Identification of Chromatin Remodeling Genes Arid4a and Arid4b as Leukemia Suppressor Genes

Mei-Yi Wu; Karen W. Eldin; Arthur L. Beaudet

Disclosures

J Natl Cancer Inst. 2008;100(17):1247-1259. 

In This Article

Methods

Animal Care

The mice (100 wild-type mice, 150 Arid4a -/- mice, 40 Arid4a ± Arid4b ± mice, and 40 Arid4a -/- Arid4b ± mice) were all bred and maintained according to a protocol approved by the Baylor College of Medicine Animal Care and Use Committee at the institution's specific pathogen-free mouse facility, which is approved by the American Association for Accreditation of Laboratory Animal Care and is operated in accordance with current regulations and standards of the US Departments of Agriculture and of Health and Human Services.

Beginning at 5 months of age, the Arid4a -/- mice and Arid4a -/- Arid4b ± mice were monitored weekly for signs of morbidity. Blood was obtained weekly for complete blood cell counts. Mice were sacrificed when they met any of the following criteria: 1) obvious morbidity, in which case mice proved to have either severe anemia or greatly increased white blood cell (WBC) counts; 2) severe anemia or high WBC counts with milder or impending morbidity; or 3) marked splenomegaly or hepatomegaly. Mice were anesthetized with isoflurane and then killed by cervical dislocation. The mice were dissected, and organs were examined for the presence or absence of tumor enlargement. The lung, spleen, and liver were removed, and bone marrow cells from both femurs were flushed with syringes.

Hematologic Analysis

Mice (52 wild-type mice, 50 Arid4a -/- mice, 40 Arid4a ± Arid4b ± mice, and 10 Arid4a -/- Arid4b ± mice) were anesthetized with isoflurane, and blood from each mouse was obtained from the retro-orbital venous plexus. Complete blood cell counts were determined with an analyzer (ADVIA 120 Hematology System; Bayer Diagnostics).

Peripheral blood and bone marrow smears were stained with Wright-Giemsa stain. On the peripheral blood smears (from 10 wild-type mice, more than 20 chronic myelomonocytic leukemia [CMML]-like Arid4a -/- mice, three acute myeloid leukemia [AML] Arid4a -/- mice, and five AML Arid4a -/- Arid4b ± mice) and bone marrow smears (from five wild-type mice, five CMML-like Arid4a -/- mice, two AML Arid4a -/- mice, and three AML Arid4a -/- Arid4b ± mice), we analyzed blasts, immature precursors, WBCs (including lymphocytes, neutrophils, monocytes, eosinophils, and basophils), red blood cells (RBCs), and platelets.

Histologic Analysis

For bone marrow examination, femurs from three wild-type mice and three Arid4a -/- mice were fixed and decalcified by immersion in Cal-EXII solution (Fisher Scientific, Pittsburgh, PA). Spleen and liver from five wild-type mice, five CMML-like mice, and five AML mice were fixed in 10% formalin (Fisher Scientific). Histology was performed on 5-µm paraffin-embedded tissue sections that were stained either with reticulin to show myelofibrosis for femur bone sections (three slides from three wild-type mice and three slides from three Arid4a -/- mice) or with hematoxylin and eosin to show extramedullary hematopoiesis in spleen and liver (one slide each from five wild-type mice, five CMML-like Arid4a -/- mice, and five AML Arid4a -/- Arid4b ± mice).

