Cell lines were obtained from the American Type Culture Collection. MCF7 cells were grown under ATCC recommended conditions. MDA-MB-468 cells were cultured in Modified Improved MEM (IMEM; Invitrogen, Carlsbad, CA) with 10% FBS and MCF10A cells were cultured and maintained as previously described (Soule et al., 1990
). Transfections were performed using TransIT-LT1 reagent (Mirus Bio, Madison, WI) and cells were assayed after 48 hours.
wild type and knockout mice (gift of Gerard Grosveld, St. Jude Children’s Hospital) were used to generate primary mouse embryonic fibroblasts (MEFs) (Wise-Draper et al., 2009a
). MEFs were explanted from day 13.5 embryos and maintained in high glucose DMEM containing 10% FBS, 2mM L-glutamine, MEM non-essential amino acids, 0.055mM β-mercaptoethanol, and gentamycin. Immortalized MEFs were obtained by 3T3 serial passaging.
Western blotting was performed as previously described using 40 μg of total protein and semi-dry transfer (Andreassen and Margolis, 1994
, Wise-Draper et al., 2006
). Membranes were probed with antibodies to DEK (1:1000, BD Biosciences, San Jose, CA), cyclin A (1:400, Santa Cruz, Santa Cruz Biotechnology, CA), E-cadherin (1:2500, BD Biosciences), p63 (1:200, Santa Cruz), total β-catenin (1:2000, BD Biosciences), activated phosphorylated (Y142) β-catenin (1:500, Abcam Inc, Cambridge, MA), activated β-catenin (1: 1000, clone 8E7 “anti-ABC”, Millipore, Billerica, MA), Histone H3 (1:2000, Abcam), γ-tubulin (1:1000, Sigma-Aldrich, St. Louis, MO) and Actin C4 (1:10,000, gift of James Lessard, Cincinnati Children’s Hospital). Fractionated lysates were prepared as previously described (Dignam et al., 1983
). Protein from tumor tissue was isolated by pulverizing snap-frozen tissue with a mortar and pestle prior to the addition of lysis buffer. Band intensities were measured using Image J software.
A breast tissue microarray (BRC961C_F, Pantomics, Inc., Richmond, CA) was stained by immunohistochemically stained for DEK as described below. The tissue microarray contained intact duplicate samples including normal breast (n=2), invasive adenocarcinomas (n=30, 28 ductal, one ductal papillary and one mucinous) and non-malignant breast disease including hyperplasia (n=3), fibrocystic changes (n=4) and fibroadenomas (n=3). All patients were female with the exception of the one case of ductal papillary adenocarcinoma. DEK staining was blindly scored as positive or negative based on the presence of any brown-stained nuclei.
Paraformaldehyde fixed and paraffin embedded tissues were sectioned, subjected to sodium citrate antigen retrieval, and stained using the M.O.M. Peroxidase kit (Vector Labs, Burlingame, CA) and 3,3′-Diaminobenzidine (DAB). The following antibodies were used: DEK (1:60, BD Biosciences), BrdU (1:100, Molecular Probes, Invitrogen), ΔNp63 (1:50, Santa Cruz), or cleaved caspase 3 (Asp175; 1:100, Cell Signaling Technology, Beverly, MA). Samples were counterstained with 0.1% Nuclear Fast Red (Poly Scientific, Bay Shore, NY) and preserved with Permount (Fisher Scientific, Pittsburgh, PA).
Retroviral and Lentiviral Transduction
Cells were transduced with retroviral constructs (R780 and R780:DEK) as described previously and sorted based on GFP expression (Wise-Draper et al., 2009a
). Alternatively, cells were transduced with the lentiviral pLKO.1 constructs (Sigma Aldrich Mission shRNA library) and selected in 2 μg/ml puromycin. DEKsh2 represents construct pLKO.1_DEK832 and DEKsh5 represents construct pLKO.1_DEK523. NTsh is a non-targeting control. DEKsh2 functionality was published previously (Kappes et al., 2008
). All cells were analyzed within four passages of selection.
Cells were plated at equal density in six-well plates, trypsinized, and counted in triplicate using a hemacytometer.
Apoptotic cells were labeled with the Caspase 3, Active Form, Apoptosis kit (BD Biosciences) according to manufacturer’s instructions. Samples were analyzed with a FACSCalibur flow cytometer (BD Bioscience). The side population was analyzed based on the exclusion of Hoecsht 33342 using an LSRII flow cytometer (BD Bioscience) as previously described (Goodell et al., 1996
). Verapamil at a final concentration of 100 μM was used as a negative control to properly identify the population.
