beta-catenin is involved in N-cadherin-dependent adhesion, but not in canonical Wnt signaling in E2A-PBX1-positive B acute lymphoblastic leukemia cells.

OBJECTIVE: The t(1;19)(q23;13) translocation, resulting in the production of the E2A-PBX1 chimeric protein, is a common nonrandom translocation in pediatric B-lineage acute lymphoblastic leukemia (B-ALL). The E2A-PBX1 chimeric protein activates expression of several genes, including Wnt16. In the present study, we explored the role of Wnt16 and beta-catenin in t(1;19) B-ALL cells. MATERIALS AND METHODS: Canonical Wnt signaling was measured by TOPflash activity. Localization of beta-catenin in the cell membrane and its involvement in leukemia-stroma interaction were studied by confocal microscopy. Adhesion to N-cadherin was analyzed by adding (3)H-thymidin-labeled cells to N-cadherin-coated wells. RESULTS: In contrast to previous reports, we detected no effects on cell viability or proliferation upon modulation of the Wnt16 levels. Moreover, despite high levels of Wnt16 and beta-catenin, the cells had very low levels of canonical Wnt signaling. Instead, beta-catenin was located in the cell membrane along with N-cadherin. E2A-PBX1-positive leukemia cells adhered strongly to bone marrow stroma cells, and we showed that adherence junctions stained strongly for both proteins. Moreover, knockdown of beta-catenin reduced the adhesion of E2A-PBX1-positive leukemia cells to N-cadherin, suggesting that beta-catenin and N-cadherin play a central role in homotypic cell-to-cell adhesion and in leukemia-stroma adhesion. Interestingly, knockdown of Wnt16 by small interfering RNA reduced the level of N-cadherin. CONCLUSION: Wnt16 does not activate canonical Wnt signaling in E2A-PBX1-positive cells. Instead, beta-catenin is involved in N-cadherin-dependent adherence junctions, suggesting for the first time that leukemia-stroma interactions may be mediated via an N-cadherin-dependent mechanism.

Bone marrow stroma cells regulate TIEG1 expression in acute lymphoblastic leukemia cells: role of TGFbeta/BMP-6 and TIEG1 in chemotherapy escape.

The bone marrow microenvironment regulates early B lymphopoiesis and protects leukemia cells against chemotherapy treatment, thus the microenvironment may serve as a sanctuary site for these cells. Yet, few factors that contribute to this process are known. We have explored the role of transforming growth factor beta (TGFbeta) and bone morphogenetic protein-6 (BMP-6) and one target gene, TGFbeta inducible early gene 1 (TIEG1), in the communication between stroma cells and acute lymphoblastic leukemia (ALL) cell lines and their escape from chemotherapy. Here, we have demonstrated TIEG1 expression in both normal B progenitor cells and ALL cells, which increased rapidly upon TGFbeta and BMP-6 treatment. Stimulation with TGFbeta or BMP-6, as well as overexpression of TIEG1 inhibited proliferation. Furthermore, interaction with stroma cells induced TIEG1 expression in ALL cells, inhibited their proliferation and protected the cells against chemotherapeutic treatment. Similarly, treatment with TGFbeta or BMP-6, as well as overexpression of TIEG1, protected ALL cells against chemotherapy-induced cell death. These data suggest that TGFbeta and BMP-6 in the bone marrow microenvironment allow leukemia cells to escape therapy. Further, the data indicate that TIEG1 might be involved in mediating this effect from the microenvironment onto the leukemia cells.

Wnt3A activates canonical Wnt signalling in acute lymphoblastic leukaemia (ALL) cells and inhibits the proliferation of B-ALL cell lines.

Acute 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 beta-catenin accumulation in both primary B-ALL cells and B-ALL leukaemia cell lines. Further, Wnt3A was shown to induce nuclear translocation of beta-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.

Wnt expression and canonical Wnt signaling in human bone marrow B lymphopoiesis.

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. RESULTS: Examination of the mRNA expression pattern of Wnt ligands, Fzd receptors and Wnt antagonists revealed that BM B progenitor cells and stromal cells express a set of ligands and receptors available for induction of Wnt signaling as well as antagonists for fine tuning of this signaling. Furthermore, different B progenitor maturation stages showed differential expression of Wnt receptors and co-receptors, beta-catenin, plakoglobin, LEF-1 and TCF-4 mRNAs, suggesting canonical Wnt signaling as a regulator of early B lymphopoiesis. Exogenous Wnt3A induced stabilization and nuclear accumulation of beta-catenin in primary lineage restricted B progenitor cells. Also, Wnt3A inhibited B lymphopoiesis of CD133+CD10- hematopoietic progenitor cells and CD10+ B progenitor cells in coculture assays using a supportive layer of stromal cells. This effect was blocked by the Wnt antagonists sFRP1 or Dkk1. Examination of early events in the coculture showed that Wnt3A inhibits cell division of B progenitor cells. CONCLUSION: These results indicate that canonical Wnt signaling is involved in human BM B lymphopoiesis where it acts as a negative regulator of cell proliferation in a direct or stroma dependent manner.