Flow Cytometry

Spleens were removed from mice and dissociated into single cells by sliding tissue between two superfrost microscope slides. RBCs were lysed by hypotonic buffer (NH4Cl, 0.14 M; Tris, 0.017 M, pH 7.2). Cells from spleen or bone marrow were gently filtered through 70-µm cell strainers (BD Falcon, VWR, Batavia, IL) and were stained with directly fluorescein-5-isothiocyanate (FitC)-conjugated antibodies to c-kit (2B8, eBioscience, San Diego, CA), Sca1 (D7, eBioscience), Mac1 (M1/70, BD Biosciences Pharmingen, San Jose, CA), Gr1 (RB6-8C5, BD Biosciences Pharmingen), B220 (RA3-6B2, BD Biosciences Pharmingen), CD19 (1D3, BD Biosciences Pharmingen), CD3 (145-2C11, BD Biosciences Pharmingen), or Ter119 (Ter119, eBioscience). A lineage antibody cocktail (Lin) included antibodies against Mac1 (M1/70), Gr1 (RB6-8C5), CD4 (L3T4, BD Biosciences Pharmingen), CD8 (53-6.7, BD Biosciences Pharmingen), CD19 (1D3), and B220 (RA3-6B2) to stain bone marrow cells. All antibodies were diluted (1:100) in phosphate-buffered saline (PBS). Hematopoietic stem cells (HSCs) (Lin-Sca1+c-Kit+), common myeloid progenitors (CMPs) (Lin-Sca1-c-Kit+), granulocytes and monocytes (Gr1+Mac1+), erythroid cells (Ter119+), T cells (CD3+), and B cells (B220+CD19+) were analyzed. For apoptosis analysis, bone marrow cells were stained with annexin V-FITC conjugates (BD Biosciences Pharmingen). Cells were then washed and analyzed by flow cytometry (Beckman-Coulter EPICS XL-MCL).

Immunofluorescence

Bone marrow cells flushed from femur bones were fixed in 4% paraformaldehyde, spread on glass slides, and permeabilized in cold acetone. Subsequently, cells were blocked with 5% bovine serum albumin in PBS, followed by incubation with primary antibodies against trimethylated H3K9, H3K4, or H4K20 (07-442 for H3K9me3, 05-745 for H3K4me3, and 07-463 for H4K20me3, Upstate, Charlottesville, VA) at a dilution of 1:200 in blocking solution. Then, cells were washed with blocking solution and incubated with Alexa 488-conjugated goat anti-rabbit secondary antibody (Molecular Probes, Invitrogen, Carlsbad, CA). Cells were mounted with Vectashield containing 4',6-diamidino-2-phenylindole (DAPI) (Vector Laboratories, Burlingame, CA) and analyzed on a deconvolution fluorescence microscope (DeltaVision Restoration Microscope, Zeiss, Jena, Germany).

Western Blotting

Bone marrow cells flushed from femurs were resuspended in lysis buffer (10 mM HEPES, pH 7.9, 1.5 mM MgCl2, 10 mM KCl, and protease inhibitors [Complete protease inhibitor cocktail tablets, Roche, Indianapolis, IN]). Histones were acid extracted by 0.2 N HCl and precipitated with 20% trichloroacetic acid. Proteins were electrophoresed on 7.5% Tris-Cl ready gels (Bio-Rad, Hercules, CA) and then transferred to nitrocellulose membranes (Bio-Rad). The incubations with the appropriate primary antibodies were performed as follows: rabbit anti-H3 (1:1000 dilution, ab1791, Abcam, Cambridge, MA), goat anti-H4 (1:100 dilution, sc-8658, Santa Cruz Biotechology, Santa Cruz, CA), rabbit anti-H2AX (1:5000 dilution, BL179, Bethyl, TX), rabbit anti-H3K4me3 (1:4000 dilution, 05-745, Upstate), rabbit anti-H3K9me3 (1:500 dilution, 07-523, Upstate), or rabbit anti-H4K20me3 (1:200 dilution, 07-749, Upstate). The membranes were then incubated with either goat anti-rabbit IgG horseradish peroxidase (HRP) (1:5000 dilution, ac-2004, Santa Cruz Biotechnology) or donkey anti-goat HRP (1:5,000 dilution, ac-2020, Santa Cruz Biotechnology). Antibody binding was detected by enhanced chemiluminesence (ECL, Amersham, Piscataway, NJ).

Reverse Transcription-Polymerase Chain Reaction

Total RNA was purified from bone marrow cells using an RNeasy plus kit (Qiagen, Hilden, Germany). Total RNA (5 µg) was used for reverse transcription to synthesize the first-strand cDNA (Superscript III First-strand synthesis system, Invitrogen). cDNA (5 µg) was used for polymerase chain reaction (PCR). PCR conditions and primer sequences have been described[28] and/or are listed in Supplementary Table 1, available online. Hprt transcripts were amplified as a control for gene expression.

Statistical Analysis

Means and the accompanying 95% confidence intervals were calculated from at least three independent experiments. Group means were compared using a two-sided Student t test. P values less than .05 were considered to be statistically significant.

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