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Blood, Vol. 113, Issue 19, 4690-4701, May 7, 2009
Nf1 haploinsufficiency and Icsbp deficiency synergize in the development of leukemias
Blood Koenigsmann et al.
113: 4690
Supplemental materials for: Koenigsmann et al
pERK/pAKT-activity. Whole bone marrow cells were stimulated with GM-CSF at indicated concentrations. The cells were fixed in 4% paraformaldehyde and afterwards permeabilized with 90% methanol. For intracellular staining, pERK antibody (phospho-p44/42, clone D13.14.4E, and pAKT, clone H193H12 Cell Signalling Technologies, Beverly, MA, USA) and fluorochrome-coated IgG antibody (Alexa Fluor 488 F(ab′)2 fragment of goat anti-rabbit IgG; Invitrogen) were used according to the manufactures protocol. GMP and CMP isolation and global gene expression array. Bone marrow derived lin–IL-7Rα–Sca-1– BM cells from ICSBP wt and −∕− mice were sorted for expression of c-Kit versus IL-3Rα as previously described.1 Sorted c-Kit+IL-3Rα+ cells were stained for expression of FcγR (CD16/CD32) versus CD41, and further separated into FcγRhiCD41–. This population has been previously reported to contain exclusivly granulocyte/monocyte progenitor cells (CFU-GM). Lineage antibodies were B220 (clone RA3-6B2), CD3ε (clones 145-2C11 or 17A2), Gr-1 (clone RB6-8C5), Mac1 (clone M1/70), and TER119 (all labeled with fluorescein isothiocyanate FITC). Further antibodies were phycoerythrin (PE)–labeled anti–IL-3Rα (clone 5B11), biotinylated anti-FcγRII/III (clone 2.4G2), FITC-labeled anti-CD41 (clone MWreg30), PE-labeled CD71 (clone C2), allophycocyanin (APC)–labeled anti-CD117 (clone 2B8), FITC-labeled or biotinylated anti–Sca-1 (clone D7) (all from BD), and FITC-labeled anti–IL-7Rα (clone A7R34) (a gift from S. Nishikawa, RIKEN Center for Developmental Biology, Kobe, Japan). Second-step reagents were streptavidin-tricolor (Caltag, Burlingame, CA), or streptavidin-APC (Molecular Probes, Eugene, OR). Total RNA was isolated from purified GMP population from 10- to 16-week-old mice using the RNeasy kit including DNase digestion (Qiagen, Hilden, Germany). Total RNA (5 µg) was used to generate cDNA according to the technical manual (Affymetrix, Santa Clara, CA). cRNA was generated with the BioArray high-yield transcript labeling kit (ENZO, Farmingdale, NY), and 15 µg cRNA was hybridized to Affymetrix mouse expression 430A arrays at 45°C for 16 hours. DNA chips were stained, washed, and scanned according to the manufacturer's protocol. Scanned GeneChip DAT files were analyzed by the GeneChip Analysis Suite Software (Affymetrix) with global scaling to 500. Further analysis of data output was carried out using Data Mining Tool and NetAffx Web-Based Database (Affymetrix) as well as Microsoft Excel. The data have been submitted to the GEO Web site (http://www.ncbi.nlm.nih.gov/geo; submission no. GSE1584 NCBI GEO). Chromosome preparation. For preparation of metaphase chromosomes, cells were treated with colcemide at a concentration of 0.035 µg/ml for 6 hours, incubated in 0.075 M KCl for 20 minutes at 37°C and fixed in a freshly prepared mixture of methanol: acetic acid (3:1) at room temperature. Cells suspensions were dropped onto glass slides in a climate chamber (Polymer, Kassel, Germany) at 22°C and 48% humidity. Spectral karyotyping (SKY). SKY was performed as previously described2 and according to the manufacture’s instructions (ASI; Applied Spectral Imaging, Ltd., MigdalHaEmek, Israel). Spectral images were acquired using an epifluorescence microscope equipped with an interferometer (SpectraCubeTM, ASI), a custom designed optical filter and the SkyViewTM software (ASI). Microarray-based comparative genomic hybridization (array CGH). Array CGH experiments were performed as previously described using a cDNA microarray platform obtained from the Stanford Functional Genomics Facility and genomic DNA isolated from murine bone marrow.3 Background-subtracted fluorescence ratios were normalized for each array by setting the average fluorescence ratio for all array elements equal to 1. Genes were considered reliably measured if the fluorescence intensity for the Cy3 reference channel was at least 1.4-fold above background. The complete microarray dataset is available at the Stanford Microarray Database (http://smd.stanford.edu).4
Files in this Data Supplement:
- Table S1. Expression of Nf1-transcripts (probe set IDs: 1438067_at and 1452525_a_at) in isolated GMP via Affymetrix gene chip expression array (PDF, 31.4 KB) -
GeneChip Analysis Suite Software (Affymetrix) with global scaling to 500 was performed from isolated GMP (Icsbp +∕+ N=2, and Icsbp −∕− N=3; first number denotes array series, second number denotes sample in series) and analysis of data output was carried out using Data Mining Tool and NetAffx Web-Based Database (Affymetrix) as well as Microsoft Excel. A: denotes absent call, P: denotes present; M: denotes intermediate. The data have been submitted to the NCBI Gene Expression Omnibus database (http://www.ncbi.nlm.nih.gov/geo; series record GSE6821).
