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Blood, Vol. 114, Issue 2, 360-370, July 9, 2009
IGF-IR tyrosine kinase interacts with NPM-ALK oncogene to induce survival of T-cell ALK+ anaplastic large-cell lymphoma cells
Blood Shi et al.
114: 360
Supplemental materials for: Shi et al
Antibodies: Antibodies obtained from Santa Cruz Biotechnology (Santa Cruz, CA) included IGF-IRβ (catalogue number: sc-9038), IGF-I (sc-9013), STAT3 (sc-8019), pSTAT3 (Tyr705; sc-7993-R), ALK (sc-6346), JAK3 (sc-513), Bcl-2 (sc-7382), cyclin B1 (sc-7393), Cdc2 (sc-52316), pCdc2 (Thr14/Tyr15; sc-12340-R), and p16 (sc-56330); from Cell Signaling Technology (Danvers, MA) were pIGF-IR (Tyr1131; 3021), Akt (9272), pAkt (Ser473; 4051), pALK (Tyr1586 equivalent to Tyr646 in pNPM-ALK; 3343), and pALK (Tyr1604; Tyr664 in pNPM-ALK; 3341); from Zymed Laboratories (South San Francisco, CA) were IGF-IRβ (39–6700) and Bcl-XL (18–0217); from Dako (Carpinteria, CA) was ALK (M7195); from R&D Systems (Minneapolis, MN) were IGF-IR blocking antibody (MAB391) and IGF-I neutralizing antibody (AF-291-NA); and from Sigma (St. Louis, MO) was β-Actin (A-2228). Relative antigen density of IGF-IR: The relative antigen density of IGF-IR was assessed using flow cytometry. PE-labeled anti-human IGF-IR antibody (555999; BD Biosciences, San Jose, CA) and PE-labeled anti-mouse IgG1 isotype control (555749; BD Biosciences) were used. IGF-IR antigen density per cell was determined according the manufacturer’s instructions by using the QuantiBRITE PE kit (340495; BD Biosciences) and was calculated considering that the PE:mAb ratio was 1:1. Cell viability and proliferation assays: Cell viability was evaluated by exclusion of staining by the trypan blue dye method. Cell proliferation analysis was performed using an ELISA-based kit (Roche, Mannheim, Germany). Absorbance was measured at 370 nm (reference wavelength: 492 nm). Apoptosis detection: Apoptosis detection was performed using flow cytometry after staining the cells with annexin V-FITC and propedium iodide (PI) using apoptosis detection kit (556547, BD Biosciences). Cells stained for annexin V or for annexin V and PI were considered apoptotic. In addition, apoptosis detection was performed by evaluating cellular morphology after staining cytospin-prepared slides with Giemsa. Cell-cycle analysis: Analysis of the cell cycle was performed using a commercially available kit (CycleTEST PLUS DNA Reagent Kit, BD Biosciences), whereby PI-stained nuclei were analyzed by flow cytometry (FACscan, BD Biosciences). The cell-cycle phases were analyzed by ModFit LT 2.0 software (Verity Software House, Topsham, ME). Cell migration assay: The effect of IGF-I on ALK+ ALCL cell migration was studied using 24-well Transwell plates with polycarbonate membranes (3421; Corning Costar, Cambridge, MA). Briefly, cells were loaded, with/out anti–IGF-IR blocking antibody, onto the upper compartment. Simultaneously, 500 ng/mL IGF-I with/out anti–IGF-I neutralizing antibody was loaded in culture medium onto the lower compartment. For control cells, reagents were not loaded onto the lower compartment. Plates were incubated for 4 h at 37°C, and cells migrating through the membrane into the lower chamber were counted using a particle counter and size analyzer (Coulter, Miami, FL). Colony formation in soft agar: Colony formation was assayed using cell transformation detection kit (Chemicon, Temecula, CA). Harvested cells were resuspended in 0.4% agar in culture medium with or without 2 µM PPP at a density of 2.5 × 103 cells/0.5 mL/plate in a 6-well plate, and seeded into solidified 0.8% agar in culture medium. Plates were incubated for 2–4 weeks at 37°C in 5% CO2. Fresh culture medium with/out 2.0 µM PPP was supplemented 1–2 times per week. Colonies were stained and photographed using the FluorChem 8800 imaging system (Alpha Innotech, San Leandro, CA). Autocrine release of IGF-I: The level of IGF-I in cell culture supernatants was detected by using an ELISA-based kit (Quantikine, R&D Systems). Because IGF-I might be present in FBS, 4.0 × 105 cells/mL were maintained in serum-free medium for 24 h before testing. Serum-free cell culture supernatant (20 mL) was concentrated using centrifugal filter devices (Centriplus, Millipore, Bedford, MA) to a final volume of 2.0 mL. Optical densities were measured at 450 nm (reference wavelength: 570 nm). IGF-I was used to create a standard curve. Tyrosine kinase activity: ALK or IGF-IR tyrosine kinase activity was measured using a commercially available kit (Takara Bio, Otsu, Shiga, Japan). Cell lysates were prepared and the specific antibody was used for immunoprecipitation. Agarose beads conjugated with protein A/G were added. Kinase reactions were initiated by adding ATP-2Na into immobilized wells coated with peptide substrate. Next, horseradish peroxidase-conjugated antiphosphotyrosine (PY20) antibody was added to each well and developed by addition of horseradish peroxidase and TMBZ. The reaction was stopped with 1N H2SO4 and absorbance was measured at 450 nm in a microplate reader (MRX II; Dynex, Frankfurt, Germany). The ATP-free reaction was used as a negative control. Confocal laser-scanning electron microscopy: Cytospin slides (ALK+ ALCL cells) or cell culture chambers (P6 or R−) were washed with PBS and subjected to a fixation-permeabilization step in 