BCR-ABL1 mediates up-regulation of Fyn in chronic myelogenous leukemia
Blood Ban et al.
111: 2904
Supplemental materials for: Ban et al
Materials and methods
Antibodies. The following antibodies were purchased: polyclonal anti-Fyn (FYN3) and anti-Lyn from Santa Cruz Biotechnology (Santa Cruz, CA); monoclonal anti-Fyn from Alexis (San Diego, CA); polyclonal anti-actin from Sigma (St. Louis, Missouri) anti-mouse IgG peroxidase conjugate (Sigma, St. Louis, Missouri), anti-rabbit IgG horseradish peroxidase linked whole antibody (from donkey) and ECL plus Western Blotting detection system were obtained from Amersham Bioscience (Amersham Bioscience, UK). BCR-ABL1 and c-abl were both detected using monoclobal anti-Abl 8e9 1.
Tissue microarray. Bone marrow from CML patients in chronic phase (n=10), accelerated phase (n=8), and blastic phase (n=16), and non-CML patients (n=5) were collected after approval from the U.T. M.D. Anderson Cancer Center Institutional Review Board. The diagnosis of CML was based on the criteria described in the World Health Organization classification. All CML specimens were fixed in formalin, routinely processed, and embedded in paraffin. To construct the tissue microarray, 3 representative cores from each sample were selected from the paraffin-embedded tissue blocks. Triplicate cores from non-CML patient bone marrows were included in the tissue microarray as internal controls. Histological sections were first deparaffinized in xylene and rehydrated using a graded series of ethanol. A 3-step streptavidin–biotin–horseradish peroxidase method was used after heat-induced epitope retrieval. Endogenous peroxidase activity was blocked for 30 minutes in 3% hydrogen peroxide, and, subsequently, the slides were incubated with Protein Blocking Solution (DakoCytomation) for 15 minutes. Thereafter, slides were incubated overnight at 4°C with polyclonal anti-Fyn (Santa Cruz, CA) diluted 1:400 in 0.1% bovine serum albumin and 50 mM Tris-HCL buffer at pH 7.6. Detection of the immunoreaction was achieved using the LSAB+ kit (DakoCytomation), which contains the secondary biotinylated antibody (incubation time, 20 minutes) and the streptavidin/horseradish peroxidase complex (incubation time, 20 minutes). 3,3′-Diaminobenzidine/H2O2 (DakoCytomation) was used as the chromogen and hematoxylin (DakoCytomation) as the counterstain. Application of mouse immunoglobulin G1 antibody (DakoCytomation) was used as a negative control to exclude unspecific cross-reactions of the primary antibodies in all experiments. For all immunostains, the percentage of positively stained cells was determined by counting at least 100 neoplastic cells in each of the triplicate cores. All immunostained slides were evaluated without knowledge of CML stage or clinical outcome.
Design of lentiviral contructs for Fyn shRNA and rescue. For design of lentivirally encoded Fyn shRNA, two 0.4 kb human U6 promoter Fyn shRNA (target #1 GCACGACAAGCTGGTCCAG and target #2 GCACTACCCCAGCTTCGGT, hereafter referred to as U613 and U67) cassettes were excised from the 6.2 kb TranSilent shRNA vectors (Panomics Inc, Redwood, CA) by BamH I/Hind III, blunted with Klenow, and then cloned into the 11 kb pLVTH lentivirus vector which had been cut by BamH I/Cla I and blunted by Klenow to exclude the H1 promoter cassette. The pLV control vector contains a non-targeted sequence as described previously2.
To generate the rescue constructs, four nucleotides in shRNA target #1 and target #2 sequence regions in wild type Fyn cDNA were replaced, but all of them are silent mutants (GCACGACAAACTCGTCCAG, GCACTACCCGAGGTTCGGT), thereby encoding the same amino acid as wild type Fyn, but containing different nucleotide sequences in the Fyn shRNA target region. The resulting Fyn cDNA was subcloned into the retroviral vector pMIGR1-DsRed2, which was constructed by replacing GFP with the DSRed2 gene.
Recombinant lentiviruses were made by calcium phosphate transfection of 50% confluent 293T cells in 10 cm plates with 20 µg of LV transfer vector, 15 µg of packaging plasmid pAX2 DNA, and 6 µg of pMD2G-VSVG envelope plasmid DNA. For the production of retrovirus for Fyn shRNA rescue, 10 µg of pMIGR1-rescue Fyn DNA, 10 µg of packaging plasmid pPCPG DNA, and 10 µg of pVSVG envelope plasmid DNA were co-transfected into 293T cells. The recombinant viruses were harvested by collecting the media 48 h post-transfection.
