MDA MB-231 human breast adenocarcinoma cells were obtained from the American Type Culture Collection (Rockville, MD). They were routinely cultured in RPMI 1640 medium. HCT116 parental and p21 (-/-) cells (kindly provided by Dr. Bert Volgestein) were kept in Dulbecco's modified Eagle's medium. All media were supplemented with 10% fetal bovine serum and 50 µg/ml penicillin/streptomycin. Cells were incubated at 37°C with 95% air and 5% CO2. The medium was changed every 3 to 4 days.
AP-2α cDNA (provided by Dr. Trevor Williams [2
]) was subcloned into a pcDNA3 mammalian expression vector (Invitrogen, Carlsbad, CA). The AP-2γ expression vector AP-2γ pcDNA3 was generously provided by Dr. Ronald J. Weigel [19
]. Ad-AP-2α and Ad-AP-2γ adenoviruses were produced at the University of Iowa's Gene Transfer Vector Core Facility. The corresponding vector control was Ad-Bgl II, which contains only the adenovirus backbone. All adenovirus stocks were maintained at the University of Iowa's Vector Core Facility, where infectious particles were also amplified, purified, and characterized.
Clonogenic Survival Assay
We infected MDA MB-231 cells with 100 multiplicity of infection (MOI) of Ad-Bgl II, Ad-AP-2α, or Ad-AP-2γ. Twenty-four hours after infection, the total number of cells was determined using a Coulter counter (Beckman Coulter, Inc., Fullerton, CA). The number of cells plated into each cell culture dish was adjusted to give about 50 to 100 surviving colonies per dish. The cells were incubated at 37°C for 14 days to form colonies. The colonies were then fixed and stained by gentle addition of crystal violet to the medium in the cell culture dish. Colonies containing more than 50 cells were counted as surviving clonogenic cells, and the surviving fraction was normalized to the plating efficiency, as given below
Nuclear Protein Extraction
Nuclear extracts were prepared according to the method of Zhu etal. [34
], as previously described. Briefly, the medium was removed from tissue culture dishes. Cells were washed twice with phosphate-buffered saline (PBS) and scrape-harvested in 500 µl of ice-cold hypotonic buffer (10 mM HEPES, 1.5 mM MgCl2
, 10 mM KCl, and 0.5 mM DTT). Cells were incubated on ice for 20 minutes, lysed by a glass dounce homogenizer with type B pestle, and centrifuged at 300g
for 5 minutes. Supernatants were saved as cytosolic proteins, and 40 µl of ice-cold high-salt buffer (20 mM HEPES, 25% glycerol, 0.42 M NaCl, 1.5 mM MgCl2
, 0.2 mM EDTA, 0.5 mM PMSF, and 0.5 mM DTT) was added to the nuclear pellets and mixed. The nuclei were incubated on ice for 15 minutes and centrifuged at maximum speed for 1 minute. Supernatants were saved as nuclear protein extracts. Protein concentrations were determined with Bio-Rad DC protein assay (Bio-Rad Laboratories, Hercules, CA), according to the manufacturer's instructions.
Total Protein Extraction
Cells were collected by trypsinization and centrifugation and incubated in RIPA buffer [150 mM NaCl; 50 mM Tris, pH 8.0; 1 mM EDTA; 0.5% NP40; 0.1 mM Na3VO4; 1% sodium deoxycholic acid; and 0.1% sodium dodecyl sulfate (SDS)] for 30 minutes on ice. After 15 seconds of sonication, cell lysates were centrifuged for 1 minute at 13,000 rpm. Supernatants were saved as total protein extracts.