- Figure S1. Distribution of lymphocytes in all four genotypes (JPG, 128 KB)
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(A) Icsbp−∕−Nf1+∕− compared to Icsbp−∕−Nf1+∕+ mice showed no reduction of lymphocytes in the peripheral blood. Leukocytes were stained after lysis of erythrocytes with antibodies against CD11b and B220 and percentages of B-lymphocytes were determined. Absolute leukocyte numbers were determined using an automated blood cell counter and absolute lymphocyte numbers were calculated. Mice were aged between 2–4 months. Dots depict individual measurements, lines identify medians, level of significance (Mann-Whitney-U) comparing Icsbp−∕−Nf1+∕− and Icsbp −∕−Nf1+∕+ mice is shown above dots (N=5). (B) No difference in B-lymphocytes between Icsbp−∕−Nf1+∕− and Icsbp −∕−Nf1+∕+ mice. Leukocytes isolated from bone marrow, spleen and peripheral blood were stained with antibodies against CD11b and B220 and percentages of B-lymphocytes were determined. Statistical significance (Mann-Whitney-U) between Icsbp−∕−Nf1+∕− and Icsbp+∕+Nf1+∕− (**) as well as between Icsbp−∕−Nf1+∕+ and Icsbp+∕+Nf1+∕+ (*) is indicated (N=5).
- Figure S2 (JPG, 134 KB)
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(A) No differences in primary CFU-GM colony formation between Icsbp −∕− Nf1 +∕− and Icsbp −∕− Nf1 +∕+ mice. Bone marrow (A) were grown in 1 ml of methylcellulose supplemented with combinations of rmGM-CSF and SCF or IL-3 and SCF as described in materials and methods. Bone marrow and spleen cells (N=3–4) (B) were grown with rmGM-CSF. Cultures were performed in duplicates, incubated at 37°C with 7% CO2 and cultured for 8 – 10 days. (N=6–7), Mann-Whitney-U-Test. (C) No difference in cytokine response between Icsbp−∕−Nf1+∕− and Icsbp−∕−Nf1+∕+ primary ckit+ cells. Primary ckit+ cells were grown in duplicates of 1 ml of methylcellulose supplemented with decreasing amounts of GM-CSF (1, 0–02, 0.1, 0.02 ng/ml) SCF (10ng/ml). ckit+ cells were incubated at 37°C with 7% CO2 and colonies were counted on day 7 post plating. Independent of cytokine combination or concentration, there was no difference in colony numbers between Icsbp−∕−Nf1+∕− and Icsbp−∕−Nf1+∕+ mice. Therefore we could neither detect a cytokine hypersensitivity nor any differences in cytokine response for Icsbp−∕−Nf1+∕− primary ckit+ cells in colony forming unit assays (N=3). (D) No increased ERK- and AKT activity Icsbp−∕−Nf1+∕− bone marrow cells. To determine differences in ERK and AKT-activity primary ckit+ cells were stimulated for 0, 15 and 45 minutes with GM-CSF at 0.1, 1 and 10 ng/ml. pERK and pAKT were detected by intracellular flow cytometry and staining is represented as Median Fluorescence Intensity (MFI). In direct comparison with Icsbp−∕−Nf1+∕+, no significant increase in MFI could be detected in Icsbp−∕−Nf1+∕− bone marrow cells (N=3).
- Figure S3. Simplified scheme of hierarchy of different hematopoietic progenitor populations and expression of Nf1 in different progenitor populations (JPG, 123 KB)
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(A) Simplified scheme of hierarchy of different hematopoietic progenitor populations modified according to Adolfsson et co-workers.5 (B) Expression of Nf1 and Gapdh (control transcript) in isolated CMP, GMP and in three independently generated myeloid committed c-kit+/(Gr-1+; CD11b+) progenitor cell samples. Nf1 and Gapdh mRNA expression was detected using reverse transcriptase PCR with sequence specific primers from total RNA isolated from FACS sorted progenitor populations from wild type and Icsbp−∕− mice. Nf1 expression was undetectable in GMP as well as in myeloid committed ckit+ progenitor cells.
