Mice carrying a loss-of-function mutation of the brk gene were previously generated by Dr. Valeri Vasioukhin. The targeting construct contained the neomycin phosphotransferase (neo) gene, which was introduced into exon 1 in order to disrupt the initiation codon of the brk gene. In addition, the targeting construct included a diphtheria toxin A-fragment (DT) gene, which was introduced into the 3’ end and used for negative selection that enriches homologous recombinants in embryonic stem (ES) cells (Yagi, et al., 1990). When non-homologous recombination occurred, the DT gene was expressed causing the death of these recombinants. Homologous recombination resulted in loss of the DT gene and the recombinant cells survived selection with G418 due to expression of the neo gene. The ES cells, derived from mouse strain 129, were screened for homologous recombination by PCR and confirmed by Southern Blotting. The cells were injected into 4-day embryos from C57BL/6 mice and embryos were fostered in pseudopregnant females. Chimeric animals were identified by coat color and males were mated with Swiss black females. Heterozygous animals were mated to generate four brk knockout lines designated 2.8, 32.7, 33.7 and 11.6. To produce a congenic strain deficient for the brk gene, knockout mice from the line 2.8 were backcrossed to C57BL/6 mice for 12 generations. This inbred mouse line was designated 2.8 G12. Mutant mice were identified by PCR and confirmed by Southern Blotting and RNase protection assays.
Adult male wild-type and knockout mice (n ≥ 5) were age-matched (age ≥ 7 weeks) and colitis was induced by feeding 3% dextran sodium sulfate (ICN), MW=40,000, dissolved in the drinking water ad libitum (Tessner, et al., 1998). 3% DSS was administered for 5 days followed by a recovery period with water for 3 days. Mice were studied at the end of 5 days of DSS treatment (5 days) and at the end of 3 days water after DSS (8 days) (Fig. 12A). Mice were monitored for rectal bleeding and weight loss for the duration of the study. The control group received distilled water without DSS. At the time of sacrifice, the distal ileum, cecum and entire colon were excised and processed for biochemical and immunohistochemical analysis (see below).
Age- and sex-matched wild-type and knockout mice (n ≥ 20) were subjected to treatment with azoxymethane (AOM) in combination with dextran sodium sulfate (DSS) in a colon carcinogenesis model (Tanaka, et al., 2003). Mice were given a single intraperitoneal injection of AOM dissolved in saline (10 µg/ g bodyweight). Starting 1 week after the injection, animals received 2% DSS in the drinking water for 7 days and then no further treatment for 18 weeks (Fig. 14A). Animals were weighed weekly and monitored for rectal bleeding. At the end of the study (week 20), mice were sacrificed and their entire colon was excised and flushed with cold PBS. The colons were cut open longitudinally and washed with PBS. The tissue was fixed in 70% ethanol and macroscopically inspected.
Adult male wild-type and knockout mice (n ≥ 4) were age-matched (age ≥ 7 weeks) and exposed to ionizing radiation in a JL Shepherd Model 6810 137Caesium γ-irradiator (JL Shepherd). The animals were whole-body γ-irradiated at a dose of 8 Gγ on a rotating platform. The mice were sacrificed at 6 and 72 hours post irradiation representing the peak of early apoptosis and the point of crypt regeneration in the small intestine respectively (Potten, 1997, Potten, 1998 #3408). The distal jejunum and ileum were excised as described below. The tissues were processed for biochemical and histological analysis.
Wild-type and knockout mice were sacrificed by CO2 anesthesia followed by cervical dislocation. The gastrointestinal tract, including the entire small and large intestine, was removed and flushed with cold PBS with a syringe and large-bore needle. The small intestine was divided into pieces of tissue representing the proximal duodenum (first 3 cm at proximal end), distal ileum (last 3 cm at distal end) and distal jejunum (3 cm distal from the center), and fixed in 4% paraformaldehyde in PBS overnight at 4°C. Specimens were dehydrated for embedding by passage through 75% ethanol for 45 minutes, 85% ethanol for 45 minutes, 95% ethanol for 45 minutes, 100% ethanol for 45 minutes (two times), xylene for 30 minutes (two times), and liquid paraffin embedding medium (Fisher) at 62°C for 1 hour (two times). Tissues were then mounted in paraffin blocks and sections of 5 µm thickness were made using a microtome. After drying, these paraffin sections were heated for 2 hours at 65°C to promote tissue adherence to the slides.
