Cell lines: MCF15 is a new human breast cancer cell line 51 (link). GI101Ap is a clone of GI101A cells kindly provided by Dr. Janet E. Price at the MD Anderson Cancer Center, the University of Texas, Houston. MCF10A cells were obtained from Karmanos Cancer Center (Detroit, Michigan), which is the former Michigan Cancer Foundation that developed the MCF series of cells, whereas MCF7, T47D and MDA-MB231 (MB231) human breast cancer cell lines were originally obtained from the American Type Culture Collection (ATCC). Among these cell lines, MCF7, MCF15 and T47D cells are estrogen receptor (ER) α negative in culture, T47D and MB231 cells have p53 mutations, whereas the ERα negative MB231 and ERα positive GI101Ap are two most commonly used metastatic cell lines, after MDA-MB435 is recently characterized to be of melanoma origin 52 (link). Moreover, MB231 cells are so-called triple-negative (estrogen receptor, progesterone receptor and Her-2 negative).
Reagents: Naphtho [2, 1-α] pyrrolo [3, 4-c] carbazole-5, 7 (6H, 12H)-dione (NPCD) (fig 1) was synthesized and purified by us at a purity of over 99% proved by HPLC. It was dissolved with dimethyl sulfoxide (DMSO) and kept at -20oC until use. Unless specified separately, primary antibodies used in this study were purchased from Santa Cruz Biotechnology Inc (Santa Cruz, CA), including mouse monoclonal anti-β-actin (sc-47778) as well as rabbit polyclonal anti-cyclin D1 (sc-718), anti-CDK4 (sc-260), anti-CDK6 (sc-7180), anti-CDK2 (sc-163), anti-cyclin E (sc-481), anti-Rb (sc-50), anti-pRb (Ser807/811; sc-16670), anti-p27 (sc-528), and anti-p21 (sc-397). The anti-pRb Ser780 (Cat#9307) rabbit polyclonal antibody was purchased from Cell Signaling Technology, Inc (Danvers, MA). Peroxidase-conjugated anti-mouse (NA931) and anti-rabbit (NA934) secondary antibodies were purchased from Amersham Biosciences (Piscataway, NJ).
3-(4, 5-Dimethylthiazol-2-yl)-2, 5-diphenylte­trazolium bromide (MTT) assay: Cells were seeded in a 96-well micro-plate at 4,000 cells per well, five wells per dose and per time point, and incubated at 37oC with 5% CO2. NPCD was added 24 hours later at indicated concentrations, with DMSO as non-treated control. The culture was continued for the indicated time period. At the end of the treatment, MTT was added into each well at a final concentration of 0.5mg/ml followed by incubation at 37oC for three hours in dark. The culture medium containing MTT was discarded and the dye crystals were dissolved in DMSO. The viable cells were detected by reading the absorbance of the metabolic MTT at wavelength 570nm using the Beckman Coulter AD340 absorbance detector (Beckman Coulter Inc, Fullerton, CA). The experiment was repeated at least three times to ensure the data reproducibility.
Clonogenic survival assay: Cells were seeded in 24-well culture plates at 1x105 cells per well and 24 hours later NPCD was added into the culture, with DMSO as non-treated control. The cells were trypsinized 72 hours after the NPCD treatment and evenly reseeded at a lower density (1x103 cells per well) in triplicate in a 6-well culture plate. Cells were maintained in culture for 10 to 15 days, with medium change every 3 days, to let the viable cells propagate to sizable colonies for quantification. The colonies were fixed with methanol-acetic acid at 3:1 ratio and then stained with 1% crystal violet for 30 minutes at room temperature. The number of colonies formed in each well was counted and photographed under the microscope. The experiment was repeated three times and the resulting data are presented after statistical processing.
Acridine orange and ethidium bromide staining: Cells were seeded in 96-well plates at 3,000 cells per well and incubated at 37oC with 5% CO2. NPCD at a final concentration of 2 or 4 µM was added 24 hours later in multiple repetitions (5 wells per dose); plates were incubated for an additional 24 or 48 hours. Prior to cell staining, the medium was discarded. The cells were washed with phosphate-buffered saline (PBS) first and then incubated with 10 µl PBS containing 10 µg/ml of Ethidium Bromide (EB) and 3 µg/ml of Acridine orange (AO). The cells stained by EB and AO were immediately visualized under the Leica DM IRB inverted fluorescence microscope (Leica Microsystems Inc. Bannockburn, IL). Multiple photos were taken at randomly-selected areas of the well to ensure that the data obtained are representative. Photographs were processed using “Image ProPlus 6.0” ultimate image analysis software (Media Cybernetics, Inc. Bethesda, MD).
