25 research outputs found

    Effect of the t(9;22) fusion proteins on the biology of murine HSCs.

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    <p>(A) Experimental strategy for studying the influence of the t(9;22) fusion proteins on the biology of murine HSCs. Sca1<sup>+</sup>/lin<sup>−</sup> BM cells were infected with the indicated retroviruses and plated in semi-solid medium supplemented with the indicated growth factors for determination of the serial replating potential. Cells from the first plating (I) round were examined for the expression of differentiation-specific surface markers. Cells plated in liquid culture supplemented with the indicated growth factors were used for cell cycle analysis and gene expression by qRT-PCR. (B) Sca1<sup>+</sup>/lin<sup>−</sup> BM cells were infected with the indicated retroviruses and expression levels of the transgenes were analyzed by Western blotting using the indicated antibodies. (C) Long-term serial replating. Sca1<sup>+</sup>/lin<sup>−</sup> cells were infected with the indicated retroviruses and plated into methyl-cellulose supplemented with the indicated growth factors to assess primary colony formation. Colony numbers were counted on days 8–10. Cells were then harvested and serially replated. Colonies were counted on days 8–10 after each replating. (D) Colony morphology during the first plating. Type A (compact colonies), type B (dense center surrounded by a halo of migrating cells) and type C (diffuse colonies with mobile differentiating cells) colonies were distinguished. (E) Expression of differentiation-specific surface markers. One representative experiment of three yielding similar results. (F) Sca1<sup>+</sup>/lin<sup>−</sup> BM cells were infected with the indicated retroviruses and, after 48 h, cell cycle progression was determined. The provided results are the average of three independent experiments +/− SD. (G) Activation of Wnt-signaling by the t(9;22) fusion proteins. BCR, p185<i><sup>BCR/ABL</sup></i>, p40<i><sup>ABL/BCR</sup></i> and p96<i><sup>ABL/BCR</sup></i> and the Topflash and Fopflash reporter constructs were co-transfected by electroporation into U937 cells. U937 cells expressing PML/RARα - positive control; pGL3basic - negative control. Luciferase activity measured at 24 h post-transfection was normalized to Renilla luciferase activity. Each experiment was performed in triplicate a total of three times with similar results. One representative experiment is given +/− SD. (H) Sca1<sup>+</sup>/lin<sup>−</sup> BM cells were infected with the indicated retroviruses and, at 48 h post-infection, the expression levels of HoxB4, Cdkn1a (p21<sup>(cip1/waf1)</sup>), c-Myc and SCL were analyzed by qRT-PCR. The relative concentration of each mRNA was normalized to the concentration of the housekeeping gene GAPDH and is represented as 2<sup>−Δ/Δ</sup> CT. Each experiment was performed in triplicate a total of three times with similar results. One representative experiment is given +/− SD.</p

    Influence of the t(9;22) fusion proteins alone or in combination on B cell commitment and expression of B cell-specific gene transcripts.

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    <p>(A) Experimental strategy for studying the influence of the reciprocal t(9;22) fusion proteins on the B cell commitment of murine HSCs. Sca1<sup>+</sup>/lin<sup>−</sup> BM cells were infected with the indicated retroviruses and plated in semi-solid medium supplemented with the indicated growth factors for determination of the serial replating potential. (B) Transgene expression in transduced Sca1<sup>+</sup>/lin<sup>−</sup> BM cells as determined by Western blotting using the indicated antibodies. (C) Long-term serial replating of Sca1<sup>+</sup>/lin<sup>−</sup> cells infected with the indicated retroviruses and plated into wells containing methyl-cellulose medium supplemented with the indicated growth factors to assess primary colony formation. Colony numbers were determined on days 8–10. Cells were harvested and replated for the determination of the serial replating potential (D–E). Influence of the t(9;22) fusion proteins on key factors involved in B cell commitment and the pre-B cell receptor complex. Sca1<sup>+</sup>/lin<sup>−</sup> BM cells were infected with the indicated retroviruses and, at 48 h post-infection, the expression of PAX5, E2A, ID2, BLNK, CD19 and CD79a were analyzed by qRT-PCR. The relative concentration of each mRNA was normalized to the concentration of the housekeeping gene GAPDH and is represented as 2<sup>−Δ/Δ</sup> CT. Each experiment was performed in triplicate a total of three times with similar results. One representative experiment is given +/− SD.</p

    Differential effects of p40<i><sup>ABL/BCR</sup></i>, p96<i><sup>ABL/BCR</sup></i> and p185<i><sup>BCR/ABL</sup></i> on the B cell commitment of murine HSCs.

