Cell cultures, chemicals, and tissue samples.
Human fibroblast GM00637 cells were obtained from the Coriell Institute for Medical Research and cultured in MEM. HUVECs were obtained from Cambrex Bio Science and cultured in EGM-2 (Cambrex Bio Science). Gastric cancer cells (AGS, SNU216, SNU484, and SNU638), glioblastomas (U87MG and U373MG), lung cancer cells (NCI-H460 and NCI-H1299), colon cancer cells (SW480, HT29, NCI-H548, NCI-H716, and DLD1), and pancreatic cancer cells (Capan-1, BXPC-3, and ASPC-1) were cultured in RPMI-1640 (Invitrogen). Human glioblastomas (M059J and M059K) were grown in DMEM/F12. DMS53 (human lung cancer) cells were grown in WayMouth’s medium (Invitrogen). Calu-1 (human lung cancer) cells were grown in McCoy’s 5A medium. SK-MES-1 (lung cancer), NCI-H747 (colon cancer), KM12SM (colon cancer), and PANC1 and MIAPaCa-2 (pancreatic cancer) cells were grown in DMEM. KM12C (colon cancer) cells were grown in MEM. All cancer cell lines were purchased from ATCC, except for the human gastric cancer cell lines SNU216, SNU484, SNU638 (Korean Cell Line Bank). All media were supplemented with 10% heat-inactivated FBS and 1% penicillin/streptomycin. Cells were maintained in 5% CO2-humidified atmosphere at 37°C. N-[N-(3,5-difluorophenacetyl)-l-alanyl]-(S)-phenylglycinet-butyl ester (DAPT) and DMSO were purchased from Calbiochem. Samples of colon adenocarcinoma and normal tissue from the same patient were provided by C. Choi (Chonnam National University Hospital, National Biobank of Korea, Gwangju, Republic of Korea).
Gene expression analysis.
Control and GM00637-APEX1 cells, control and APEX1-shRNA/DLD1, and control and SW480-APEX1 cells were harvested, and total RNA was extracted using TRIzol
(Invitrogen) and purified using RNeasy columns (Qiagen) according to the manufacturers’ instructions. RNA quality and purity were assessed by measuring OD260/280
using an Agilent 2100 Bioanalyzer (Agilent Technologies). Total RNA was amplified and purified using Agilent’s Low RNA Input Linear Amplification Kit (Agilent Technologies) to yield cyanine-labeled cRNA. After purification, the cRNA was quantified using the ND-1000 Spectrophotometer (NanoDrop) and hybridized to microarrays. Gene expression profiling was conducted using A Whole Human Genome Microarray 44K (Agilent Technologies) containing more than 40,000 60-mer polynucleotide probes. Analyses were performed according to the manufacturers’ recommendations. 2 repetitions were performed. After hybridization, arrays were scanned using a DNA microarray scanner (Agilent Technologies) and quantified using Feature Extraction Software (Agilent Technologies). All data normalization and selection of “fold-changed genes” were performed using GeneSpringGX 7.3 (Agilent Technologies). Normalized ratio averages were calculated by dividing the average normalized signal channel intensity by the average normalized control channel intensity. Biological pathway– and ontology-based analysis was performed using the Gene Ontology Database (
) and Gene Cards Database (
). All microarray data were deposited in GEO (accession no. GSE45912).
Plasmid constructs, RNAi, and generation of stable cell lines.
