Suzanne Lyman

Integrin-mediated Activation of Focal Adhesion Kinase Is Independent of Focal Adhesion Formation or Integrin Activation STUDIES WITH ACTIVATED AND INHIBITORY β3CYTOPLASMIC DOMAIN MUTANTS

Integrin αIIbβ3functions as the fibrinogen receptor on platelets and mediates platelet aggregation and clot retraction. Among the events that occur during either “inside-out” or “outside-in” signaling through αIIbβ3 is the phosphorylation of focal adhesion kinase (pp125FAK) and the association of pp125FAK with cytoskeletal components. To examine the role of pp125FAK in these integrin-mediated events, pp125FAK phosphorylation and association with the cytoskeleton was determined in cells expressing two mutant forms of αIIbβ3: αIIbβ3(D723A/E726A), a constitutively active integrin in which the putative binding site for pp125FAK is altered, and αIIbβ3(F727A/K729E/F730A), in which the putative binding site for α-actinin is altered. Both mutants were expressed on the cell surface and were able to bind ligand, either spontaneously or upon activation. Whereas cells expressing αIIbβ3(D723A/E726A) were able to form focal adhesions and stress fibers upon adherence to fibrinogen, cells expressing αIIbβ3(F727A/K729E/F730A) adhere to fibrinogen, but had reduced focal adhesions and stress fibers. pp125FAK is recruited to focal adhesions in adherent cells expressing αIIbβ3(D723A/E726A) and is phosphorylated in adherent cells or in cells in suspension in the presence of fibrinogen. In adherent cells expressing αIIbβ3(F727A/K729E/F730A), pp125FAK was phosphorylated despite reduced formation of focal adhesions and stress fibers. We conclude that activation of pp125FAK can be dissociated from two important events in integrin signaling, the assembly of focal adhesions in adherent cells and integrin activation following ligand occupation.

Ski Promotes Tumor Growth Through Abrogation of Transforming Growth Factor-β Signaling in Pancreatic Cancer

Objective:We hypothesized that human pancreatic cancer resists TGF-β signaling and cell death through increased Ski expression. Summary Background Data:Ski is an oncogenic protein that acts as a TGF-β repressor and prevents related gene transcription. Previous work suggests that Ski acts as an oncoprotein in melanoma and esophageal cancer. Ski expression and function have not been determined in human pancreatic cancer. Methods:Immunohistochemistry and immunoblots assessed Ski expression in human pancreatic cancer. Panc-1 cells were treated with or without Ski siRNA, and Ski and Smad protein expression, transcriptional reporter activation, and growth assays were determined. Panc-1 cells were inoculated in the flank of nude mice and tumor volume and histology assessed after administration of Ski siRNA or control vector. Results:Ski was abundantly expressed in human pancreatic cancer specimens assessed by immunohistochemistry (91%) and immunoblot analysis (67%). Panc-1 cells exhibited nascent Ski expression that was maximally inhibited 48 hours after transfection with Ski siRNA. TGF-β transcriptional activity was increased 2.5-fold in Ski siRNA-treated cells compared with control (P < 0.05). Ski siRNA increased TGF-β-induced Smad2 phosphorylation and p21 expression. Panc-1 growth in culture was decreased 2-fold at 72 hours. A Ski siRNA expression vector injected into nude mice resulted in a 5-fold decrease in growth. Conclusion:Inhibition of Ski through RNA interference restored TGF-β signaling and growth inhibition in vitro, and decreased tumor growth in vivo.

Recombineering-Based Procedure for Creating BAC Transgene Constructs for Animals and Cell Lines

The use of BAC/P1 as a vector for the generation of a transgene has gained popularity after the genomic annotation of many organisms was completed (often based on the respective BAC library). Large‐scale generation of BAC transgenic mice has proven that BAC transgene approaches have less integration position effects and dosage artifacts when compared with traditional transgenic approaches. Also, a BAC can achieve the same tissue‐specific expression as a knock‐in of the same gene with less effort and shorter time of establishment. The λ‐RED recombinogenic system has been used to manipulate DNA constructs with site‐directed mutagenesis, truncation, and tagging with an epitope tag or as a fusion protein by homologous recombination, as well as used here to modify many BACs with various transgenes. The recombineering plasmid, pKD46, is used to fabricate BAC transgenic constructs that can be used in generating transgenic organisms as well as used in mammalian cell culture. Curr. Protoc. Mol. Biol. 95:23.14.1‐23.14.28.

Making BAC transgene constructs with lambda-red recombineering system for transgenic animals or cell lines.

The genomic DNA libraries based on Bacteria Artificial Chromosomes (BAC) are the foundation of whole genomic mapping, sequencing, and annotation for many species like mice and humans. With their large insert size, BACs harbor the gene-of-interest and nearby transcriptional regulatory elements necessary to direct the expression of the gene-of-interest in a temporal and cell-type specific manner. When replacing a gene-of-interest with a transgene in vivo, the transgene can be expressed with the same patterns and machinery as that of the endogenous gene. This chapter describes in detail a method of using lambda-red recombineering to make BAC transgene constructs with the integration of a transgene into a designated location within a BAC. As the final BAC construct will be used for transfection in cell lines or making transgenic animals, specific considerations with BAC transgenes such as genotyping, BAC coverage and integrity as well as quality of BAC DNA will be addressed. Not only does this approach provide a practical and effective way to modify large DNA constructs, the same recombineering principles can apply to smaller high copy plasmids as well as to chromosome engineering.