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    <p>(A) Experimental strategy for studying the influence of the reciprocal t(9;22) fusion proteins on the B cell commitment of murine HSCs. Sca1<sup>+</sup>/lin<sup>−</sup> BM cells were infected with the indicated retroviruses and plated in semi-solid medium supplemented with the indicated growth factors for determination of the serial replating potential. Cells from the first plating (I) round and cells plated in liquid culture supplemented with the indicated growth factors were examined for the expression of differentiation-specific surface markers. (B) Long-term serial replating. Sca1<sup>+</sup>/lin<sup>−</sup> cells were infected with the indicated retroviruses and plated into methyl-cellulose medium supplemented with the indicated growth factors to assess primary colony formation. Colony numbers were counted on days 8–10. Cells were then harvested and serially replated. Colonies were counted on days 8–10 for each replating. I-IV - number of the plating round. (C) Expression of differentiation-specific surface markers. (D) Colony morphology of the second plating. Type A (compact colonies), type B (a dense center surrounded by a halo of migrating cells) and type C (diffuse colonies with mobile differentiating cells) colonies were distinguished. p185<i><sup>BCR/ABL</sup></i> exhibited a high number of viable cells that were not organized into colonies. (E) Determination of the maturation stage of B220<sup>+</sup> lymphocytes by CD43 expression analysis. CD43 positivity is characteristic of immature pro-B cells.</p

    Effect of the t(9;22) fusion proteins on the re-population capacity of murine HSC.

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    <p>(A) The schematic indicates the experimental procedure for determination of the re-population potential of murine HSCs expressing t(9;22) fusion proteins. Sca1<sup>+</sup>/lin<sup>−</sup> BM cells from CD45.1<sup>+</sup> mice were infected with the indicated retroviruses and, at 48 h post-infection, the cells were transplanted together with CD45.2<sup>+</sup> BM cells into lethally (11 Gy) irradiated CD45.2<sup>+</sup> recipient mice. Analysis of donor chimerism was performed 12 weeks after transplantation. (B) Plots show representative donor-derived chimerisms from individual mice at 12 weeks post-transplantation. (C) Graph of donor-derived chimerism in each mouse after long-term reconstitution (4–5 mice/group).</p

    The t(9;22) fusion proteins and their expression in Ph+ cells.

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    <p>(A) Modular organization of the translocation partners and the fusion proteins in t(9;22). A schematic representation of the fusion proteins encoded by derivative 9 (der9) and 22 (der22), the Philadelphia chromosome, as well as of their combination in m-BCR-positive Ph+ ALL and M-BCR-positive CML, respectively. CC - coiled coil oligomerization interface; Y177 - Tyrosine phosphorylation site at aa 177; S/T kinase - serine/threonine kinase domain; DH - dbl homology domain; PH - pleckstrin homology domain; GAP - Rac-GAP domain; STEV - PDZ-domain binding motif; SH2 and SH3 - Src homology domains 2 and 3; Y kinase - tyrosine kinase domain; AB - actin binding domain. (B) Expression of the reciprocal ABL/BCR fusion proteins in Ph+ leukemia cell lines. Negative control: Phoenix cells; Tom-1, SD-1 and SupB15 - cell lines derived from m-BCR-positive ALL patient samples; BV173 - cells derived from a sample obtained from a CML patient in lymphatic BC; K562 - cells derived from a sample obtained from a CML patient in myeloid BC. (C) Expression of the reciprocal ABL/BCR fusion proteins in samples from Ph+ leukemia patients. Negative controls - three samples from AML patients and one sample from a CML patient for p96<i><sup>ABL/BCR</sup></i> in Ph+ ALL samples; one sample from an AML patient for p40<i><sup>ABL/BCR</sup></i> in CML samples.</p

    The leukemogenic potential of p40<i><sup>ABL/BCR</sup></i> and p96<i><sup>ABL/BCR</sup></i>.

