ASB2 is an Elongin BC-interacting protein that can assemble with Cullin 5 and Rbx1 to reconstitute an E3 ubiquitin ligase complex.

International audience; The ankyrin repeat-containing protein with a suppressor of cytokine signaling box-2 (ASB2) gene was identified as a retinoic acid-response gene and a target of the promyelocytic leukemia-retinoic acid receptor-alpha oncogenic protein characteristic of acute promyelocytic leukemia. Expression of ASB2 in myeloid leukemia cells inhibits growth and promotes commitment, recapitulating an early step known to be critical for differentiation. Here we show that ASB2, by interacting with the Elongin BC complex, can assemble with Cullin5.Rbx1 to form an E3 ubiquitin ligase complex that stimulates polyubiquitination by the E2 ubiquitin-conjugating enzyme Ubc5. This is a first indication that a member of the ASB protein family, ASB2, is a subunit of an ECS (Elongin C-Cullin-SOCS box)-type E3 ubiquitin ligase complex. Altogether, our results strongly suggest that ASB2 targets specific proteins to destruction by the proteasome in leukemia cells that have been induced to differentiate

Proteinase 3 mRNA expression is induced in monocytes but not in neutrophils of patients with cystic fibrosis

AbstractProteinase 3 (PR3), a serine proteinase which can degrade lung tissue, is present in the cystic fibrosis (CF) sputum. In the present study, PR3 protein and mRNA expression was determined in circulating neutrophils and monocytes. CF neutrophils contained similar PR3 concentrations as healthy controls and poorly expressed PR3 mRNA. In contrast, CF monocytes showed significantly higher PR3 concentrations than controls, together with an upregulation of PR3 mRNA expression especially during pulmonary exacerbation. Interestingly, antibiotic treatment fully abrogated PR3 mRNA expression and decreased PR3 protein in monocytes. Our findings highlight a potential role of monocyte-derived PR3 in CF-associated airway inflammation

Dasatinib inhibits the growth of molecularly heterogeneous myeloid leukemias.

PURPOSE: Dasatinib is a dual Src/Abl inhibitor recently approved for Bcr-Abl+ leukemias with resistance or intolerance to prior therapy. Because Src kinases contribute to multiple blood cell functions by triggering a variety of signaling pathways, we hypothesized that their molecular targeting might lead to growth inhibition in acute myeloid leukemia (AML). EXPERIMENTAL DESIGN: We studied growth factor-dependent and growth factor-independent leukemic cell lines, including three cell lines expressing mutants of receptor tyrosine kinases (Flt3 or c-Kit) as well as primary AML blasts for responsiveness to dasatinib. RESULTS: Dasatinib resulted in the inhibition of Src family kinases in all cell lines and blast cells at approximately 1 x 10(-9) mol/L. It also inhibited mutant Flt3 or Kit tyrosine phosphorylation at approximately 1 x 10(-6) mol/L. Mo7e cells expressing the activating mutation (codon 816) of c-Kit were most sensitive to growth inhibition with a GI(50) of 5 x 10(-9) mol/L. Primary AML blast cells exhibited a growth inhibition of \u3c1 x\u3e10(-6) mol/L. Cell lines that showed growth inhibition at approximately 1 x 10(-6) mol/L showed a G(1) cell cycle arrest and correlated with accumulation of p21 and p27 protein. The addition of rapamycin or cytotoxic agents enhanced growth inhibition. Dasatinib also caused the apoptosis of Mo7e cells expressing oncogenic Kit. CONCLUSIONS: Although all of the precise targets for dasatinib are not known, this multikinase inhibitor causes either growth arrest or apoptosis in molecularly heterogeneous AML. The addition of cytotoxic or targeted agents can enhance its effects

Caractérisation fonctionnelle de PRAM-1 (un nouvel adaptateur des cellules myéloïdes)

LE KREMLIN-B.- PARIS 11-BU Méd (940432101) / SudocPARIS-BIUP (751062107) / SudocSudocFranceF

Caractérisation fonctionnelle d'ASB2 (une protéine impliquée dans la différenciation myéloïde)