For the quantitative examination of villus height and cryptal depth, control and mutant adult mice (n ≥ 3) were age-matched (7-10 weeks). Paraffin-embedded cross sections of distal ileum or jejunum were stained with hematoxylin and eosin. Samples were examined by light microscopy and images of the sections were captured. The measurements of villus height and cryptal depth were made for each animal using Adobe Photoshop 7.0 image analysis software. For each animal, 6 well-oriented villi and crypts from at least 5 quadrants (for a total of at least 30) of each cross section were measured. The average of each animal was used to determine an average villus height and cryptal depth for wild-type and knockout mice. The data gathered for each parameter assessed were compared by subjecting the results to Student’s two-tailed t-test to determine the P-values.
Proliferation was detected by the incorporation of BrdU into S-phase cells during a 1-2 hours pulse just before death. Mice were injected intraperitoneally with 5-bromo-2-deoxyuridine (BrdU, Sigma) in PBS at 50 µg/ g bodyweight. Gastrointestinal tissues were dissected as described above and fixed in Carnoy’s (10% glacial acetic acid, 60% ethanol, 30% chloroform) solution for 3 hours at 4°C. Tissues were then washed and processed for embedding as described above. BrdU immunohistochemistry was performed using a M.O.M. mouse on mouse Immunodetection Kit (Vector) following manufacturer’s instructions. Briefly, sections were deparaffinized in xylene two times for 5 minutes and rehydrated by sequential passage through 100%, 100%, 70%, 50%, and 30% ethanol for 3 minutes each. After washing in PBS, antigen retrieval was performed in antigen demasking solution (Vector) for 20 minutes at 90°C. After washing in TNT (0.1 M Tris-HCl pH 7.5, 0.15 M NaCl, 0.05% Tween 20), sections were treated with 1 µg/ml Proteinase K for 10 minutes followed by incubation with 3% H2O2 in methanol for 10 minutes and incubation with 70 mM NaOH for 3 minutes. Samples were blocked using an Avidin/Biotin blocking Kit (Vector) according to manufacturer’s instructions followed by incubation with M.O.M. mouse IgG blocking reagent for 1 hour at room temperature. The blocking reagent was removed and slides were pretreated with M.O.M. diluent (Vector) for 5 minutes before incubation with anti-BrdU monoclonal antibody (Becton Dickinson) 1:75 in M.O.M. diluent at room temperature for 1 hour. Slides were washed in TNT buffer three times for 5 minutes and incubated with M.O.M. biotinylated anti-mouse IgG reagent (Vector) for 30 minutes at room temperature. The BrdU-signal was visualized by incubation of the slides with avidin-horseradish peroxidase (Vector) for 30 minutes followed by incubation with the chromogenic substrate 3,3’-diaminobenzidine tetrahydrochloride (DAB, Sigma) for 10 minutes. Nuclei were counterstained with hematoxylin. The sections were dehydrated by sequential passage through 30%, 50%, 70%, 100%, and 100% ethanol, then passed through xylene and mounted with cover slips and Permount (Vector).
For further immunohistochemical analyses, tissue sections were processed using the Vectastain ABC Kit (Vector) according to manufacturer’s instructions. Briefly, after deparaffinizing and rehydrating the 5 µm tissue sections, the slides were washed in TNT and antigen retrieval was performed by microwaving the samples in 10 mM sodium-citrate buffer (C6H7O7Na) pH 6.0 four times for 5 minutes each, replacing evaporated buffer as needed. To quench endogenous peroxidase activity, slides were incubated with 3% H2O2 in methanol for 10 minutes followed by washing in TNT for 5 minutes. Samples were then blocked in goat or horse serum in PBS (Vector) for 30-60 minutes at room temperature. Tissues were incubated in blocking buffer with the primary antibody for 1 hour at room temperature for anti-Brk polyclonal antibody (Santa Cruz) 1:50, anti-PCNA polyclonal antibody (Santa Cruz) 1:200, anti-β-catenin monoclonal antibody (BD Biosciences) 1:200, and overnight at 4°C for anti-c-myc polyclonal antibody (Santa Cruz). Controls were performed with equal dilutions of rabbit or mouse IgG (Santa Cruz). Detection of the primary antibody was performed by incubation with goat anti-rabbit or horse anti-mouse biotinylated secondary antibody (Vector) for 30 minutes followed by incubation with avidin-horseradish peroxidase (Vector) for 30 minutes according to the Vectastain ABC Kit (Vector). To visualize the signal, sections were incubated in DAB (Sigma) for 5-10 minutes at room temperature. Reactions were stopped by washing in water for 10 minutes. Counterstaining with hematoxylin was performed as indicated. The sections were dehydrated and mounted as described above.