Human Jagged1 cDNA was amplified from GM00637 cells by RT-PCR (forward primer, 5′-ATGCGTTCCCCACGGAC-3′; reverse primer, 5′-CTATACGATGTACTCCATTCGGTTTAAGCTC-3′) and cloned into the pcDNA3.1 mammalian expression vector (Invitrogen). The APEX1-expressing vector (pcDNA3-APEX1) was described previously (29
). Cells were transfected with siRNA using RNA max-i (Invitrogen). siRNA target sequences were as follows: APEX1, 5′-AAGTCTGGTACGACTGGAGTA-3′; Jagged1, 5′-AAATGGGATGATGACTGTAAT-3′; Notch3, 5′-AACTGCGAAGTGAACATTGAC-3′; EGR1, 5′-AAGAGGCATACCAAGATCCACTT-3′. GFP siRNA (sc-45924; Santa Cruz Biotechnology) was used as a negative control. For generating cell lines stably overexpressing APEX1 shRNA or Jagged1 shRNA, oligonucleotides encoding the target sequence for APEX1 (forward, 5′-GATCCGTCTGGTACGACTGGAGTATTCAAGAGATACTCCAGTCGTACCAGACTTTTTTGGAAA-3′; reverse, 5′-AGCTTTTCCAAAAAAGTCTGGTACGACTGGAGTATCTCTTGAATACTCCAGTCGTACCAGACG-3′) or Jagged1 (forward, 5′-GATCCATGGGATGATGACTGTAATTTCAAGAGAATTACAGTCATCATCCCATTTTTTTGGAAA-3′; reverse, 5′-AGCTTTTCCAAAAAAATGGGATGATGACTGTAATTCTCTTGAAATTACAGTCATCATCCCATG-3′) were annealed and cloned into psilencer2.1-U6 (Ambion). For control shRNA, a nontargeting scramble sequence was cloned into psilencer2.1-U6. DLD1 and KM12SM cells were transfected with APEX1 shRNA or scrambled control shRNA using Lipofectamine 2000 (Invitrogen) and cultured in selection medium containing 400 μg/ml hygromycin for 4–5 weeks. To generate stable GM00637-APEX1, SW480-APEX1, and HT29-APEX1 cells, the cells were transfected with empty vector, pcDNA3, or APEX1-expressing vector using Lipofectamine 2000 (Invitrogen). Cells were then incubated in medium containing 400 mg/ml G418 for 4–5 weeks.
Quantitative real-time RT-PCR.
Total RNA was purified from cells using TRIzol (Invitrogen) according to the manufacturer’s protocol. cDNA was synthesized using 1 μg RNA, M-MLV RT (Invitrogen), and random hexamers (Promega). Real-time PCR analysis was performed using the SYBR Premix Ex Taq kit (TaKaRa Bio) and the Mx3000P Kit (Stratagene) with specific primers. Primers used for real-time RT-PCR were as follows: JAG1 forward, 5′-GAAACAGCTCGCTGATTGCT-3′; JAG1 reverse, 5′-ACCAAGCAACAGATCCAAGC-3′; APEX1 forward, 5′-TGAAGCCTTTCGCAAGTTCCT-3′; APEX1 reverse, 5′-TGAGGTCTCCACACAGCACAA-3′; HES1 forward, 5′-ACAGAAAGTCATCAAAGCCT-3′; HES1 reverse, 5′-AGAGCATCCAAAATCAGTGT-3′; q18S rRNA forward, 5′-CGCCGCTAGAGGTGAAATTC-3′; q18S rRNA reverse, 5′-TTGGCAAATGCTTTCGCTC-3′. Each sample was analyzed in triplicate, and target genes were normalized relative to the reference housekeeping gene, 18S rRNA.
Cells were washed with 1× PBS and lysed in lysis buffer (20 mM HEPES, pH 7.4; 2 mM EGTA; 50 mM glycerol phosphate; 1% Triton X-100; 10% glycerol; 1 mM DTT; 1 mM phenylmethylsulfonyl fluoride; 10 μg/ml leupeptin; 10 μg/ml aprotinin; 1 mM Na3VO4; and 5 mM NaF). Protein content was determined using a dye-binding microassay (Bio-Rad), and 10–160 μg protein per lane was electrophoresed on 8–12% SDS polyacrylamide gels. Proteins were blotted onto Hybond ECL membranes (Amersham Pharmacia Biotech), and immunoblotting was carried out using the following antibodies: mouse anti-APEX1 (sc-17774), rabbit anti-Jagged1 (sc-8303), goat anti-Notch1 (sc-6014), rabbit anti-Notch2 (sc-5545), rabbit anti-Notch3 (sc-5593), goat anti-Notch4 (sc-8644), mouse anti–β-actin (sc-47778), and mouse anti–α-tubulin (sc-23948) from Santa Cruz Biotechnology; rabbit anti–cleaved Notch1 (Val1744) (D38B8), rabbit anti-Notch1 (3608), and rabbit anti-Notch3 (2889P) from Cell Signaling Technology. 4 protein ladders (PM1001, SM0671, P8500, and P8502) were used for molecular weight determination (Fermentas, GenDEPOT). The blotted proteins were detected using an enhanced chemiluminescence detection system (iNtRON).
