DEK is an ∼45-kD phosphoprotein that is fused to the nucleoporin CAN as a result of a (6;9) chromosomal translocation in a subset of acute myeloid leukemias (AMLs). It has also been identified as an autoimmune antigen in juvenile rheumatoid arthritis and other rheumatic diseases. Despite the association of DEK with several human diseases, its function is not known. In this study, we demonstrate that DEK, together with SR proteins, associates with the SRm160 splicing coactivator in vitro. DEK is recruited to splicing factor-containing nuclear speckles upon concentration of SRm160 in these structures, indicating that DEK and SRm160 associate in vivo. We further demonstrate that DEK associates with splicing complexes through interactions mediated by SR proteins. Significantly, DEK remains bound to the exon-product RNA after splicing, and this association requires the prior formation of a spliceosome. Thus, DEK is a candidate factor for controlling postsplicing steps in gene expression that are influenced by the prior removal of an intron from pre-mRNA.

gp150 is a membrane glycoprotein which has been implicated in cell-cell adhesion in the postaggregation stages of Dictyostelium development. An analysis of its tryptic peptides by mass spectrometry has identified gp150 as the product of the lagC gene, which was previously shown to play a role in morphogenesis and cell-type specification. Antibodies raised against the GST-LagC fusion protein specifically recognized gp150 in wild-type cells and showed that it is missing in lagC-null cells. Immunolocalization studies have confirmed its enrichment in cell-cell contact regions. In mutant cells that lack the aggregation stage-specific cell adhesion molecule gp80, gp150 is expressed precociously. Moreover, these cells acquire EDTA-resistant cell-cell binding during aggregation, suggesting a role for gp150 in this process. Cells in which the genes encoding gp80 and gp150 are both inactivated do not acquire EDTA-resistant cell adhesion during aggregation. Strains transformed with an actin 15::lagC construct express gp150 precociously, but do not show EDTA-resistant adhesion during early development. However, vegetative cells expressing gp150 can be recruited into aggregates of 16-h lagC-null cells. These results, together with those obtained with the cell-to-substratum binding assay, indicate that gp150 mediates cell-cell adhesion via heterophilic interactions with another component that accumulates during the aggregation stage.

To develop a novel strategy of nonautologous somatic gene therapy, we now demonstrate the feasibility of culturing genetically modified fibroblasts within an immunoprotective environment and the optimal conditions required for their continued survival in vitro. When mouse Ltk(-) fibroblasts transfected with the human growth hormone gene were enclosed within permselective microcapsules fabricated from alginate-polylysine-alginate, they continued to secrete human growth hormone at the same rates as the nonencapsulated cells. They also continued to proliferate in vitro for at least 1 month even though their viability gradually declined to about 50%. The viability can be improved by controlling for (a) temperature during encapsulation, (b) duration of treatment with polylysine, (c) duration of liquefying the core alginate with sodium citrate, and (d) cell density at the time of encapsulation. The best conditions leading to improved survival and maximum proliferation of cells within the microcapsules were obtained by encapsulating the cells at 4 to 10 degrees C instead of room temperature, coating the microspheres with polylysine for 6 to 10 min instead of 20 min, liquefying the core alginate by treating with citrate for 20 min instead of 6 to 10 min, and using a concentration of 2 x 10(6) cells/mL of alginate for encapsulation. Under such conditions, normally adherent and genetically engineered mouse fibroblasts survived and proliferated optimally within the microcapsule environment. The encapsulated fibroblasts maintained their level of transgene expression while recombinant gene products such as human growth hormone could diffuse through the microcapsule membrane without impediment. The demonstration that genetically modified fibroblasts can survive and continue to deliver recombinant gene products from within these microcapsules and the optimization for their maximal viability and growth within microcapsules should increase the potential for success in using such microencapsulated recombinant cells for somatic gene therapy. (c) 1994 John Wiley & Sons, Inc.

Polo-like kinase 4 or PLK4 is a member of a conserved family of serine/threonine protein kinases that regulate multiple cellular processes, such as cell division and checkpoint regulation of mitosis. These kinases are often deregulated in cancer. PLK4 is the most structurally divergent PLK, localizes to centrosomes and is a critical regulator of centriole duplication. Over-expression of PLK4 leads to centrosome amplification and results in chromosome instability (CIN), a common characteristic observed in many types of cancers. We found that PLK4 is upregulated in breast cancer, specifically in the basal-like subtype. PLK4 expression is induced by hypoxia and suppressed by p53 in cancer cells. Consistent with this observation, PLK4 expression in cancer cells is upregulated when they are implanted and grown as xenografts in vivo. Furthermore, RNAi-mediated depletion of PLK4 inhibits the growth of cancer cells, but not normal cells (HMEC), in vitro, and tumor growth in vivo. Interestingly, siRNA knockdown of PLK4 sensitizes cancer cells to hypoxia. These findings suggest that targeting PLK4 may be a good therapeutic strategy in treating certain cancers. To this end, we initiated a discovery program that resulted in the identification of potent PLK4 inhibitors. These novel inhibitors are potent anti-prolifeartives, cause loss of mitotic checkpoint followed by apoptotic cell death, and suppress tumor growth in xenograft models. Mechanistically, inhibition of PLK4 suppresses phosphorylation of PLK4 and Histone H3, leads to failure of centrosome clustering and formation of multipolar spindles. Interestingly, breast cancer cell response to PLK4 inhibition appears to differ among subtypes of breast cancer cells and to be influenced by receptor and mutation status, such as ER and PTEN. Since multipolar division in cancer cells is not viable, due to massive missegregation of chromosomes, inhibition of PLK4 and formation of multipolar division followed by cell death may be a unique strategy for killing cancer cells. Implications of these findings in treating cancer will also be discussed. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr LB-215. doi:10.1158/1538-7445.AM2011-LB-215

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