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SummaryAcute lymphoblastic leukaemia (ALL) is the most common malignancy in children. Recently, there has been a growing interest in Wnt signalling in several aspects of cellular development, including cancer formation. Little is known about Wnt signalling in B-ALL. We investigated whether activation of canonical Wnt signalling could occur in B-ALL cells and thereby play a potential role in cellular growth and/or survival. This study found that Wnt3A induced b-catenin accumulation in both primary B-ALL cells and B-ALL leukaemia cell lines. Further, Wnt3A was shown to induce nuclear translocation of b-catenin and TCF/Lef-1 dependent transcriptions in the B-ALL cell line Nalm-6. Examination of the mRNA expression pattern of WNT ligands, FZD receptors and WNT antagonists in Nalm-6 cells identified a set of ligands and receptors available for signalling, as well as antagonists potentially available for modulating the response. Functional analyses showed that Wnt3A inhibited the proliferation of several, but not all, B-ALL cell lines studied. Finally, microarray analysis was used to identify several Wnt3A target genes involved in a diverse range of cellular activities, which are potential mediators of the Wnt3A-restrained proliferation.

erythroid progenitors stimulated with Epo in the presence or absence of LY294002 were subjected to gene expression profiling. Several novel target genes of Epo were identified, and the majority were regulated in a PI3K-dependent manner, including KIT (CD117) and CDH1 (E-cadherin). FACS analysis of Epo-stimulated erythroid progenitors showed that the increased mRNA expression of KIT and CDH1 was accompanied by an induction of the corresponding proteins CD117 and E-cadherin.Keywords: Erythropoietin, phosphatidylinositol 3-kinase, KIT, erythropoiesis, microarray. molecules. The lipid products of PI3K activate a plethora of targets, including the protein kinase B/Akt. The PI3K-Akt pathway has been shown to play a central role in regulation of apoptosis and proliferation in several systems, including normal erythroid progenitors (Haseyama et al, 1999;Uddin et al, 2000). In the context of Epo signalling it has been shown that Akt kinase, which is activated by PI(3,4,5)P3 or PI(3,4)P2, phosphorylates both GATA-1 and Foxo3a, transcription factors of crucial importance in erythropoiesis (Uddin et al, 2000;Bouscary et al, 2003;Kadri et al, 2005).In the present study we have investigated how Epo, by activating PI3K, exerts its effects on cell cycle progression and differentiation in human CD34+ progenitor cells. By use of genome wide expression profiling of Epo-stimulated CD34 + CD71 + CD45RA) GPA ) erythroid progenitors, we identified novel Epo target genes and confirmed their differential expression by fluorescent-activated cell sorting (FACS) analysis, as well as hypothesise novel roles for signalling pathways that have no previously established function in erythropoiesis. Materials and methods DNA expression vectorsExpression vectors for HA-tagged wild type (WT-Akt) or myristylated Akt1 (Myr-Akt) created in pCMV6, were provided as a gift (Ahmed et al, 1997). pcDNA3 without insert was used as control vector (Control). Reagents and antibodiesRecombinant human (rh) Epo was obtained from Boehringer Mannheim (GmbH, Germany) and rh Scf was from R&D Systems (Abingdon, UK). Cytokines were used at predetermined optimal concentrations: 5 U/ml for Epo and 50 ng/ml for Scf or as specified. LY294002 was from Calbiochem (Darmstadt, Germany). The following antibodies (mAbs, antihuman) were used in flow-cytometry analysis: anti-CD34 phycoerythrin (PE), anti-CD117 PE-cyanin 5 (CY5) and anti-CD45RA PE were from Becton Dickinson (San Jose, CA, USA), anti-glycophorin A-PE (GPA PE), and anti-CD71 fluorescein isothiocyanate (FITC) were from DakoCytomation AS (Glostrup, Denmark). Anti E-cadherin was from Calbiochem (San Diego, CA, USA; mouse IgG1, #205601) and anti mouse IgG1 PE (Southern Biotech, Birmingham, AL, USA) was used as secondary layer. The following antibodies were used in immunoblot analysis: anti-cyclin D 3 , anti-cyclin A (sc-596), anti-cyclin E (sc-247), anti p21 (sc-397), anti-p27 (sc-528), anti-pRB (Ser780) and from Santa Cruz Biotechnologies (Santa Cruz, CA, USA), anti-Akt (#9272) and anti phospho Akt1 (Ser473, #9271) were ...

Background: The early B lymphopoiesis in mammals is regulated through close interactions with stromal cells and components of the intracellular matrix in the bone marrow (BM) microenvironment. Although B lymphopoiesis has been studied for decades, the factors that are implicated in this process, both autocrine and paracrine, are inadequately explored. Wnt signaling is known to be involved in embryonic development and growth regulation of tissues and cancer. Wnt molecules are produced in the BM, and we here ask whether canonical Wnt signaling has a role in regulating human BM B lymphopoiesis.

We have identified and characterized the novel human transmembrane protein 9 (TMEM9). TMEM9 encodes a 183 amino-acid protein that contains an N-terminal signal peptide, a single transmembrane region, three potential N-glycosylation sites, and three conserved cys-rich domains in the N-terminus, but no hitherto known functional domains. The protein is highly conserved between species from Caenorhabditis elegans to man and belongs to a novel family of transmembrane proteins. The TMEM9 gene consists of at least 6 exons and is localized to chromosome 1q41. TMEM9 mRNA is expressed in a wide range of tissues and cells. COS-1 cells transfected with a TMEM9 expression plasmid gave three bands of about 28, 31, and 33kDa representing glycosylated forms of TMEM9 with a protein backbone of about 26kDa. In COS-1 cells transfected with a TMEM9-GFP expression construct,TMEM9-GFP is co-expressed with LAMP1 on late endosomes and lysosomes as well as on ER. Thus, TMEM9 is a phylogenetically conserved, widely expressed transmembrane protein with a potential, but unknown function in intracellular transport.