Jagged1 promoter cloning and reporter assay.
The Jagged1 promoter (nucleotides –1,473 to +14) was amplified from genomic DNA of HeLa cells. The approximately 1.5-kb PCR product was cloned into the pGL3-Basic vector (Promega) to create a pGL-Jagged1 luciferase reporter (pA) vector, and the deletion fragments of the Jagged1 promoter were amplified by PCR using the pA Jagged1 promoter as template. The pB Jagged1 promoter construct encompassed nucleotides –1,091 to +14; the pC Jagged1 promoter construct encompassed nucleotides –850 to +14; the pD Jagged1 promoter construct encompassed nucleotides –1,476 to –850; the pE Jagged1 promoter construct encompassed nucleotides –490 to +14; and the mEGR1-1, mEGR1-2, and mEGR1-3 Jagged1 promoter constructs contained 3 mutated putative EGR1 binding sites (E1, E2, and E3, respectively), which were generated using the pC Jagged1 promoter construct as a template with a Muta-Direct Site Directed Mutagenesis Kit (Intron Biotechnology). The mutated nucleotide sequences (denoted by underline) were as follows: mutated E1, 5′-GTTGGAAATCGCC-3′; mutated E2, 5′-GGTAGAGTCTCCG-3′; mutated E3, 5′-CCCGGAGTTGGCT-3′.
Luciferase reporter assay.
Cells were cotransfected with pGL3-Basic vector, Jagged1 promoter constructs, or CBF-1 promoter constructs (provided by S.D. Hayward, Johns Hopkins School of Medicine, Baltimore, Maryland, USA) and pSV-gal (Promega) using Fugene6 (Roche Diagnostics) for 24 hours. Luciferase activity and β-gal activity were analyzed using the Luciferase assay system and β-gal Enzyme Assay System (Promega), respectively. Luciferase activity was normalized based on β-gal activity and adjusted using the empty pGL3-Basic vector to determine relative luciferase activity. Each sample was analyzed in triplicate, and experiments were performed at least 3 times.
Nuclear lysates were prepared from parent, pcDNA3, and GM00637-APEX1 cells for the EGR1 EMSA assays. Double-stranded oligonucleotides corresponding to nucleotides –794 to –778 (E1, 5′-CCCAGGGTGAGCCCCTCTCATGAATATTAA-3′), –638 to –622 (E2, 5′-GAGCATCCCGCTCCCAACCCCTTCCAAGTTC-3′), and –556 to –540 (E3, 5′-GCCCGGGGCGCCCGAGGGGGCGGTCCCCGCTGGG-3′) of the human Jagged1 promoter containing the putative EGR1 binding sites were annealed and end-labeled with [γ-32P]ATP and T4 polynucleotide kinase (New England Biolabs). The labeled probe was incubated with nuclear extracts in binding buffer containing 10 mM Tris-HCL, 50 mM NaCl, 1 mM EDTA, 4% glycerol, and 2 μg polydI-dC in a total volume of 20 μl at room temperature for 30 minutes. For competition experiments, unlabeled competitor DNA was added to the mixture before adding the labeled probe. For supershift assay, 1 μM anti-EGR1 antibody (sc-189; Santa Cruz Biotechnology) was added to the reaction mixtures and incubated for 30 minutes prior to separating the DNA-protein complex. DNA-protein complexes were resolved on a 6% polyacrylamide gel with 1× Tris-borate/EDTA buffer at 150 V for 2 hours at room temperature. Gels were dried, and label complexes were detected by autoradiography.
