The murine hemangioma cell line (EOMA), CRL-2586™, was obtained from the American Type Culture Collection (ATCC, Manassas, VA). EOMA cells were maintained in Dulbecco's Modified Eagle's Medium (DMEM) (Life Technologies, Rockville, MD) with 10% Fetal Bovine Serum (FBS) and 50 µg/ml gentamicin sulfate (Life Technologies). Immortalized human dermal microvascular endothelial cell line, HMEC-1,19
were cultured in MCDB131 media (Life Technologies), and fortified with 10% FBS, 2mM glutamine (Life Technologies) and 50 µg/ml gentamicin.
Three-dimensional (3D) cultures were prepared using a modified procedure (described in ref. 20
) using 3D BM (Matrigel). Cultures were analyzed after 24 or 72 hours of cultivation. Morphology of the 3D cultures was assessed examining the degree of colony organization visually by phase contrast microscopy.
When indicated, four hours before release from the 3D BM, cells were preincubated at 37°C with 30 µM of LY294002 or 1 µM of MG-132 (Calbiochem, EMD Bioscience Inc., San Diego, CA) or DMSO as a control (Sigma Chemical CO, St. Louis, MO). After preincubation at 24 or 72 hours, cells were release as described below.
Retroviral vectors and transduction.
The pBabe HA-HoxA5 retroviral vector was constructed as described in reference 12
. The myr-Akt pWZL retroviral construct myr a-129 was provided by Mina Bissell (Lawrence Berkeley National Laboratory, Berkeley, CA).
Retroviral vectors were transfected into the amphotropic packaging cell line Phoenix Ampho (ATCC) using the calcium/phosphate-DNA precipitation method, and 48 hours after transfection, viral supernatant was collected, passed through 0.45 µm filters and used for transduction of EOMA cells.
EOMA cells were transduced with control plasmid (pBabe), and pBabe HA-A5 using 2 ml of virus-containing media in the presence of 8 µg/ml of polybrene (Sigma) for 16 hours at 37°C. Twenty-four hours later cells were selected in 1 µg/ml puromycin (Sigma) for seven days, and the pooled cell population was used for subsequent experiments. EOMA cells transduced with pWZL and myr-Akt pWZL virus were selected in 60 µg/ml hygromycin (Sigma) for two weeks.
Release of cellular structures and RNA extraction from 3D cultures.
Cultures grown in 3D were isolated from 3DBM using a modified procedure (described in Weaver et al. ref. 20
). Briefly, cells were washed with cold PBS without Ca2+
and containing 5 mM EDTA, and scraped into a centrifuge tube with a minimum volume of 30 ml cold PBS/EDTA. Cells were incubated on ice for 45 min, until the Matrigel dissociated, and centrifuged at 115 g for 2 min. The pellet was resuspended in an appropriate amount of lysis buffer for mRNA or protein isolation. Soluble and insoluble subcellular fractions were obtained as described (ref. 21
Reverse-transcriptase-polymerase chain reaction (RT-PCR).
One microgram of RNA from cell lysates, extracted using the RNeasy isolation kit (Qiagen,) was reverse transcribed using MMuLV RT (Invitrogen) for one hour at 42°C in a total volume of 25 µl. 1/25 of this reaction was linearly amplified for 30 cycles (HoxA5 and TSP2), 27 cycles (Efna1) and 25 cycles (β-catenin and Akt), following denaturation (30 sec 95°C), annealing (30 sec at 55°C for Akt, 56°C for HoxA5, 58°C for TSP2 and β-catenin), and finally extension (30 sec at 72°C) in a thermal cycler (PTC-200 Peltier Thermal cycler, MJ Research). RNA was normalized using 18s internal standards at a 1:3 ratio (Ambion, Austin, TX). The expected products were visualized by electrophoresis on 1% agarose gels containing ethidium bromide.
PCR primer sequences.
The following primers were used:
forward 5′ AAT GAG CTC TTA TTT TGT AAA CTC 3′
reverse 5′ TCA GAT ACT CAG GGA CGG AAG GC 3′
forward 5′ AAG GAG AAA GGA GGG CTT CT 3′
reverse 5′ TGC AGG TGC ATG TGG TAC AAC T 3′
forward 5′ TCT GGA ATC CAT TCT GGT GC 3′
reverse 5′ ACC ACT GGC CAG AAT GAT GA 3′
forward 5′ GTC GCC AAG GAT GAG GT 3′
reverse 5′ TCT CGT GGT CCT GGT TGT AG 3′
The delivery of the siRNA into the HMEC-1 cells was done with a Nucleofector device and its corresponding kits (Amaxa, Inc. Cologne, Germany). Transfection protocols were performed following the manufacturer's instructions using the T23 program. The annealed predesigned siRNA against HoxA5 was purchased from Ambion (Austin, TX) and has the following sequence 5′gcugcacauaagucaugactt3′. As a control we used a siRNA against pMAXGFP (Amaxa, Inc). After the transfection, cells were plated into six well plates coated with 3D Matrigel as described above.