Le gène ASB2, identifié par criblage différentiel dans une lignée dérivant d'un patient atteint de leucémie aiguë promyélocytaire (LAP), code une protéine comportant onze répétitions ankyrine et une boîte SOCS contenant un motif BC. Via ce motif BC, ASB2 interagit avec le complexe des ELONGINES B/C suggérant qu'ASB2 appartiendrait à un complexe E3 à activité ubiquitine ligase permettant la dégradation par le protéasome de protéines cibles. Sa surexpression dans des cellules de leucémie myéloïde induit des phénomènes caractéristiques des étapes d'engagement vers la différenciation. ASB2 présente toutes les caractéristiques d'un gène critique de la différenciation myéloïde : il est réprimé par PML-RARa dans les cellules de LAP et réactivé après traitement à l'acide rétinoïque. Ces travaux suggèrent qu'ASB2 pourrait être impliqué dans les étapes précoces de la différenciation myéloïde en ciblant des régulateurs clés de l'hématopoïèse vers le système de dégradation par le protéasome.ASB2 gene, identified by a subtractive screening in a cell line derived from a patient with acute promyelocytic leukemia (APL), encodes a protein with eleven ankyrin repeats and a SOCS box including a BC box. ASB2 interacts, via its BC box, with ELONGIN B/C complex suggesting that it could belong to an E3 complex with an ubiquitine ligase activity leading to proteasomal degradation of target proteins. ASB2 overexpression in myeloid leukemic cells induces phenomena characteristics of commitment to differentiation. ASB2 presents all the characteristics of a critical gene for myeloid differentiation : it is repressed by PML-RARa in APL cells and reactivated after retinoic acid tratment. These results suggest that ASB2 could be involved in early steps of myeloid differentiation targeting key hematopoietic regulators to proteasomal degradation.PARIS5-BU Saints-Pères (751062109) / SudocSudocFranceF

Ciblage pharmacologique et peptidique de la voie de signalisation des Src Kinases (une contribution à de nouvelles perspectives thérapeutiques dans les leucémies aiguës de la lignée)

LE KREMLIN-B.- PARIS 11-BU Méd (940432101) / SudocSudocFranceF

Myeloblastin is an Myb target gene: mechanisms of regulation in myeloid leukemia cells growth-arrested by retinoic acid

International audienceA pivotal role has been assigned to Myb in the control of myeloid cell growth. Although Myb is a target of retinoic acid, little is known about the mechanisms by which it may contribute to induced growth arrest in leukemia cells. Indeed, few Myb target genes are known to be linked to proliferation. Myeloblastin is involved in the control of proliferation in myeloid leukemia cells. It is expressed early during hematopoiesis and is a granulocyte colony-stimulating factor-responsive gene. Myeloblastin can confer factor-independent growth to hematopoietic cells, an early step in leukemia transformation. The myeloblastin promoter contains PU.1, C/EBP, and Myb binding sites, each of which are critical for constitutive expression in myeloid cells. Inhibition of myeloblastin expression in leukemia cells growth-arrested by retinoic acid is demonstrated to depend on Myb down-regulation. Myb is shown to induce myeloblastin expression and abolish its down-regulation by retinoic acid. Altogether, the data offer a clue as to how a myeloid-specific transcriptional machinery can be accessible to regulation by retinoic acid and point to myeloblastin as a novel target of Myb. This link between Myb and myeloblastin suggests a previously nonidentified Myb pathway through which growth arrest is induced by retinoic acid in myeloid leukemia cells

Microfluidic device with integrated microfilter of conical-shaped holes for high efficiency and high purity capture of circulating tumor cells

International audienceCapture of circulating tumor cells (CTCs) from peripheral blood of cancer patients has major implications for metastatic detection and therapy analyses. Here we demonstrated a microfluidic device for high efficiency and high purity capture of CTCs. The key novelty of this approach lies on the integration of a microfilter with conical-shaped holes and a micro-injector with cross-flow components for size dependent capture of tumor cells without significant retention of non-tumor cells. Under conditions of constant flow rate, tumor cells spiked into phosphate buffered saline could be recovered and then cultured for further analyses. When tumor cells were spiked in blood of healthy donors, they could also be recovered at high efficiency and high clearance efficiency of white blood cells. When the same device was used for clinical validation, CTCs could be detected in blood samples of cancer patients but not in that of healthy donors. Finally, the capture efficiency of tumor cells is cell-type dependent but the hole size of the filter should be more closely correlated to the nuclei size of the tumor cells. Together with the advantage of easy operation, low-cost and high potential of integration, this approach offers unprecedented opportunities for metastatic detection and cancer treatment monitoring

ASB-2 Inhibits Growth and Promotes Commitment in Myeloid Leukemia Cells*

International audienceIn acute promyelocytic leukemia (APL) cells harboringthe promyelocytic leukemia retinoic acid receptor (PML-RAR) chimeric protein, retinoic acid (RA)-induceddifferentiation is triggered through a PML-RARsignaling resulting in activation of critical target genes.Induced differentiation of APL cells is always precededby withdrawal from the cell cycle and commitmentevents leading to terminal differentiation. Here we haveidentified the human ankyrin repeat-containing proteinwith a suppressor of cytokine signaling box-2 (ASB-2)cDNA, as a novel RA-induced gene in APL cells. PMLRAR strongly enhanced RA-induced ASB-2 mRNA expression.In myeloid leukemia cells, ASB-2 expressioninduced growth inhibition and chromatin condensationrecapitulating early events critical to RA-induced differentiationof APL cells