Western Blot Analysis
Equal amounts of proteins from differently treated cells were size-fractionated on a 10% (pRb) or a 12% (all other proteins) Tris-HCI polyacrylamide ready gel (Bio-Rad Laboratories). Separated proteins were then electrotransferred to a nitrocellulose membrane (Schleicher and Schuell, Keene, NH) by running at 100 V for 1 hour (6 hours for pRb). For AP-2α and AP-2γ Western blot analyses, the primary antibodies were mouse anti-AP-2α IgG and mouse anti-AP-2γ-IgG, respectively (Santa Cruz Biotechnology, Santa Cruz, CA), both used at 1:400 dilution. For p21 Western blot analyses, the primary antibody was mouse anti-p21 IgG (Pharmingen, San Diego, CA), used at a dilution of 1:250. For phospho-Rb (pS780) Western blot analyses, the primary antibody was rabbit anti-pRb (pS780) IgG (Cell Signaling Technology, Beverly, MA), used at a dilution of 1:1000. For cyclin D1 Western blot analyses, the primary antibody was mouse anti-cyclin D1 IgG (Pharmingen), used at a dilution of 1:1500. For β-actin Western blot analyses, the primary antibody was mouse anti-human β-actin IgG (Santa Cruz Biotechnology), used at a dilution of 1:3000. For AP-2, cyclin D1, and actin Western blot analyses, the secondary antibody was goat anti-mouse IgG (Pharmingen), used at a 1:10,000 dilution. For p21 and pRb (pS780), the secondary antibody was used at 1:3000 dilution. Blots were washed with TTBS (0.02 M Tris-HCI buffer, pH 7.5; 0.137 M NaCl; and 0.1% Tween 20). Detection by chemiluminescence reaction was carried out using an enhanced chemiluminescence kit (Amersham Pharmacia Biotech, Piscataway, NJ), followed by exposure to Kodak X-ray film (Kodak, Rochester, NY).
Electrophoretic Gel Mobility Shift Assay (EMSA)
We used double-stranded oligodeoxynucleotides containing a consensus AP-2-binding sequence (Promega, Madison, WI). The probe was made by labeling these double-stranded oligonucleotides with [γ-32P] ATP using T4 polynucleotide kinase. After that, 10 µg of nuclear protein was incubated with the 32P-radiolabeled oligonucleotide probe in the presence of 1 mg of poly(dIdC) (Amersham Pharmacia Biotech) and 1 x gel shift buffer (10 mM Tris, pH 7.5; 50 mM NaCl; 1 mM MgCl2; 0.5 mM EDTA; 0.5 mM dithiothreitol; and 4% glycerol) at room temperature for 20 minutes. Binding reactions were loaded onto a 5% polyacrylamide gel and run at 100 V in 1x TBE (90 mM Tris, 90 mM boric acid, and 2 mM EDTA, pH 8.0). The gels were wrapped in plastic wrap and exposed to Kodak X-ray film overnight at -80°C. To assess the specificity of the binding reaction, antibodies specific to AP-2α and AP-2γ were incubated with each binding reaction for 20 minutes before loading onto the gel.
Plasmid Constructs and Reporter Gene Assays
An E2F-responsive reporter construct, pE2F-Luc (Clontech, Mountain View, CA), containing four E2F consensus-binding sites in the enhancer region upstream of the firefly luciferase gene was used to characterize E2F-transactivating activity in cells expressing AP-2. Firefly luciferase activities were determined using the Dual Luciferase assay system (Promega) and were normalized relative to renilla luciferase activity. Reproducibility was ensured by transfection performed in triplicate.
Cell Cycle Analysis
Cells were plated at 5 x 105 cells/dish and infected with 100 MOI of various adenoviruses for 24 hours. After 24-hour infection, the medium was changed, and cells were pulse-labeled with BrdU (final concentration, 1 µM) for 30 minutes. Cells were washed with 1x PBS and fixed with 70% cold ethanol. Cells were washed with PBT (1x PBS, 1 mg/ml BSA, and 0.1% Tween 20) on the next day and digested with pepsin for half an hour. After neutralization with 0.1 M borax, nuclei were washed again and incubated with BrdU primary antibody (Becton Dickinson Immunocytometry Systems, San Jose, CA) and fluorescein isothiocyanate (FITC) GAM secondary antibody (Becton Dickinson), following the manufacturer's protocol. Samples were analyzed at the Flow Cytometry Facility at the University of Iowa. Data were analyzed with CellQuest software (Becton Dickinson) for combined BrdU/propidium iodide (PI) staining, and with ModFit software (Verity Software House, Topsham, ME) for PI staining only.
