Translocations involving the abl locus on chromosome 9 fuses the tyrosine kinase c-ABL to proteins harboring oligomerization interfaces such as BCR or TEL, enabling these ABL-fusion proteins (X-ABL) to transform cells and to induce leukemia. The ABL kinase activity is blocked by the ABL kinase inhibitor STI571 which abrogates transformation by X-ABL. To investigate the role of oligomerization for the transformation potential of X-ABL and for the sensitivity to STI571, we constructed ABL chimeras with oligomerization interfaces of proteins involved in leukemia-associated translocations such as BCR, TEL, PML, and PLZF. We assessed the capacity of these chimeras to form high molecular weight (

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To better understand the transcriptional program that accompanies orderly lineage-specific hematopoietic differentiation, we performed serial oligonucleotide microarray analysis of human normal CD34 + bone marrow cells during lineage-specific differentiation. CD34 + bone marrow cells isolated from healthy individuals were selectively stimulated in vitro with the cytokines erythropoietin (EPO), thrombopoietin (TPO), granulocyte colony-stimulating factor (G-CSF), and granulocyte macrophage colony-stimulating factor (GM-CSF). Cells from each of the lineages were harvested after 4, 7, and 11 days of culture for expression profiling. Gene expression was analyzed by oligonucleotide microarrays (HG-U133A; Affymetrix, Santa Clara, CA). Experiments were done in triplicates. We identified 258 genes that are consistently upregulated or downregulated during the course of lineage-specific differentiation within each specific lineage (horizontal change). In addition, we identified 52 genes that contributed to a specific expression profile, yielding a genetic signature specific for successive stages of differentiation within each of the three lineages. Analysis of horizontal changes selected 21 continuously upregulated genes for EPO-induced differentiation (including GTPase activator proteins RAP1GA1 and ARHGAP8, which regulate small Rho GTPases), 21 for G-CSF-induced/GM-CSF-induced differentiation, and 91 for TPO-induced differentiation (including DLK1, of which the role in normal hematopoiesis is not defined). During the lineage-specific differentiation, 58 (erythropoiesis), 30 (granulopoiesis), and 37 (thrombopoiesis) genes were significantly downregulated, respectively. The expression of selected genes was confirmed by real-time polymerase chain reaction. Our data encompass the first extensive transcriptional profile of human hematopoiesis during in vitro lineage-specific differentiation.

Since the 19th century, arsenic (As 2 O 3 ) has been used in the treatment of chronic myelogenous leukemia (CML) characterized by the t(9;22) translocation. As 2 O 3 induces complete remissions in patients with acute promyelocytic leukemia. The response to As 2 O 3 is genetically determined by the t(15;17)-or the t(9;22)-specific fusion proteins PML/RARa or BCR/ABL. The PML portion of PML/RARa is crucial for the sensitivity to As 2 O 3 . PML is nearly entirely contained in PML/RARa. PML is upregulated by oncogenic RAS in primary fibroblasts. The aberrant kinase activity of BCR/ABL leads to constitutive activation of RAS. Therefore, we hypothesized that BCR/ ABL could increase sensitivity to As 2 O 2 -induced apoptosis by modifying PML expression. To disclose the mechanism of As 2 O 3 -induced apoptosis in PML/RARaand BCR/ABL-expressing cells, we focused on the role of PML for As 2 O 3 -induced cell death. Here we report that (i) sensitivity to As 2 O 3 -induced apoptosis of U937 cells can be increased either by overexpression of PML, or by conditional expression of activated RAS; (ii) also the expression of the t(8;21)-related AML-1/ETO increased sensitivity to As 2 O 3 -induced apoptosis; (iii) both BCR/ ABL and AML-1/ETO activated RAS and modified the PML expression pattern; (iv) the expression of either BCR/ABL or AML-1/ETO rendered U937 cells sensitive to interferon a-induced apoptosis. In summary, these data suggest a crucial role of factors able to upregulate PML for As 2 O 2 -induced cell death.

Background: The reciprocal (9;22) translocation fuses the bcr (breakpoint cluster region) gene on chromosome 22 to the abl (Abelson-leukemia-virus) gene on chromosome 9. Depending on the breakpoint on chromosome 22 (the Philadelphia chromosome -Ph+) the derivative 9+ encodes either the p40 (ABL/BCR) fusion transcript, detectable in about 65% patients suffering from chronic myeloid leukemia, or the p96 (ABL/BCR) fusion transcript, detectable in 100% of Ph+ acute lymphatic leukemia patients. The ABL/BCRs are N-terminally truncated BCR mutants. The fact that BCR contains Rho-GEF and Rac-GAP functions strongly suggest an important role in cytoskeleton modeling by regulating the activity of Rho-like GTPases, such as Rho, Rac and cdc42. We, therefore, compared the function of the ABL/BCR proteins with that of wild-type BCR. Methods:We investigated the effects of BCR and ABL/BCRs i.) on the activation status of Rho, Rac and cdc42 in GTPase-activation assays; ii.) on the actin cytoskeleton by direct immunofluorescence; and iii) on cell motility by studying migration into a three-dimensional stroma spheroid model, adhesion on an endothelial cell layer under shear stress in a flow chamber model, and chemotaxis and endothelial transmigration in a transwell model with an SDF-1α gradient.Results: Here we show that both ABL/BCRs lost fundamental functional features of BCR regarding the regulation of small Rho-like GTPases with negative consequences on cell motility, in particular on the capacity to adhere to endothelial cells. Conclusion:Our data presented here describe for the first time an analysis of the biological function of the reciprocal t(9;22) ABL/BCR fusion proteins in comparison to their physiological counterpart BCR.