Many receptor and nonreceptor tyrosine kinases activate phosphoinositide 3-kinases (PI3Ks). To assess the role of the ␦ isoform of the p110 catalytic subunit of PI3Ks, we derived enzyme-deficient mice. The mice are viable but have decreased numbers of mature B cells, a block in pro-B-cell differentiation, and a B1 B-cell deficiency. Both immunoglobulin M receptor-induced Ca 2؉ flux and proliferation in response to B-cell mitogens are attenuated. Immunoglobulin levels are decreased substantially. The ability to respond to T-cellindependent antigens is markedly reduced, and the ability to respond to T-cell-dependent antigens is completely eliminated. Germinal center formation in the spleen in response to antigen stimulation is disrupted. These results define a nonredundant signaling pathway(s) utilizing the ␦ isoform of p110 PI3K for the development and function of B cells.Lymphocyte development and function are regulated through the coordinated action of receptors of the cytokine receptor superfamily and the B-cell antigen-specific receptor (BCR) or T-cell antigen-specific receptors (TCR). Engagement of either receptor complex initiates tyrosine phosphorylation of a variety of intracellular substrates, including receptor chains, resulting in the initiation of cellular responses. Members of the cytokine receptor superfamily utilize JAK family cytoplasmic kinases (14), while the BCR and TCR complexes utilize members of the Src, Tec, and Zap70/Syk families of tyrosine kinases. In BCR signal transduction, the Tec family kinase Btk plays a critical role as evidenced by the loss of a proliferative response to BCR engagement in Btk-deficient B cells (15,16). Among the substrates typically phosphorylated and recruited to hematopoietic receptor complexes are the regulatory subunits for phosphoinositide 3-kinases (PI3Ks) (9,10,30). In mammals there are three genes that encode adapter proteins for PI3K catalytic subunits, including p85␣, p85␤, and p55␥. The adapter proteins facilitate association of the catalytic subunits with the receptor complex and are proposed to regulate enzyme activity. The disruption of the p85␣ gene, in a manner that deletes the p85 isoform as well as two splice variants of p55 and p50, results in defective BCR signaling comparable to that seen with Btk deficiency (11,26). This strongly suggests that PI3K activity is critical in BCR signal transduction.Three of the known PI3Ks, i.e., PI3K␣, PI3K␤, and PI3K␦, are regulated through their interaction with regulatory subunits (30). The fourth PI3K, PI3K␥, functions in the context of heterotrimeric G-protein-coupled receptors and is essential for leukocyte function (12). The critical, nonredundant role that PI3K␣ plays in cellular responses has been demonstrated through the derivation of mice lacking the gene. This deficiency results in an embryonic lethality at E9.5 to E10.5 due to a severe proliferative defect in many tissues (2). Similarly, deletion of the PI3K␤ gene alone results in a very early embryonic lethality (1, 2). In contrast to PI3K␣ and PI...

Activation of Stat5 is frequently found in leukemias. To study the mechanism and role of Stat5 activation, we introduced a constitutively activated Stat5a mutant, cS5F, into murine bone marrow (BM) cells. BM transplantation with cS5F-transfected cells caused development of multilineage leukemias in lethally irradiated wild-type or nonirradiated Rag2(-/-) mice. The leukemic cells showed strongly enhanced levels of cS5F tetramers but unchanged cS5F dimer levels in a DNA binding assay. Moreover, Stat5a mutants engineered to form only dimers, but not tetramers, failed to induce leukemias. In addition, Stat5 tetramers were found to accumulate in excess compared to dimers in various human leukemias. These data suggest that Stat5 tetramers are associated with leukemogenesis.

Three-dimensional (3D) tumor cell cultures grown in laminin-rich-extracellular matrix (lrECM) are considered to reflect human tumors more realistic as compared to cells grown as monolayer on plastic. Here, we systematically investigated the impact of ECM on phenotype, gene expression, EGFR signaling pathway, and on EGFR inhibition in commonly used colorectal cancer (CRC) cell lines. LrECM on-top (3D) culture assays were performed with the CRC cell lines SW-480, HT-29, DLD-1, LOVO, CACO-2, COLO-205 and COLO-206F. Morphology of lrECM cultivated CRC cell lines was determined by phase contrast and confocal laser scanning fluorescence microscopy. Proliferation of cells was examined by MTT assay, invasive capacity of the cell lines was assayed using Matrigel-coated Boyden chambers, and migratory activity was determined employing the Fence assay. Differential gene expression was analyzed at the transcriptional level by the Agilent array platform. EGFR was inhibited by using the specific small molecule inhibitor AG1478. A specific spheroid growth pattern was observed for all investigated CRC cell lines. DLD-1, HT-29 and SW-480 and CACO-2 exhibited a clear solid tumor cell formation, while LOVO, COLO-205 and COLO-206F were characterized by forming grape-like structures. Although the occurrence of a spheroid morphology did not correlate with an altered migratory, invasive, or proliferative capacity of CRC cell lines, gene expression was clearly altered in cells grown on lrECM as compared to 2D cultures. Interestingly, in KRAS wild-type cell lines, inhibition of EGFR was less effective in lrECM (3D) cultures as compared to 2D cell cultures. Thus, comparing both 2D and 3D cell culture models, our data support the influence of the ECM on cancer growth. Compared to conventional 2D cell culture, the lrECM (3D) cell culture model offers the opportunity to investigate permanent CRC cell lines under more physiological conditions, i.e. in the context of molecular therapeutic targets and their pharmacological inhibition.