Real-Time Reverse Transcription-Polymerase Chain Reaction (RT-PCR) Analysis
Total RNA was extracted using Qiagen RNeasy kit (Qiagen, Inc., Valencia, CA). RT was performed using high-capacity cDNA archive kit (Applied Biosystems, Foster City, CA), according to the manufacturer's protocol. PCR conditions were as follows: 95°C for 10 minutes, followed by 40 cycles of denaturation at 95°C for 30 seconds, and annealing and extension at 60°C for 1.5 minutes. PCR was performed, and data were collected using the ABI Prism 7000 real-time sequence detection system (Applied Biosystems). The target gene was detected using SYBR green PCR master mix (Applied Biosystems). Differences in expression were determined using relative quantity calculated by standard curve. p21 mRNA level was determined by SYBR Green master mix (Applied Biosystems) using forward primer 5′-ATCCCGTGTTCTCCTTT and reverse primer 5′-GCTGGCATGAAGCC. Cyclin D1 was detected by using the forward primer 5′-GAGGTCTGCGAGGAACAGAAGT and the reverse primer 5′-CCTTCATCTTAGAGGCCACGA.
Chromatin Immunoprecipitation (ChIP) Assays
MDA MB-231 cells were infected with 100 MOI of Ad-Bgl II, Ad-AP-2α, or Ad-AP-2γ for 24 hours. Cells were treated with 1% formaldehyde for 10 minutes. The cells were then gently scraped and collected by centrifugation at 4°C. Cells pellets were resuspended in 500 µl of sonication buffer (50 mM Tris-CI, pH 8.0; 10 mM EDTA; 1% SDS; and proteinase inhibitors) and incubated on ice for 10 minutes. DNA-protein complexes were sonicated to lengths between 200 and 1000 bp, as determined by gel electrophoresis. Samples were centrifuged at 16,000g at 4°C to spin out cell debris, and then the supernatant was diluted 10-fold with ChIP dilution buffer. One tenth of the sample was set aside for input control, and the remaining sample was precleared with protein A/G PLUS Agarose (Upstate Biotech, Charlottesville, VA). Following preclearing, the sample was split in half and the two portions were incubated with 30 µl of anti-AP-2α antibody (Santa Cruz Biotechnology) and 30 ml of anti-AP-2γ antibody (Santa Cruz Biotechnology), respectively, plus protein A/G PLUS agarose. Following overnight incubation at 4°C, the beads were washed with low-salt, high-salt, and LiCl wash buffers, and then twice with Tris-EDTA, pH 8.0. Chromatin-antibody complexes were eluted, and DNA-protein crosslinks were reversed with 0.4 M NaCl (final concentration) at 65°C for 4 hours. Samples were subsequently treated with proteinase K, and genomic DNA was recovered by Qiagen DNeasy kit (Qiagen, Inc.) and quantitated with a Bio-Photometer (Eppendorf Scientific, Hamburg, Germany).
Conventional genomic PCR was used to analyze ChIP DNA using PCR Master Mix (Qiagen, Inc.) following manufacturer's protocol (20% Q-solution was added). PCR was carried out on 70 ng of ChIP DNA using the following conditions: 95°C for 15 minutes, 94°C for 30 seconds, 57°C for 30 seconds, and 72°C for 30 seconds (40 cycles in total). The corresponding PCR primers used were as follows: p21 forward primer 5′-GCCAGATTTGTGGCTCACTTCG and p21 reverse primer 5′-ACGCTTGGCTCGGCTCTGG.
In Vivo Tumorigenicity Assays
All animal experiments were conducted in strict accordance with an approved IACUC protocol from the Office of Animal Research at the University of Iowa. Athymic nude mice were housed in specific pathogen-free conditions at the animal care facility at the University of Iowa. Under sterile conditions in a laminar flow hood, 7- to 8-week-old female nude mice were injected subcutaneously with 5 x 106 MDA MB-231 cells into each flank. The animals were examined every 4 days for the status of tumor growth. Tumor sizes (Tumorvolume = length x width x height/2) were measured as a function of time. Animals were sacrificed when any tumor reached 1 cm3. To determine the tumorigenic capability of differently treated cells, MDA MB-231 cells were infected with 100 MOI of vector control or Ad-AP-2 for 24 hours before injection into mice. To infect established tumors in mice, 1 x 109 pfu of adenoviruses was injected into each tumor when the average diameter was 3 to 4 mm. Animals were grouped at the beginning to achieve the same average tumor volume within each group. Adenoviral injections were performed every 5 days (a total of three times).