For immunohistochemical analysis of tight junctions, tissue sections were stained with anti-ZO-1 monoclonal antibody conjugated to FITC (Zymed laboratories). Briefly, after deparaffinizing and rehydrating, slides were washed in TNT and antigen retrieval was performed by microwaving the samples in 10 mM sodium-citrate buffer (C6H7O7Na) as described above. Samples were then blocked in horse serum in PBS (Vector) for 60 minutes at room temperature. Tissues were incubated with the primary antibody anti-ZO-1-FITC 1:200 in 1% BSA in PBS overnight at 4°C. Nuclei were counterstained with 2 µg/ml 4’,6-diamidino-2-phenylindole (DAPI, Sigma). Sections were coverslipped with Vectashield mounting medium for fluorescence (Vector) and examined by fluorescence microscopy.
To detect cleavage of caspase-3 in the gastrointestinal tract, tyramide signal amplification was performed. Sections were deparaffinized and rehydrated as described above. Antigen retrieval was performed by boiling sections in 1mM EDTA pH 8.0 for four times 5 minutes each. Samples were blocked with TNB buffer (NEN, 0.1 M Tris, pH7.5, 0.15 M NaCl, 0.5% Blocking Reagent) for 1 hour at room temperature and incubated with anti-cleaved-Caspase-3 polyclonal antibody (Cell Signaling) 1:100 in TNB overnight at 4°C. As a control, sections were incubated with rabbit IgG (Santa Cruz) 1:100. After washing and incubation with biotinylated goat anti-rabbit antibody (Vector) for 60 minutes, the TSA-indirect kit (NEN) was used according to manufacturer’s instructions. Briefly, sections were treated with streptavidin-horseradish peroxidase (1:100, NEN), reacted for 8 minutes with biotinylated tyramide reagent (NEN), and visualized with FITC-Avidin DCS (Vector). Nuclei were counterstained with DAPI (Sigma). Sections were coverslipped with Vectashield mounting medium for fluorescence (Vector) and examined by fluorescence microscopy. The number of cleaved Caspase 3-positive cells per crypt-villus unit was scored for each animal. For quantification, positive cells were counted for at least 10 crypt-villus units from at least 5 quadrants (a minimum of total 50) for each animal and used to determine the average of each animal. The data gathered were compared by subjecting the results to Student’s two-tailed t-test to determine the P-values.
Wild-type Brk (Brk WT) and Brk Y-F coding sequences were cloned into the retroviral expression vector pLXSN (Vasioukhin and Tyner, 1997). Brk Y-F has a substitution of the regulatory tyrosine at position 447 of wild-type mouse Brk to phenylalanine, resulting in a constitutively activated mutant of the kinase. Phoenix cells and Rat1A fibroblasts were cultured in Dulbecco’s modified Eagle medium (DMEM, Invitrogen) supplemented with penicillin (100 U/ml) (Invitrogen), streptomycin (100 µg/ml) (Invitrogen) and 10% fetal bovine serum (Atlanta Biologicals) at 37°C in 5% CO2.
Retroviral transduction was performed using the Phoenix ecotrophic packaging cell line (Kennedy, et al., 1997). Phoenix cells were plated at approximately 50-75% confluence in 15-cm-diameter tissue culture plates and transfected with the control neomycin vector pLXSN, wild-type Brk and activated Brk Y-F constructs by calcium phosphate co-precipitation in the presence of 0.2 M chloroquine (Pear, et al., 1993, Honigwachs, 1989 #3500). Cells were washed after 24 hours, and the high-titer retroviral supernatants were collected at 48 and 72 hours post transfection. Retroviral infection of Rat1A fibroblasts was performed by incubation of a 50% confluent 10-cm dish with 1.5 ml of retrovirus-containing supernatant in the presence of 8 µg/ml Polybrene (Fisher) for 24 hours. After the incubation, retrovirus expressing cells were selected with 300 µg/ml G418 (Invitrogen). The cells were pooled, expanded, and maintained as polyclonal populations. After establishment, cell lines were frozen and stored in liquid nitrogen, and a fresh aliquot of cells was used for each set of experiments.
