10 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

    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

    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

    Shear Stress experiments.

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    <p>2A Analysis of shear stress-dependent adhesion of RP1 mutants on endothelial cells under flow 1×10<sup>5</sup> HEK293 cells stably transfected with different RP1 mutants were allowed to settle for 3 min on parallel plate flow chambers with pre grown confluent HUVECs. Subsequently, preheated HBSS/0.1% BSA was flushed through the chambers at the indicated calculated shear stress, and shear stress levels were increased every 30 s. Photographs were taken and adherent cells were counted in four fields for every condition. Cell line with empty vector (black squares), RP1 wild type (wt) (black circles), RP1-ALA<sup>236</sup> (ALA) mutant (white circles), RP1-ASP<sup>236</sup> (ASP) mutant (white squares). Values are means of n = 5–6+/− SEM. Asterisks denote statistically significant differences *p<0.05 or **p<0.01 between parental cell line and ASP mutant as determined by a two-tailed t-test. <b>2B Analysis of RP1 expression under fluid shear stress</b> Native HEK293 cells were exposed to fluid shear stress or simply cultured (control). Thereafter, cells were lysed and total protein from the lysates was employed in immunoprecipitation of RP1. Endogenous RP1 was detected by an RP1 specific antibody. α-tubulin served as a loading control. RP-1protein detected by Western blot was quantified using the ImageJ software. Asterisks mark statistically significant differences **p<0.01 between sheared and non-sheared cells as determined by a two-tailed t-test. <b>2C </b><b>Analysis of RP1 phosphorylation under fluid shear stress</b> HEK293 cells overexpressing RP1-wt were exposed to 1dynes/cm<sup>2</sup> shear stress. From cell lysate RP1 was immunoprecipitated and subjected to Western blotting. In parallel the phosphorylation status of RP1 was detected with an anti phospho-serine antibody (anti PS). As control HEK293 cells overexpressing RP1-wt were cultured without shear stress and otherwise processed alike. Total RP1 (RP1) expression served as loading control. The difference between phosphorylation intensity in sheared versus control cells was statistically significant (**, p<0.05). <b>2D Analysis of RP1 shRNA regulated cells and RP1 phosphor-mutants under fluid shear stress</b> HEK293 containing empty vector or various mutants were exposed to increasing shear stress. The curves show the percentage of adhering cells under different shear stress intensity (0, 1.5, 4.5 and 8.5 dynes/cm2) on control cells transfected with irrelevant shRNA served as a reference (black boxes) and were compared to RP1 specific shRNA bearing cells and the mutant cell lines RP1-ALA236, RP1-ASP236. A significant gain of cell adhesion is seen for RP1 specific shRNAs (white boxes). The statistical differences of adherent cells between the depicted cell lines and the control cells are marked with Asterisks *p<0.05 or **p<0.01.</p

    G- and F-Actin in RP1-expressing cells.

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    <p><b>3A</b> G- and F-actin content of 10×10<sup>5</sup> constitutively RP1 expressing cells were measured by FACS analysis. G-Actin (green) was measured in the FL-1 channel (Fluor 488, green) and F-Actin in the FL-3 channel (phalloidin rodamine staining, red). The upper left quadrant of each panel represents the F-actin pool, the upper right quadrant the G-Actin pool. The top three panels are the controls: Upper left panel, unstained control cells. Upper middle panel: Boiled fluoresceine conjugated DNAse I unable to bind G-Actin serving as a negative control. Upper right panel: Double staining of HEK293 cells with Fluor 488 conjugated DNAse I and Phalloidin-Rhodamine carrying the empty pEAK8 vector to determine the general content of G-actin and F-actin pools as reference. The bottom panels show the respective degree of G-actin decrease seen in the RP1-wt, ALA, ASP containing cell lines. <b>3B</b> Quantification of G-Actin content in RP1 expressing HEK293 cells compared to mock transfected cells from <b>3A</b>. Differences marked by asterisks were statistically significant using the two-tailed Fisher’s exact test (**p = 0.0001;*p = 0.0003).</p

    Binding and Phosphorylation of RP1 by CK2.