ChIP assay was performed using the EZ ChIP kit (Upstate Chemicon) according to the manufacturer’s instructions. The cell pellets were suspended in lysis buffer and sonicated to shear DNA. After sonication, the lysate was centrifuged, and the supernatant was diluted 10-fold with ChIP dilution buffer (0.01% SDS; 1% Triton X-100; 2 mM EDTA; 20 mM Tris–HCl, pH 8.0; 150 mM NaCl; and protease inhibitors). Rabbit anti-EGR1 (sc-189, 1:250; Santa Cruz Biotechnology) or normal rabbit IgG (negative control, 1:500; Upstate Chemicon) were added to the supernatant and incubated overnight at 4°C with rotation. The immunocomplex was precipitated with protein A/G-agarose, washed, and eluted with elution buffer (1% SDS; 0.1M NaHCO; and 200 mM NaCl). Reversal of cross-linking was performed by heating at 65°C overnight in the presence of NaCl. The DNA was purified using a spin column. The region amplified was the promoter region of the human Jagged1 promoter (–638 and –622), which contains a putative EGR1-binding site, E2. Primer sequences were as follows: forward, 5′-GCCCTGGTTCTTCTACGC-3′; reverse, 5′-TAGTGCGAGGAGGAACTTGG-3′.
7-μm tumor cryosections were stained with APEX1 (sc-17774, 1:500; Santa Cruz Biotechnology) or Jagged1 (sc-8303, 1:200; Santa Cruz Biotechnology) antibodies. For immunohistochemistry, a biotinylated goat anti-mouse or rabbit antibody (Vector Laboratories) followed by horseradish peroxidase–conjugated streptavidin (Vector Laboratories) was used. Protein expression was scored in the nucleus for APEX1 and in the cytoplasmic membrane and cytoplasm for Jagged1. APEX1 and Jagged1 immunoreactivity was determined by scoring for staining intensity (0, none; 1, weak; 2; moderate; 3, strong) and percent positive cells (0, <5%; 1, 6%–25%; 2, 26%–50%; 3, 50%–75%; 4, >76%) and expressed as the product of both scores. For immunofluorescence, the FITC-conjugated secondary antibodies goat anti-rabbit Alexa Fluor 594 and goat anti-mouse Alexa Fluor 488 were used (both from Invitrogen), and nuclei were counterstained with DAPI (Sigma-Aldrich). Immunofluorescence was detected by confocal microscopy (Carl Zeiss).
Soft agar colony formation assay.
Soft agar assays were performed in 6-well plates. The base layer of each well consisted of 2 ml with final concentrations of 1× medium and 0.6% low–melting point agarose (Duchefa). Plates were chilled at 4°C until solid. Next, a 1-ml growth agar layer was poured, consisting of 5 × 104 cells suspended in 1× medium and 0.3% low–melting point agarose; plates were again chilled at 4°C until the growth layer congealed. A further 1 ml of 1× medium without agarose was added on top of the growth layer. Cells were allowed to grow at 37°C and 5% CO2 for 14 days, and total colonies were stained with 0.005% crystal violet (Sigma-Aldrich) and counted. Images were analyzed using Image-Pro Plus 4.5 software (Media Cybernetics). Assays were repeated a total of 3 times.
Cell migration and invasion assays.
In vitro cell migration assays were performed in a 24-well Transwell plate with 8-μm polyethylene terephthalate membrane filters (BD Biosciences) separating the lower and upper culture chambers. Cells were grown to subconfluence (~75%–80%) and serum starved for 24 hours. After detachment with trypsin, cells were washed with PBS and resuspended in serum-free medium, after which the cell suspension (2 × 104 cells) was added to the upper chamber. Complete medium was added to the bottom wells of the chamber. The cells that had not migrated were removed from the upper face of the filters using cotton swabs, and the cells that had migrated to the lower face of the filters were fixed with 4% formaldehyde and stained with 0.1% crystal violet. Images of 3 random ×10 fields were captured from each membrane, and the number of migratory cells was counted. The mean of triplicate assays for each experimental condition was used. The cell invasion assay was essentially the same as the cell migration assays, except that the membrane filters were coated with Matrigel.
GM00637 cells transfected with the APEX1 expression vector or with the control vector were seeded in 6-well culture dishes at a density of 15 × 104 cells/well. A wound was incised 24 hours later in the central area of the confluent culture, which was incubated for a further 28 hours, following careful washing to remove detached cells and addition of fresh medium. Cultures were observed at 0, 12, 24, 28, and 36 hours, and phase-contrast pictures were taken on the wounded area using an inverted microscope.
Cell growth assay.