Polyclonal Rat1a fibroblasts stably expressing wild-type (Brk WT) and activated Brk (Brk Y-F), and the control neomycin vector pLXSN, were generated as described above. For growth rate determinations, 1 x 105 cells seeded in 60 mm dishes were fed and counted daily for 5 days using a Hematocytometer. Three plates were counted for each time point and cell line to determine the average cell number.
For apoptotic assays, the cells were plated on 6-well culture dishes at 1 x 105 cells/ well 16 hours before treatment. Cells were then either serum starved or subjected to a combination of serum starvation and UV-irradiation. For serum starvation experiments, cells were grown in 0% FBS for 24 hours. For UV-irradiation experiments, cells were placed in PBS and irradiated with UV light (50 J/m2) (Kennedy, et al., 1999). After UV-irradiation, PBS was replaced with DMEM containing no serum and cells were incubated for 3, 3.5, 4 and 4.5 hours at 37°C in 5% CO2. After the indicated treatment, cells were fixed by the addition of formaldehyde at a final concentration of 18.5% and incubated for 24 hours at room temperature. Cells were then rinsed with PBS and stained with 2 µg/ml DAPI for 3 minutes. The percentage of apoptotic cells was visualized under an inverted fluorescence microscope. Fragmented and condensed nuclei were scored as apoptotic with at least 200 cells per experimental group. All treatments were done at least in triplicate, and the data for each experiment was used to determine the average amount of cell death. Results were subjected to Student’s two-tailed t-test to determine the P-values.
For cell cycle analysis, combined detection of BrdU incorporation and DNA content using propidium iodide (PI) was performed, using CellQuest software on a FACSCalibur flow cytometer (Becton Dickinson) and a BrdU flow kit (BD Biosciences) according to the manufacturer’s recommendations. Polyclonal Rat1A fibroblasts stably overexpressing the empty expression vector pLXSN (control), wild-type Brk (Brk WT) and the activated mutant of Brk (Brk Y-F) were synchronized in DMEM containing 0.1% FBS for 48 hours. The medium was replaced with DMEM containing 10% FBS, and after the indicated time-points (0, 2, 4, 8 and 16 hours) the cells were pulse labeled with 100 µM BrdU (Sigma) for 40 min. Cells were harvested by trypsinizing, pelleted by centrifugation for 5 min at 1200 rpm, and 1 x 106 cells were resuspended in 0.5 ml ice cold PBS. Cells were then fixed by drop-wise addition to 70% ice-cold Ethanol and stored overnight at 4°C. Subsequently, cells were pelleted by centrifugation, washed in PBS containing 0.5% BSA and denatured in 2N HCl/0.5% Triton X-100 solution for 20 minutes at room temperature. The cells were washed in PBS/0.5% BSA and residual acid was neutralized by incubation of the cell pellet with 0.1M sodium borate, pH 8.5 for 2 minutes at room temperature. After an additional wash cells were immunostained with 0.5 µg FITC-conjugated mouse anti-BrdU monoclonal antibody (BD Biosciences) for 30 minutes at room temperature, protected from light, and centrifuged after addition of 1 ml of PBS/0.5% BSA. The cell pellet was then resuspended in 0.5 ml staining solution containing 20 µg/ml of propidium iodide (Molecular Probes) and 200 µg/ml of RNase A (Sigma) in 0.1% Triton X-100. After at least 30 minutes of incubation at room temperature, the cell suspension was passed through a 35-µm-pore-size cell strainer (Fisher) and analyzed by two-parameter flow cytometry using a Becton Dickinson FACSCalibur flow cytometer.