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    <p>(A) Experimental strategy to model leukemia induced by the reciprocal t(9;22) fusion proteins. Sca1<sup>+</sup>/lin<sup>−</sup> BM cells were infected with the indicated retroviruses and inoculated into sub-lethally irradiated mice. As positive and negative controls, we used γ-catenin and empty vector-transduced cells, respectively. (B) Survival curves show the frequency of recipients succumbing to disease after receiving the transduced cells. Statistical relevance was set at p<0.05. (C) May-Grünwald-Giemsa staining of cytospins from BM and spleen of one representative mouse in each group. (D) Relative splenomegaly of p40<i><sup>ABL/BCR</sup></i>-, p96<i><sup>ABL/BCR</sup></i>- or γ-catenin-positive leukemia. (E) Expression of differentiation-specific surface markers.</p

    TNF-α dose response of h-GF for SAOS-2 binding.

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    <p>A graph shows the dose response for TNF-α of h-GF stimulated for 24 hrs with regard to subsequent SAOS-2 binding. The effect of TNF-α was seen by 0.06 nM of cytokine, and was maximal by 1.16 nM (p<0.05).</p

    ICAM-1 was involved with TNF-α stimulated h-GF binding of SAOS-2.

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    <p>FACS analysis for ICAM-1 expression in h-GF with or without TNF-α (1.16nM) pre-treatment is shown (A), as well as at increasing times over 24 hr of TNF-α (1.16nM) treatment (B), compared with graphs showing the time-course of increased SAOS-2 binding by TNF-α (1.16nM) treated h-GF (C), and a histogram of the effect of blocking antibody against ICAM-1 (D). (A) h-GF expressed ICAM-1 and this was increased by TNF-α with maximal expression by 6 hr of cytokine treatment (B). (C) In an experiment performed at the same time as that shown in (5B), and using h-GF from the same donor, Maximal ICAM-1 expression correlated with maximal binding of SAOS-2 (p<0.001). As seen in the insert (C) examining the first 5 hr of cytokine stimulation in a separate time course experiment, increased SAOS-2 binding occurred between 0.5 and 1 hr of TNF-α (1.16nM) h-GF stimulation, and was maximal by 1.5 hrs (p<0.001). (D) Blocking antibody against ICAM-1 reduced binding of SAOS-2 to both untreated and TNF-α (1.16nM) stimulated h-GF (p<0.04).</p

    TNF-α increased ICAM-1 expression in h-GF and HUVEC, but only increased VCAM-1 in HUVEC.

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    <p>Fluorescence distribution profiles are shown proportionate to the peak incidence of fluorescence in each cell population studied. Background levels of ICAM-1 in both h-GF and HUVEC were appreciably increased following 24 hrs of stimulation with TNF-α (1.16nM). Although a similar effect of the cytokine was seen in HUVEC with regard to VCAM-1 expression, negligible VCAM-1 in h-GF was not significantly increased by TNF-α (1.16nM) stimulation.</p

    Decaying effect of TNF-α stimulation on h-GF binding of SAOS-2.

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    <p>A graph is shown of SAOS-2 binding to h-GF, in which h-GF were first stimulated with TNF-α (1.16nM) for 24 hrs, and then washed before further culture for from 0 to 24 hrs in M199 with BSA (4%), either with or without fresh TNF-α (1.16nM). There was decreased SAOS-2 attachment between 6 hr and 12 hrs post-cytokine stimulation (p<0.005), and this reduced to a plateau by 18 hrs (p<0.001).</p
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