K562 cells were infected by plating 200,000 cells per well in twelve well plates in 2 ml of 0.45 µm filtered virus medium. Polybrene was added to a final concentration of 10 µg/ml and spinoculated at 1000 g for 90 min at room temperature. After a 6 h incubation, cells were resuspended and cultured in fresh RPMI 1640 media. For lentivirus-mediated Fyn shRNA, the target #1 lentivirus infected K562 cells were sorted by GFP and then infected again by the target #2 lentivirus.
Western blotting. Cells were lysed in RIPA lysis buffer containing protease inhibitors and rotated at 4°C for 1 hour. Lysates were clarified by centrifugation. Protein content in lysates was measured using the Bio-Rad DC protein assay (Hercules, CA). Equal amounts of protein were loaded onto a 12% gel SDS-PAGE gel. Proteins in the gel were transferred to nitrocellulose. Membranes were incubated overnight at 4°C with primary antibody and ECL anti-rabbit IgG, horseradish peroxidase linked whole antibody or anti-mouse IgG peroxidase conjugate for 1 hour at room temperature. Proteins were visualized with ECL and analyzed by either the Gel Documentation and Analysis System (Bio-Rad laboratories, Hercules, CA) or NIH Image J3.
BCR-ABL-1 and Fyn kinase assays. Lysates from BaF3 vector, BaF3 p210, and K562 cells infected with lentivirus containing non-targeted (LV) or Fyn specific shRNA (U613 + U67) sequences were generated using lysis buffer containing 100 mM NaCl, 50 mM Tris pH 7.6, 1 mM EDTA, 0.1% Triton-x 100, 1% NP-40, and 1% 2-mercaptoethanol. BCR-ABL-1 and Fyn were immunoprecipitated from 500 µg of lysate for BCR-ABL1 or 1 mg lysate for Fyn using antibodies mentioned above and Protein A beads (Santa Cruz Biotechnology). Immunoprecipitates were washed twice with 500 µl of lysis buffer and once with kinase assay buffer (25 mM Tris Hcl pH 7.4, 10 mM MgCl2, 1 mM DTT). Kinase reaction was performed using 1 µCi 
-32PATP (6,000 Ci/mmol; GE Healthcare) 10 µg of either Crk (for BCR-ABL1) or Sam 68 (for Fyn), 5 µM of cold ATP, and kinase buffer for a total of 20 µl of reaction volume. The immunoprecipitates were boiled in SDS sample buffer and separated by electrophoresis on a 15% SDS-polyacrylamide gel. Phosphorylated protein was subsequently detected by autoradiography.
Detection of Fyn mRNA. Total RNA of cells was extracted using the RNeasy mini kit (Qiagen, Hilden, Germany). First-strand cDNA was synthesized using an Omniscript RT kit (Qiagen, Hilden, Germany). A PCR approach was used to amplify Fyn. The outer and inner primers for Fyn were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). For the first round of PCR, cDNA generated from 2 µg of total RNA was added to PCR master mix (AB Applied Biosystems, Branchburg, New Jersey) with 1 µM of outer primers. PCR cycles were performed with the following temperature profile: 95°C for 2 min, then 60 cycles consisting of 30 s at 94°C, 30 s at 55°C, 1 min at 72°C, and a final elongation step at 72°C for 10 min. Actin was amplified under the same conditions.
In vivo mouse experiments. A CML mouse transplant model was set up as described previously4. Briefly, two four month old wild type C57/Bl6 mice were injected with 200 mg/kg 5-fluorouracil. Mice were sacrificed and marrow cells from the femur and tibia were flushed and cultured with MSCV p210 BCR-ABL1. Strain and age matched recipient C57/Bl6 mice were irradiated with 600 cGy and one million donor virus infected cells were injected into the tail vein.
For assessment of Fyn knockdown in vivo, 20 million K562 cells transfected with scrambled shRNA or Fyn shRNA were introduced by tail vein injection into four week old female SCID mice (Taconic Labs, Albany, NY). Injected cells were > 90% viable as determined by trypan blue staining. Mice were followed for four weeks and peripheral blood was drawn by tail vein bleeds. Blood was submitted to the Veterinary Lab Medicine Department at M.D. Anderson Cancer Center for assessment of complete blood counts (CBC). Mice were followed until morbid and then euthanized by IACUC approved methods.
Statistical analyses. Significance was calculated using the Student’s t-test for all in vitro experiments. For CML specimens, the Wilcoxon test was used. The Bonferroni correction to the significance level (alpha = 0.05/4 = 0.0125) was used to control the overall significance level to 0.05. All Western blots and RT-PCR were conducted at least three times and depicted results are representative of several independent experiments.
REFERENCES:
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