For analysis of apoptosis, serum starved or irradiated/starved stable Rat1A populations (treatments as above) expressing vector only (control), Brk WT and Brk Y-F were subjected to FACS analysis. After collection of floating cells and trypsinization of attached cells, the pooled fractions (1 x 106) were rinsed with PBS, resuspended in 0.5 ml of PBS and fixed by drop-wise addition to ice-cold 70% ethanol and stored for at least 16 hours at 4°C. Subsequently, cells were pelleted by centrifugation and incubated in 0.5 ml staining solution containing 20 µg/ml of propidium iodide (Molecular Probes) and 200 µg/ml of RNase A (Sigma) in 0.1% Triton X-100 for 30 min at room temperature. The cell suspension was passed through a 35-µm-pore-size cell strainer (Fisher) and flow cytometry was performed using a Beckton Dickinson FACSCalibur flow cytometer. To determine the cell cycle distribution by propidium iodide staining, data collected from the FACsort was analyzed using CellQuest and ModFit software (Becton Dickinson). For the purpose of analysis, acquired events were gated to eliminate cell aggregates and debris. A gated population of single diploid cells was analyzed.
Total RNA was prepared after the guanidine iso-thiocyanate method (Chomczynski and Sacchi, 1987) using TRIzol reagent (Invitrogen). Mouse tissues were dissected quickly and immediately homogenized in 3 ml TRIzol reagent. Total RNA was isolated following the manufacturer’s instructions.
Brk expression in the gastrointestinal tract was analyzed as described previously (Siyanova, et al., 1994), using [32P]-CTP-labeled antisense RNA probes. A pBlueScript SK II+ plasmid containing a 205-bp fragment encoding a portion of the mouse Brk catalytic domain was linearized at an XbaI site in the polylinker. As control for RNA levels and integrity, a pTRI plasmid (Ambion) containing a fragment of mouse cyclophilin was linearized. In vitro transcription was performed using T7 polymerase (Promega).
Expression of various cytokines in the murine gastrointestinal tract was analyzed by RNase protection assay using the RiboQuant multiprobe RNase protection assay system (BD Biosciences) according to the manufacturer’s instructions. Briefly, the mCK-2b multiprobe DNA templates were used for synthesis of [32P]-α-UTP-labeled anti-sense RNA probes by T7 polymerase-directed in vitro transcription. The highly-specific RNA probes can hybridize with target mouse mRNAs encoding interleukin-12p35 (IL-12p35), IL-12p40, IL-10, IL-1α, IL-1β, IL-1Ra, IL-18, IL-6, IFN-γ, migration inhibition factor (MIF) as well as housekeeping genes L32 and glyceraldehyde-3-phosphate-dehydrogenase (GAPDH) for assessment of RNA levels.
Twenty µg of total RNA for each sample or of yeast tRNA were precipitated with ethanol and resuspended in 30 µl hybridization buffer containing 3.95 x 105 cpm/µl of probe. Samples were hybridized for 12-16 hours at 56°C followed by treatment with 100 µl of ribonuclease digestion buffer containing 192 x 10-3 ng/ml of RNase A and 0.6 units/µl RNase T (BD Biosciences) for 45 minutes at 30°C. Samples were subjected to proteinase K digestion, ethanol precipitation, and subsequently resolved on denaturing polyacrylamide gels as described. The gel was dried and protected fragments were visualized by exposing to X-ray film (Midwest Scientific).
Mouse tissues were dissected as described above and immediately homogenized in 1.5 ml of cold lysis buffer (20 mM Hepes, pH 7.4, 1% Triton X-100, 150 mM NaCl, 1 mM EDTA, pH 8.0, 1 mM EGTA, pH8.0, 10 mM Na-pyrophosphate, 100 mM NaF, 5 mM Iodoacetic Acid, 0.2 mM PMSF, 10 µg/ml aprotinin, 10 µg/ml leupeptin, 10 µg/ml pepstatin A) using a polytron (Kinematica AG) at 5,000 rpm. After incubation on ice for 20 minutes, samples were sonicated for 15 seconds and centrifuged for 30 minutes at 30,000 rpm at 4°C.
Cells were grown as previously described. For total cell lysates, the cells were washed with ice-cold PBS and incubated in ice-cold lysis buffer (see above) for 20 minutes on ice with constant agitation. The cells were harvested with a cell scraper (Fisher Scientific), and the cell extracts were cleared by centrifugation at 14,000 rpm for 10 minutes.