- Figure S4. Diagnostic criteria for leukemias (JPG, 107 KB)
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Leukemias were diagnosed according to a consensus protocol.6 Acute leukemia (in contrast to chronic MPD) was defined as a disease with expansion of the hematopoietic system causing the imminent death (within 24 hours) of the mouse. MPD like myeloid leukemia was defined as acute leukemia with predominantly granulocytic (g) differentiation up to mature granulocytes and less than 20% blast (b). Acute Myelomonocytic leukemia was defined as acute leukemia showing at least residual maturation into the myeloid lineage and a blast content of more than 20%. Myelomonocytic blasts expressed only marker of the myeloid lineage: Gr-1, CD11b, but not B220. Lymphatic leukemia was defined as acute leukemia with little to no obvious myeloid differentiation, blast content above 20% and expression of B220, but not Gr-1. In some instances a coexpression of CD11b was observed and diagnosed as biphenotypic leukemia. (A) shows a representative immunphenotypic example for each leukemia. (B) shows a representative morphological example (May-Gruenwald-Giemsa staining) for each leukemia. (b: denotes blast; g: denotes granulocyte; l: denotes lymphocyte).
- Figure S5 (JPG, 173 KB)
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(A) Location of primers used to identify allelic imbalanced expression of Nf1 and LOH. Shown are the genomic locations of the primers and the respective amplification products within the Nf1-gene for either cDNA or genomic DNA. To detect the Nf1 wt-allele (194bp) and the Nf1 neo-allele (340bp) the primers Nf15.0 (5′-gtattgaattgaagcaccttt gtttgg-3′), Nf13.0 (5′-ctgcccaaggctcccccag-3′) and Nf1neo (5′-gcgtgttcgaattcgccaatg-3′) were used in combination. For the RT-PCR the primers Nf15.B (5′-gctgtcacaacctgactcca-3′), Nf13.0 (5′-ctgcccaaggctcccccag-3′) and Nf1neo (5′-gcgtgttcgaattcgccaatg-3′) were used and generated a 511bp Nf1 wt transcript fragment and a 340bp Nf1neo transcript fragment. The real-time PCR was set up with the primers Nf15.0 (5′-gtattgaattgaagcacctttgtttgg-3′) and Nf13.2 (5′-tcgcactggtcttgatgaaa-3′) which amplify a 201bp Nf1 wt transcript fragment. The Nf1 neo transcript fragment is too long (1200bp) to be amplified under those conditions. Gapdh (primers: Gapdh-up 200bp (5′-ctgacgtgccgcctggag aaac-3′) and Gapdh-low 200bp (5′-cccggcatcgaaggtggaagag-3′) was amplified as the reference transcript. (B) Array CGH identifies CNAs in lymphatic leukemias. Shown are array CGH profiles for selected chromosomes including chromosomes harboring CNAs. (1) Chromosomes 8 through 10 of a normal control. (2) Leukemia #4430 displaying a genomic gain on chr 9. (3) Chromosomes 13 through 18 of a normal control. (4) Leukemia #4115 with chromosomal gains of chr 14 and chr 17. Fluorescence ratios are plotted on a log10 scale, as a moving average of 5 adjacent genes, according to chromosome position where red and green indicate positive and negative ratios, respectively.
REFERENCES
1. Terszowski G, Waskow C, Conradt P et al. Prospective isolation and global gene expression analysis of the erythrocyte colony-forming unit (CFU-E). Blood. 2005;105:1937–1945.
2. Frank O, Rudolph C, Heberlein C et al. Tumor cells escape suicide gene therapy by genetic and epigenetic instability. Blood. 2004;104:3543–3549.
3. Sander S, Bullinger L, Karlsson A et al. Comparative genomic hybridization on mouse cDNA microarrays and its application to a murine lymphoma model. Oncogene. 2005;24:6101–6107.
4. Ball CA, Awad IA, Demeter J et al. The Stanford Microarray Database accommodates additional microarray platforms and data formats. Nucleic Acids Res. 2005;33:D580–D582.
5. Adolfsson J, Mansson R, Buza-Vidas N et al. Identification of Flt3+ lympho-myeloid stem cells lacking erythro-megakaryocytic potential a revised road map for adult blood lineage commitment. Cell. 2005;121:295–306.
6. Kogan SC, Ward JM, Anver MR et al. Bethesda proposals for classification of nonlymphoid hematopoietic neoplasms in mice. Blood. 2002;100:238–245.
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