Cell growth assay was performed using the WST-1 Assay (Roche Molecular Biochemicals). Equal numbers of cells were seeded in triplicate wells in 48-well plates and maintained in low-serum medium (0.1% FBS). WST-1 reagent was then added to the cells at the indicated times and incubated for 2 hours at 37°C. The spectrophotometric absorbance of the samples was measured using an Ultra Multifunctional Microplate Reader (Tecan) at 450 nm.
Tumor formation in nude mice.
The 6-week-old male BALB/c nude mice (Orient Bio Inc.) used in this study were housed in our pathogen-free facility and handled in accordance with standard-use protocols and animal welfare regulations. DLD1 and SW480 cells, stably transfected with the indicated shRNA or expression vector, and control cells were harvested and resuspended in DMEM. Next, 2 × 106 SW480 cells and 5 × 106 DLD1 cells were injected subcutaneously into the right flank of a BALB/c nude mouse. Tumor size was measured with a caliper every 3 days; after 36 days, mice were killed, and tumors were excised.
Peritoneal injection for the metastasis assay.
For the colon cancer cell metastasis assay, all cells were stably transfected with a RFP expression plasmid, pCMV-DsRed (Clontech). To study APEX1 deficiency or Jagged1 overexpression in a colon cancer metastasis model, mock DLD1, control-shRNA/DLD1, APEX1-shRNA/DLD1, vector-transfected APEX1-shRNA/DLD1, or Jagged1-transfected APEX1-shRNA/DLD1 cells were injected into the peritoneal cavity of Balb/c nude mice. After 5 weeks, 6 mice per group were killed, and lung tissue was analyzed for metastasis occurrence. To study the role of APEX1 overexpression or Jagged1 deficiency in metastasis to the lung, vector-transfected SW480, APEX1-transfected SW480, control shRNA–transfected SW480-APEX1, or Jagged1 shRNA–transfected SW480-APEX1 cells were injected into the peritoneal cavity of Balb/c nude mice. 8 weeks later, 6 mice per group were anesthetized and put in Kodak MI 2000 imaging system (MS FX PRO; Carestream Health). The excitation filter was set at 465 nm; exposure time per image was 30 seconds. Animals were subsequently killed, and the lungs excised for imaging analyses under the same conditions. After whole imaging, the lungs were frozen and sectioned at 4 μm for confocal laser scanning fluorescence microscopy and hematoxylin and eosin staining.
48-well culture plate wells were coated with Matrigel (BD Biosciences), which was allowed to polymerize for 30 minutes at 37°C. HUVECs cultured for 24 hours in endothelial cell basal medium (Cambrex Bio Science) were seeded on coated plates at a density of 2 × 104 cells/well in endothelial cell basal medium, then incubated at 37°C with 5% CO2 for 4–6 hours. Cells were washed with Hanks balanced salt solution and stained with Calcein-AM (Molecular Probes) in Hanks buffered saline at 37°C for 1 hour. Photography of the matrices using a fluorescence microscope (Olympus) and a CoolPix 4500 digital camera (Nikon) was used to assess network formation. Quantification of tube formation was done using Adobe Photoshop 7.0 (Adobe Systems) or Image-Pro Plus 4.5 software (Media Cybernetics).
Unless otherwise indicated, data are presented as mean ± SD of 3 or 6 independent results. Significance of differences was assessed by 2-tailed paired Student’s t test; a P value less than 0.05 was considered statistically significant. Correlation of APEX1 expression with Jagged1 levels was assessed using the Pearson correlation test with P value; a P value of 0.01 or less was considered statistically significant.
The tissue microarray slides included human colon tumor of different grades and adjacent normal colon tissues taken from BioChain and Super Bio-Chips. Normal colon tissues and adenocarcinoma and adenoma patient samples were obtained from the Chosun University Department of Pathology tissue bank and Chonnam National University Hwasun Hospital, a member of the National Biobank of Korea, which is supported by the Ministry of Health, Welfare, and Family Affairs. Informed consent was not required, as we used archival samples from a tissue bank that remained anonymous. Protocols for human studies were approved by the Institutional Review Board of Chosun University School of Medicine. All animal procedures were reviewed and approved by the Institutional Animal Welfare and Use Committee of Chosun University School of Medicine.