Thirty micrograms of total cell lysate or 50-100 µg of tissue lysate were resuspended in SDS gel-loading buffer (0.15 M Tris-Cl, pH 6.8, 15% SDS, 50% glycerol, 1.5% bromphenol blue, 15% β-mercaptoethanol). Samples were boiled for 5 minutes and separated by SDS-PAGE at 60 V (0.4 V/cm2) through the separating and 120 V (0.7 V/cm2) through the resolving gel. The proteins from the SDS-polyacrylamide gels were transferred to polyvinylidene-difluoride (PVDF) membranes (Immobilon), as described (Sambrook, et al., 1989). Transfer was performed in transfer buffer (25 mM Tris, 190 mM glycine, 20% methanol) overnight at 20 V at 4 °C.
For detection of proteins, the membranes were incubated in blocking solution (5% nonfat dry milk in TBS-T (10 mM Tris-HCl, pH 7.5, 100 mM NaCl or 200 mM NaCl for Brk, 0.1% Tween 20) for 1 hour at room temperature. The filters were then incubated with primary antibody for 1 hour at room temperature (anti-mouse Brk polyclonal antibody 1:2000 and anti-Erk1/2 polyclonal antibody 1:2000 (Santa Cruz), anti-β-actin monoclonal antibody 1:5000 (Sigma)) or overnight at 4°C (anti-Phospho-Erk1/2 polyclonal antibody 1:1000, anti-Phospho-Ser473-Akt polyclonal antibody 1:1000, anti-Akt polyclonal antibody 1:1000, anti-Phospho-GSK-3α/β (Ser21/9) polyclonal antibody 1:1000, anti-GSK-3β polyclonal antibody 1:1000 (Cell Signaling)). Primary antibodies were detected using horseradish peroxidase-conjugated donkey anti-rabbit or sheep anti-mouse secondary antibodies (Amersham) at a dilution of 1:5000 for 1 hour at room temperature. Peroxidase reaction was visualized with SuperSignal West Dura Extended Duration Substrate for chemiluminescence (Pierce) and membranes were exposed to X-ray film.
Three hundred to 500 µg of tissue lysate were used per immunoprecipitation reaction. The volume of each sample was adjusted to 1 ml with lysis buffer and the samples were precleared by incubation with 1 µg of rabbit IgG (Santa Cruz) and 75 µl of 50% Protein A sepharose slurry (Amersham) for 1 hour at 4°C. After centrifugation for 10 minutes at 13,000 rpm at 4°C, the supernatant was transferred to a fresh eppendorf tube and incubated with 1 µg of anti-Akt polyclonal antibodies (Cell Signaling) overnight at 4°C on a rotary shaker. As controls, the lysate was incubated with 1 µg of rabbit IgG (Santa Cruz). The next day, 50 µl of 50% Protein A sepharose slurry were added to the lysate and incubated for 1 hour at 4°C, rotating. After incubation, immune complexes were collected by centrifugation for 10 minutes at 13,000 rpm at 4°C. The supernatant was aspirated and the beads were washed three times in lysis buffer. After the last wash, the beads were resuspended in 20 µl 2x sample buffer and the proteins denatured by boiling for 5 minutes. Proteins were resolved by 9% SDS-PAGE, transferred to membranes, and analyzed by Western blotting with the indicated antibodies.
In vitro Akt kinase assays were performed using a Nonradioactive Akt Kinase Assay Kit (Cell Signaling) according to the manufacturer’s instructions. Briefly, 300-500 µg of tissue lysates were incubated with immobilized Akt (1G1) monoclonal antibody overnight at 4°C. The resulting immune complexes were washed two times in cell lysis buffer and two times in kinase buffer. Then, in vitro kinase assay was performed using recombinant GSK-3 fusion protein as a substrate. The Akt immunoprecipitates were incubated with 0.2 mM ATP and 1 µg GSK-3 fusion protein in kinase buffer for 30 minutes at 30°C. Reactions were terminated with 25 µl 3x sample buffer, boiled for 5 minutes and the supernatant was separated by 12% SDS-PAGE. Phosphorylation of GSK-3 was measured by Western blotting using Phospho-GSK-3α/β (Ser21/9) polyclonal antibody.
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