Cells were cultured in 3D Matrigel for 24 or 72 hours, and colonies were isolated using ice-cold PBS/EDTA as described (ref. 22
). A small aliquot of pellets were smeared gently across a small area of a glass slide and were fixed in methanol: acetone (1:1) at room temperature for ten minutes. Immunofluorecence staining was performed as described (ref. 22
). Antibodies used were β-catenin (1:1000) (BD Biosciences). The slides were mounted in fluoromount (Southern Biotechnology Associates, Inc. Birmingham, AL) and analyzed under a Nikon Eclipse TE300 fluorescence microscope and under a Nikon D-Eclipse C1 confocal microscope.
Total protein or soluble and insoluble fractions were isolated from lysates of HoxA5 or control-transfected EOMA cells at 24 or 72 hours after culturing on 3D Matrigel. Equal amounts of protein were separated on a 10% SDS-PAGE gels and transferred to PVDF membranes. After blocking for nonspecific binding, western-blots were probed with a monoclonal β-catenin antibody (1:1000, clone 14 BD Biosciences), monoclonal PTEN antibody (1:250) (BD Biosciences, San Jose, CA) or polyclonal phosphor-Akt (Ser473) (1:1000) (Cell Signaling Technology, Inc. Beverly, MA), followed by sheep anti-mouse-HRP at 1:2000 or donkey anti-rabbit-HRP at 1:5000. Excess of antibody was removed by extensive washing and blots were developed by ECL system (Amersham Biosciences, Piscataway, NJ). The membranes were then stripped and treated with polyclonal Akt antibody (1:1000) (Cell Signaling Technology, Inc. Beverly, MA), and polyclonal β-actin antibody (1:1000) (Abcam Inc. Cambridge, MA), followed by donkey anti-rabbit-HRP at 1:5000 and detected by ECL system.
Control or HoxA5 expressing EOMA cells were cultured for 72 hours and solubilized in ice-cold lysed buffer.23
Following removal of insoluble material by centrifugation at 14000 g for one min, at 4°C, aliquots of the lysates containing equal amounts of protein (500 µg) were precleared on Untralink Protein A/G beads (Pierce) for one hour at 4°C. Supernatant was incubated one hour with 2 µg of anti VE-cadherin antibody (C-19, Santa Cruz). Immune complexes were captured with 10 µl of Ultralink Protein A/G beads at 4°C for 1.5 hours under continuous mixing. This was followed by four washings with ice-cold PBS containing 300 µM sodium orthovanadate. Immunoprecipitates were recovered in Laemmli's buffer under reducing conditions, subjected to SDS-PAGE and transferred on to nitrocellulose membranes. Immunoblotting was carried out using antibodies against VE-cadherin and membranes were stripped and probed with antibodies against β-catenin or monoclonal phosphor-tyrosine (1:2000), or blotted with a polyclonal phospho-(Ser/Thr) Akt substrate (1:1000) antibody.
Transient transfections and luciferase assay.
To measure transcriptional activity of β-catenin, control or HoxA5 expressing EOMA cells were grown to 70% confluence in six well dishes. Transfections were carried out in DMEM 10%FBS using the Effectene® transfection reagent (Qiagen) according to the manufacturer's instructions with a 1/25 ratio of DNA/ Effectene reagent. Cells were incubated with 0.4 µg of pTOPFLASH (reporter plasmid containing two sets TCF binding site, thymidine kinase minimal promoter and luciferase open reading frame) or 0.4 µg of pFOPFLASH (containing TCF mutant sites as a negative control) (both were a gift from Dr. Clevers). Luciferase assays were performed using the Dual-Luciferase reporter assay system (Promega, Madison, WI, USA) and their activities in the cell extracts were measured using a Wallac Victor2
1420 Multilabel Counter luminometer (PerkinElmer Life Sciences, Boston, MA). The transcriptional activity of each construct was evaluated by cotransfection with CMV- βgal to normalize transfection efficiency. All transfections were done on plastic and in some cases, after 24 hours cells were transferred and cultured on 3D Matrigel. Transcriptional activity was measured at 72 hours after transfection. Results were expressed as means ± s.d. of four to six independent experiments performed in triplicate.
Permeability across the endothelial cell monolayer was measured by using type I collagen-coated transwell units (6.5 mm diameter, 8 µm-pore-size PET filter, BD Falcon, NJ). HMEC-1 or HMEC-1 A5 expressing cells plated at 2 x 105 cells in each well were cultured for two days before the experiments and serum starved in Fibroblast Basal Media (FBM) (Lonza, MD) containing 0.5% BSA ON. Permeability was measured by adding 1 mg of FITC-labeled dextran (molecular weight 70,000)/ml in FBM with 0.5% BSA to the upper chamber and 500 µl FBM to the lower chamber. After incubation for different periods of time, between 30 min and three hrs, 50 µl of the sample from the lower compartment was diluted with 50 µl of PBS and measured for fluorescence at 535 nm when excited at 485 nm with a Wallac Victor Multilabel Counter.
Tumor formation in vivo.
A total of 2.5 x 106 of control or HoxA5 expressing EOMA cells were prepared in DMEM media and loaded into a 1 ml tuberculin syringe. Cells were injected subcutaneously in the dorsal midline region of nude mice (n = 6 mice/group) (The Jackson Laboratory, Bar Harbor, ME). All mice were euthanized at seven days post-injection and tumor tissue harvested by snap-freezing in liquid nitrogen.
In all experiments statistical analysis was performed using Student's t distribution with significance reported when p < 0.05.