Cell cycle analysis: Cells were cultured in 6-cm culture dishes until they reached 70~80% confluence. NPCD was given at an indicated concentration with DMSO as non-treated control; the cells were harvested 36 or 48 hours later. At the cessation of the treatment, both floating and adherent cells were collected, combined, washed with cold PBS, and then fixed overnight with 70% ethanol in PBS at -20oC. The cells were then washed with PBS again and incubated in the dark with in a PBS solution containing 20µg/ml propidium iodide (PI) and 200µg/ml RNase for 30 minutes at room temperature. The cells at different stages of the cell cycle were detected with a Becton Dickinson FACS Calibur flow cytometer (BD Biosciences, San Jose, CA). Intact cells were gated in the FSC/SSC plot to exclude small debris. The population of cells at different stages of the cell cycle was quantified using ModFit LT software (Verity Software House, Inc., Topsham, ME).
Cell death analysis: Annexin V and PI dual staining was used to visualize apoptotic and necrotic cells in a similar procedure as described above but the cells were not pre-fixed with 70% ethanol. Apoptotic cells were stained using the Annexin V-FITC Apoptosis Detection Kit (MBL International Corporation, Watertown, MA) following the manufacturer's instruction. All dead cells, i.e. both apoptotic and necrotic cells stained by Annexin V and PI, were analyzed by a Becton Dickinson FACS-Calibur flow cytometer (BD Biosciences, San Jose, CA), which places the Annexin V-FITC signal that detects apoptosis in FL1 and the PI-FITC signal that detects necrosis in FL2. Therefore, cells in the upper-left quadrant of the FL1/FL2 dot plot (labeled with PI-FITC only) as shown in figure 3 were necrotic, whereas cells in the bottom-right quadrant were early apoptotic. Cells in the upper-right quadrant (labeled with Annexin V-FITC and PI) were late apoptotic or necrotic.
Protein extraction and western blotting assay: Total protein samples or nuclear and cytoplasmic proteins were extracted as described by Andrew and Faller 53 (link), followed by determination of protein concentration using a Bradford assay (Bio-Rad Laboratories Inc. Hercules, CA). An equal amount of protein from each sample was fractioned in SDS-PAGE and then transferred onto an Immobilon-P Nylon membrane (Millipore, Bedford, MA) in a tank transfer system. After being blocked with 5% milk, the membrane was incubated with specific primary antibody at an optimized concentration and then with horseradish peroxidase-conjugated secondary antibody, with three washes between each antibody. The signal was visualized with ECL chemiluminescent substrates (Pierce, Rockford, IL) on X-ray film (ISC BioExpress, Kaysville, UT). The expression levels were quantified as the band density on X-film using ImageJ software, calculated as the ratio to the β-actin expressed in the same sample, and presented as number of “+”. +++, ++ and + indicate that the ratio to β-actin is larger than 0.75, between 0.5~0.75, and less then 0.5, respectively, whereas ± and (-) indicate a very low level of expression and the absence of the signal on the X-film, respectively. The Rb protein was used as a loading control for nuclear proteins in some of the western blot assays as reported in the literature 54 (link),55 (link).
Co-immunoprecipitation assay: D1-CDK4 complex formation was analyzed with co-immunoprecipitation assay. Total protein lysates (500µg) from each sample were immunoprecipitated (IP) in 400µl lysate buffer containing 2µl anti-cyclin D1 (sc-718) or anti-CDK4 (sc-260) rabbit polyclonal antibody and inhibitors of various proteases, phosphotases and kinases at 4oC for 4 hours with rotation. Protein A-conjugated agarose beads (25 µl) were then added into the IP reaction with an additional 5 hours of rotation at 4oC. The antigen-antibody complexes were precipitated by a quick centrifugation, followed by four times of wash with cold PBS. Proper controls included an aliquot of rabbit serum to replace the D1 or CDK4 antibody in the IP reaction. The pellets were suspended in 20µl 2xSDS reducing western blot loading buffer and boiled for 5 minutes, followed by SDS-PAGE. The D1- or CDK4-immunoprecipitates were subjected to western blot assay to detect the D1 or CDK4 in the immunoprecipitates.
RT-PCR assay: Total RNA samples were extracted from cultured cells using TRIzol (Invitrogen, Cat. 15596-026) in a routine procedure, followed by DNaseI treatment to get rid of DNA residual. An aliquot (2.5µg) of the RNA samples was then reverse transcribed (RT) in a 25µl reaction solution to the first strand of cDNA using hexamer primer. The RT products were diluted with water to the final volume of 50 µl. For PCR amplification of each target gene, 1 µl of the diluted RT products was used as template, but the template was further diluted 10 times for PCR amplification of β-actin so as to control the PCR amplification of this highly expressed gene at the linear portion. The PCR condition (table 1) was optimized for each gene and was stopped within the linear portion of the amplification. The forward and reverse primers for each gene, listed in table 1, were designed in such a way that they are localized at two different exons of the given gene with one or several large introns in between. In this way if there still is a traceable amount of DNA residual, it either cannot be amplified due to large intron(s) or is amplified as a molecule larger than the expected size indicated in table 1. PCR products were separated and visualized in 1% agarose gel.
Statistic analyses: All MTT, FACS and clonogenic survival assays were performed in multiple repetitions in each experiment, and the experiments were repeated at least three times. The data were presented as mean ± SE. Statistical comparisons between groups were made with the student's t-test. A P value < 0.05 is considered as significant.