MCF7 cells were seeded at a density of 10,000 cells per ml in 6-well plates coated with 1% agarose and cultured in DMEM:F12 (1:1) media with 2% BSA, 10 ng/ml EGF, and 1.0 μg/ml each of hydrocortisone and insulin. The total number of mammospheres were counted after 10 days. Mammospheres were passaged as previously described and replated at a density of 2,500 cells per ml (Zhang et al., 2008
). Images were obtained with a Leica DMIL microscope (Leica Microsystems, Bannockburn, IL) and SPOT imaging software (Diagnostic Instruments, Sterling Heights, MI).
Migration and Invasion Assays
Transwell assays, with or without Matrigel, were used to determine cell migration and invasive potential in vitro according to manufacturer’s instructions (BD Biosciences). Cells were seeded in supplement-free media and complete media was placed into the bottom chamber as a chemoattractant. Cells attached to the outer surface of the transwell chamber were alcohol-fixed and stained with Giemsa after 20 hours.
In vivo Tumorigenesis
Cells were orthotopically injected into the inguinal mammary fat pads of nulliparous, 12-week old female athymic nude mice as described (Allan et al., 2006
). Tumors were measured with calipers and volume was calculated as [(π/6)xLxW2
] (Euhus et al., 1986
, Tomayko and Reynolds, 1989
). Two hours prior to sacrifice, the mice were intraperitoneally injected with 600 μg of BrdU (200 μl of a 10 mM solution; GE Healthcare/Amersham, Piscataway, NJ). At necropsy, the mice were weighed and the tumors with any adjacent mammary gland and/or lymph nodes were excised, measured, weighed, fixed and embedded in paraffin. Lungs and livers were visually inspected for metastases. The usage and handling of mice were performed with the approval of the University of Cincinnati Institutional Animal Care and Use Committee.
Tissue sections from paraffin-embedded MDA-MB-468 NTsh xenograft tumors from the mammary gland were deparaffinized, underdwent antigen retrieval with 10 mM sodium citrate, and were blocked with 5% normal donkey serum and immunostained with antibodies for DEK (1:50, Aviva Systems Biology, San Diego, CA) and PCNA (1:50, BD Biosciences). DEK and PCNA were visualized with rhodamine- and FITC-conjugated secondary antibodies, respetively (1:200, Jackson Immunoresearch, West Grove, PA) then counterstained with ProLong Gold antifade reagent with 4',6-diamidino-2-phenylindole (DAPI; Invitrogen Molecular Probes).
Cultured cells were seeded into 8-well chambered slides (BD Biosciences) and labeled as previously described (Prahalad et al., 2004
) except 0.1% normal goat serum was used for blocking. Cells were labeled with anti-E-cadherin antibody (1:50, BD Biosciences) and FITC-conjugated anti-mouse secondary antibody (1:50, Jackson Immunoresearch, West Grove, PA), then counterstained with Vectashield plus DAPI (Vector Labs). All immunofluorescnce images were obtained with a Leica DMI 6000b microscope (Leica Microsystems) and OpenLab v5.5 imaging software (Perkin Elmer, Waltham, MA)
TOP/FOP Luciferase Reporter Assay
MDA-MB-468 cells were transfected as described above with pRL-TK (Renilla), and either pSUPER(8x)TOPFlash or pSUPER(8x)FOPFlash then assayed with the Dual-Luciferase Reporter Assay System (Promega, Madison, WI). Rescue of β-catenin signaling was performed by transfecting cells with the constitutively active S37A β-catenin construct cloned into the pcDNA6.V5-His plasmid. All plasmids were a gift from Aaron Zorn (Cincinnati Children’s Hospital). Data is presented as the ratio of relative light units (RLUs) of TOPFlash to FOPFlash.
mRNA was isolated from sub-confluent cells using Trizol reagent (Invitrogen, Carlsbad, CA) and one microgram of total mRNA was used to produce cDNA using the QuantiTect Reverse Transcription kit (Qiagen, Valencia CA). Expression was tested using SYBR Green detection and an ABI7300 Real Time PCR system (Applied Biosystems, Carlsbad, CA). Data was analyzed using the comparative Ct method (Schmittgen and Livak, 2008
). The following primers were used: (1) E-cadherin Forward: 5-CAGAAAGTTTTCCACCAAAG-3 (2) E-cadherin Reverse: 5- AAATGTGAGCAATTCTGCTT -3 (3) GAPDH Forward: 5-GGTCTCCTCTGACTTCAACA-3 and (4) GAPDH Reverse: 5-ATACCAGGAAATGAGCTTGA-3.
Statistical significance was assayed using two-tailed Student’s t-test or Chi Square test. All in vitro experiments represent the average of triplicate experiments and errors bars depict standard error. In the figures, one asterisk (*) indicates p<0.05, and two asterisks (**) indicates p<0.001.