcoal-dextran treated FBS (CSS), 100 units/ml penicillin, 100 µg/ml streptomycin and 1 mM sodium pyruvate (“stripped medium”).
For hormone treatment, MCF-7 cells were treated with 10 nM 17ß-Estradiol and A549 cells either with 100 nM Dexamethasone or solvent (100% EtOH). For blocking proteasome activity, cells were pre-treated for 15 min with one of the three following chemical proteasome inhibitors: 50 nM Bortezomib, 20 µM MG-132 or 1 µM Epoxomicin.
3.1.2 Liposome-mediated plasmid transfection
A day before transfection cells were plated in 6-well plates so that they were approximately 70-80% confluent on the day of transfection. The cells were transfected with LipofectamineTM 2000 as recommended by the manufacturer. Briefly, cell growth medium was removed and after washing twice with PBS, 2 ml of Opti-MEM without any supplements were added. Cells were put back in the incubator. For each transfection 200 µl of Opti-MEM and 2.4 µg of total plasmid DNA were mixed in a reaction tube. In a second tube 200 µl Opti-MEM and 8 µl of LipofectamineTM 2000 were mixed and incubated for 5 min at RT. After combining the contents of both tubes and mixing gently by inverting, the samples were incubated for another 20 min at RT. The transfection mix was then added to the respective wells containing the cells and 2 ml of Opti-MEM. After incubating the cells for 4 h at 37 °C, transfection medium was removed, the cells washed twice with PBS and hormone-deprived “stripped medium” was added. 24 h following transfection, the cells were treated with proteasome inhibitors or hormones for the indicated time periods.
Methods
38 3.1.3 Reverse-transfection with siRNA
Reverse-siRNA transfections were performed using LipofectamineTM RNAiMAX according to the manufacturer‟s instructions. Briefly, 30 pmol of the respective siRNA were diluted in 500 µl of Opti-MEM medium in a well of a 6-well plate. 5 µl LipofectamineTM RNAiMAX were added and incubated for 10-20 min at RT. In the meantime MCF-7 or A549 cells were trypsinized and diluted in “normal medium” without antibiotics (DMEM supplemented with 10% FBS and 1 mM sodium pyruvate) so that 2.5 ml contain 250,000 cells (MCF-7) or 100,000 cells (A549). 2.5 ml of the diluted cells were added to each well already containing the siRNA-LipofectamineTM RNAiMAX complexes. After 24 h, medium was changed to hormone-deprived “stripped medium” and after another one or two days, hormone treatment was performed.
3.1.4 Colony formation assay
20,000 MCF-7 cells per well were seeded in a 6-well plate and cultured overnight in “normal medium”. In order to analyze the effects of estrogen on the proliferation of breast cancer cells, growth medium was replaced by hormone-deprived “stripped medium” the next day. Then, cells were treated with 10 nM 17ß-Estradiol and 50 nM Bortezomib for 6 days. Subsequently, colonies were fixed with 70% methanol for 30 min on ice and then stained with 0.1% crystal violet solution overnight. Stained colonies were rinsed thoroughly with water and then scanned with CanoScan 8600F (Canon).
3.1.5 Measurement of DNA of single cells by flow cytometry
In order to analyze the effects of various treatments on cell cycle progression, DNA content was measured via flow cytometry. MCF-7 cells were washed twice with PBS, harvested by trypsinization and pelleted by centrifugation. After resuspending the cells in 0.5 ml of PBS++, ice-cold ethanol was added dropwise to a final concentration of 75% ethanol. Cells were fixed overnight at 4 °C. Before staining, the cells were centrifuged, rehydrated in PBS++ for 10 min, resuspended in PBS++ containing 0.5 mg/ml RNAse A and incubated at 37 °C for 30 min.
Cells were stained with 15 µl propidium iodide (1 mg/ml) and flow cytometry analysis was performed using the Guava EasyCyte plus FACScan. About 10,000 cells were analyzed for each sample. Distribution of cells in distinct cell cycle phases was determined and graphically displayed using ModFIT cell cycle analysis software.
Methods
39 3.1.6 Apoptosis assay
The Guava Nexin® assay (Millipore) is based on two distinct cellular alterations of the apoptotic process. Shortly after induction of apoptosis, phosphatidylserine is translocated from the inner side of the cell membrane to the outer cell surface when the cell membrane still remains intact. Annexin V-PE, an annexin group protein labeled with phycoerythrin, binds in a calcium-dependent manner to phosphatidylserine. Alteration in plasma membrane integrity can be evaluated by staining with 7-AAD which is excluded from intact cells.
Cell growth medium was collected in a 15 ml falcon tube. Cells were washed twice with PBS, trypsinized and detached. Free as well as trypsinized cells were added to the Falcon tube containing the growth medium. After centrifuging at 500×g for 7 min, the supernatant was discarded and cell pellets were resuspended in 500 µl of medium. Cells were counted and diluted to a concentration of 2× 105 - 1× 106 cells/ml. 100 µl of each diluted cell suspension and 100 µl of Guava Nexin solution were mixed in a well of a 96-well plate. After incubation in the dark for 20 min, the samples were analyzed using the Guava FACScan.
3.1.7 Fluorescence recovery after photobleaching (FRAP)
MCF-7 cells were seeded onto 18 mm glass coverslips so that they reached a confluence of 70-80% the next day when transfection with an EGFP-hERα fusion construct using LipofectamineTM 2000 reagent was performed. 4 h after transfection, the medium was changed to hormone-deprived “stripped” medium. The actual FRAP analysis was carried out together with Dr. Chieh Hsu in the laboratory of Prof. Dr. Simons at the MPI for Experimental Medicine, Göttingen. One day before FRAP analysis, the transfected cells were transferred to the MPI and put in an incubator at 37 °C under 7% CO2 atmosphere. Cells were pre-treated with 50 nM Bortezomib or vehicle for 15 min followed by a 6 h treatment with 10 nM 17ß-Estradiol and then analyzed via FRAP assay. The 8-bit time-lapse images of the cells were acquired at a frame rate of 3 frames/s at 37 °C in 500 µl of medium with a confocal microscope set-up and its software (TCS SP2 AOBS; Leica) with a 40× oil immersion objective (Type HCX PL Apo CS, NA 1.25; Leica). The pinhole was set at 81.40 µm and the zoom-factor was adjusted to obtain 62.5 × 62.5 µm images with voxel size of 244.26 × 244.26 nm. After recording 10 images, the GFP signal in a 13.2 × 2.9 µm region within a nucleus was bleached with 488 nm laser beam and 120 post-bleaching images were taken. To correct the intensity from imaging bleaching and background signal in the bleached region of the post-bleaching series, the formula
Methods
40
was used where represents the corrected intensity at a certain time point,
represents the original intensity, is the average of background signal in the series and
is the GFP signal from another nucleus in the image field. Using the software, SigmaPlot (Sytstat Software), the fluorescence recovery of the bleached region was then fitted with a simple exponential function with three variables, , where is the theoretical maximum intensity and is the time constant. The significance of the data was evaluated with R (software environment for statistical computing and graphics).