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    <p><b>1A</b> Identification of CK2 phosphorylation site - RP1-sequence (amino acid), three potential CK2 kinase sites S59, S72, S236 (underlined) were identified by prosite scan (<a href="http://www.expasy.ch" target="_blank">www.expasy.ch</a>). The peptides used for in vitro experiments (1C) are marked in bold. S236 the actual CK2 phosphorylation site is shown in red. <b>1B</b> Interaction–assay RP1/CK2 - Endogenous RP1 (first panel) was co-precipitated with its potential binding partners. RP1/CK2 kinase interaction could be detected by specific α/ß CK2 subunit antibodies. The black wedges in this panel indicate increasing stringency of washing procedure (% Tween20/PBS). In a reverse experiment (right side panel), endogenous RP1 was verified as genuine CK2 binding substrate. By using CK2 subunits as baits, RP1 could be detected in the pulldowns by its specific RP1 antibody (right panel). No signal was seen when an insignificant IgG antibody was used. On the far right 1/10 of cell lysate of the foregoing experiments is depicted as an input control. The black wedges in this panel indicate stringency of the washing procedure (0.01% and 0.3% Tween/PBS). <b>1C</b> Biotinylated peptides (A: aa54–65, B: aa70–80, C: aa229–240) containing the potential CK2 phosphorylation sites S<i><sup>59</sup></i>, S<i><sup>72</sup></i>, S<i><sup>236</sup> were</i> synthesized and tested as CK2 phosphorylation substrates (A, B, C, 3 µg each) in an <i>in vitro</i> phosphorylation assay. A known positive CK2 kinase site peptide (DDDDSDDDDD, 3 µg) served as a control. The black wedge indicates incubation times (minutes). <b>1D</b> CK2 kinase assay - Recombinant CK2 and <sup>33</sup>P-gamma-ATP were incubated in vitro with different amounts of RP1-wt protein (first panel shows a coomassie stain of his-tagged purified RP1 protein used for the assay) and phosphorylation was measured by autoradiography (middle panel). The amounts of RP1 protein used are indicated above the middle panel. Autophosphorylation of CK2 at its subunit ß served as positive control RP1-ALA236 mutated protein (ALA) was almost non-phosphorylated in comparison to the wild type protein (right side upper panel). The lower panel on the right side shows a coomassie stain representing the amount of RP1 used for this experiment.</p

    Cadherin expression in RP1-mutants.

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    <p><b>4A</b> N-cadherin levels of HEK293 cells containing empty vector (c), wt, ALA and ASP were determined by immunoblotting with an N-terminal N-cadherin antibody. The middle panel shows a processed N-cadherin fragment (named CTF1) detected by a fragment specific antibody in respective lysates. The lower panel shows the β-tubulin loading control. <b>4B</b> The Western blot signals of <b>4A</b> were quantified using the Image-J software (Rasband, W.S., ImageJ, U. S. National Institutes of Health, Bethesda, Maryland, USA, <a href="http://rsb.info.nih.gov/ij/" target="_blank">http://rsb.info.nih.gov/ij/</a>, 1997–2008). Differences marked by asterisks were statistically significant using the two-tailed Fisher’s exact test (**p<0.001) comparing control versus wt and mutants regarding complete N-cadherin and comparing wt versus mutants regarding N-cadherin cleavage fragment (CTF1). In the right panel, the empty c lane indicates no detectable CTF in control cells. <b>4C</b> N-cadherin levels were measured by incubation with a monoclonal antibody directed against the cytoplasmic tail and subsequent FACS analysis. The negative control (yellow line) was incubated with secondary antibody only. The positive control (red) was empty vector containing HEK293 cells. The results for HEK293 expressing RP1-wt are depicted in black, RP1-ALA<sup>236</sup> in green and RP1-ASP<sup>236</sup> in blue. <b>4D</b> Quantification of N-cadherin levels from FACS analysis<b>.</b> Differences marked by asterisks were statistically significant using the two-tailed Fisher’s exact test (**p<0.001).</p
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