4% paraformaldehyde for 20 min, followed by 20 min in 0.1% Triton X-100 at room temperature. After blocking with 10% FBS in PBS for 45 min, cells were incubated with a mixture of polyclonal goat anti-ALK antibody (diluted 1:25 in PBS) and rabbit anti–IGF-IRβ antibody (1:100) overnight at 4°C and stained for 90 min with a mixture of secondary antibodies, including FITC-conjugated anti-goat IgG (1:300) and Cy3-conjugated anti-rabbit IgG (1:800) (Jackson ImmunoResearch Laboratories, West Grove, PA). Images were visualized by confocal laser-scanning microscopy (LSM 510; Carl Zeiss MicroImaging, Thornwood, NY). Lysis buffer: Lysis buffer contained 25 mM HEPES (pH 7.7), 400 mM NaCl, 1.5 mM MgCl2, 2 mM EDTA, 0.5% Triton X-100, 0.1 mM PMSF, 3 mM DTT, phosphatase inhibitor cocktail (20 mM β-GP, 1 mM Na3VO4; Roche), and protease inhibitor cocktail (10 µg/ml leupeptine, 2 µg/ml peptsatin, 50 µg/ml antipain, 1× benzamidine, 2 µg/ml aprotinin, 20 µg/ml chymostatin; Roche). Patients, tissue microarray, and immunohistochemical staining: Tissue samples from ALK+ ALCL patients and the tissue microarray have been previously described.51 Approval of the ethical research committee at Cross Cancer Institute, Edmonton, AB, Canada, was secured prior to the studies. Patients granted informed consents in accordance with the Declaration of Helsinki. Immunohistochemical staining was performed on sections from the tissue microarray as well as on formalin-fixed and paraffin-embedded sections from cellblocks prepared from cell lines using standard techniques.51 Antibody dilution was 1:30 for IGF-IR and 1:150 for IGF-I. Photomicrographs were obtained using a Nikon Microphot FXA microscope (Nikon Instruments, Melville, NY) and an Olympus DP70 camera (Olympus America, Melville, NY).
Files in this Data Supplement:
- Figure S1. Expression of IGF-IR and IGF-I in primary tumors from ALK-negative ALCL patients (JPG, 288 KB)
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Using immunohistochemical staining, we detected the expression of IGF-IR in 3 (60%) of 5 ALK-negative ALCL primary human tumors. An example of a positive tumor is shown in (A) and of a negative tumor in (B). None of 6 primary ALK-negative ALCL tumors expressed IGF-I. Two different examples of these tumors are shown in (C) and (D) (original magnification ×400).
- Figure S2. Additional studies support the physical association between IGF-IR and NPM-ALK (JPG, 223 KB)
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(A, left panel) Additional co-immunoprecipitation studies confirm the physical association between NPM-ALK and IGF-IR or pIGF-IR. (A, right panel) To rule out nonspecific binding of the protein of interest, a control was employed in all experiments where beads were used without an antibody. The results from the DEL cell line are shown as a representative example. (B) Confocal electron microscopy of Karpas 299 and SUP-M2 cells shows subcellular co-localization of IGF-IR and NPM-ALK (original magnification ×600). P6 cells were used as positive and negative controls for IGF-IR and NPM-ALK, respectively, whereas R− cells were used as a negative control for the 2 proteins. The red fluorescence (Cy3) represents IGF-IR and is located in the cell membrane and cytoplasm. The green fluorescence (FITC) represents NPM-ALK, which is localized in the cell membrane, cytoplasm, and nucleus. The yellow signal represents co-localization of IGF-IR and NPM-ALK (overlap), which is restricted to the cell membrane and cytoplasm and absent from the nuclei.
- Figure S3. NPM-ALK increases the phosphorylation of IGF-IR (JPG, 56.6 KB)
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Densitometry of the Western blot bands of NPM-ALK, pIGF-IR, and β-Actin shown in Figure 4H demonstrates that the increase in pIGF-IR levels is approximately 100% of its baseline levels after transfecting the P6 cells with WT NPM-ALK. In contrast, after transfection with NPM-ALKK210R, pIGF-IR levels remain similar to its levels after transfection with empty vector.
- Figure S4. Blockade of IGF-IR decreases the viability and induces apoptosis and cell cycle arrest of ALK+ ALCL cells (JPG, 220 KB)
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(A) Compared with control cells, treatment with PPP (1.0 µM for 24 h) induces morphologic changes (nuclear condensation and fragmentation; black arrowheads) consistent with apoptotic cell death in the 5 ALK+ ALCL cell lines. In addition, PPP markedly increased atypical mitotic forms (green arrowheads), consistent with G2/M-phase cell-cycle arrest. Notably, PPP did not induce similar effects in benign human T-lymphocytes (original magnification ×600 for ALK+ ALCL and ×1000 for the T-lymphocytes). Specific targeting of IGF-IR using siRNA at 48 h decreases the viability (B) and induces apoptosis (C) of ALK+ ALCL cell lines. Apoptotic cells increased by 92%, 71%, and 60% above their baseline levels in the Karpas 299, SU-DHL-1, and SR786 cell lines, respectively.
- Figure S5. Supershift EMSA (JPG, 118 KB)
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Anti-STAT3 antibody, but not an irrelevant anti-JAK3 antibody, diminished the density of the band corresponding to the STAT3-oligonucleotide complex in Karpas 299 cells and led to the formation of the slower migrating “supershifted” band.
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