ABSTRACT
Focal adhesion kinase is a non-receptor protein tyrosine kinase with signaling functions downstream of integrins and growth factor receptors. In addition to its role in adhesion, migration, and proliferation it also has non-kinase scaffolding functions in the nucleus. Focal adhesion kinase (FAK) activation involves the following: (1) ligand bound growth factors or clustered integrins activate FAK kinase domain; (2) FAK autophosphorylates tyrosine (Y) 397; (3) Src binds pY397 and phosphorylates FAK at various other sites including Y861; (4) downstream signaling of activated FAK elicits changes in cellular behavior. Although many studies have demonstrated roles for the kinase domain, Y397 and Y861 sites, in vitro much less is known about their functions in vivo. Here, we report the generation of a series of FAK-mutant knockin mice where mutant FAK, either kinase dead, non-phosphorylatable mutants Y397F and Y861F, or mutant Y397E-containing a phosphomimetic site that results in a constitutive active Y397, can be expressed in a Cre inducible fashion driven by the ROSA26 promoter. In future studies, intercrossing these mice with FAKflox/flox mice and inducible cre-expressing mice will enable the in vivo study of mutant FAK function in the absence of endogenous FAK in a spatially and temporally regulated fashion within the whole organism.
Subject(s)
Enzyme Activation/physiology , Focal Adhesion Kinase 1/genetics , Models, Animal , Point Mutation/genetics , Signal Transduction/genetics , Animals , Base Sequence , Blotting, Western , Enzyme Activation/genetics , Fluorescent Antibody Technique , Gene Knock-In Techniques , Genetic Vectors/genetics , Immunoprecipitation , Mice , Molecular Probes/genetics , Molecular Sequence Data , Mutagenesis, Site-Directed , Phosphorylation , Real-Time Polymerase Chain Reaction , TamoxifenABSTRACT
BACKGROUND: Progressive tumour growth is dependent on the development of a functional tumour vasculature and highly regulated by growth factors and cytokines. Nitric oxide (NO) is a free radical, produced both by tumour and host cells, and functions as a signalling molecule downstream of several angiogenic factors. Both pro- and antitumourigenic properties have been attributed to NO. METHODS: The expression of the inducible isoform of NO synthase (iNOS) was knocked down in the C6 glioma cell line using constitutive expression of antisense RNA, and the effect of tumour-derived NO on tumour progression and angiogenesis was investigated. RESULTS: Tumours in which iNOS expression was decreased displayed significantly reduced growth rates compared with tumours derived from parental C6 cells. Quantitative non-invasive magnetic resonance imaging and fluorescence microscopy of tumour uptake of Hoechst 33342, and haematoxylin and eosin staining, revealed significantly impaired vascular development and function in antisense iNOS tumours compared with control in vivo, primarily associated with the more necrotic tumour core. Decreased iNOS expression had no effect on tumour VEGF expression. CONCLUSION: Nitric oxide derived from tumour iNOS is an important modulator of tumour progression and angiogenesis in C6 gliomas and further supports the therapeutic strategy of inhibiting iNOS for the treatment of cancer.
Subject(s)
Brain Neoplasms/blood supply , Brain Neoplasms/enzymology , Glioma/blood supply , Glioma/enzymology , Neovascularization, Pathologic/enzymology , Nitric Oxide Synthase Type II/physiology , Animals , Blotting, Western , Brain Neoplasms/pathology , DNA, Antisense/pharmacology , Disease Progression , Female , Gene Silencing/physiology , Glioma/pathology , Immunoenzyme Techniques , Magnetic Resonance Imaging , Mice , Mice, Nude , Microscopy, Fluorescence , Nitric Oxide/metabolism , Rats , Tumor Cells, Cultured , Vascular Endothelial Growth Factor A/metabolism , Xenograft Model Antitumor AssaysABSTRACT
Angiogenesis is a prerequisite for tumour progression and is highly regulated by growth factors and cytokines a number of which also stimulate the production of nitric oxide. Asymmetric dimethylarginine is an endogenous inhibitor of nitric oxide synthesis. Asymmetric dimethylarginine is metabolised by dimethylarginine dimethylaminohydrolase. To study the effect of dimethylarginine dimethylaminohydrolase on tumour growth and vascular development, the rat C6 glioma cell line was manipulated to overexpress the rat gene for dimethylarginine dimethylaminohydrolase I. Enhanced expression of dimethylarginine dimethylaminohydrolase I increased nitric oxide synthesis (as indicated by a two-fold increase in the production of cGMP), expression and secretion of vascular endothelial cell growth factor, and induced angiogenesis in vitro. Tumours derived from these cells grew more rapidly in vivo than cells with normal dimethylarginine dimethylaminohydrolase I expression. Immunohistochemical and magnetic resonance imaging measurements were consistent with increased tumour vascular development. Furthermore, dimethylarginine dimethylaminohydrolase activity was detected in a series of human tumours. This data demonstrates that dimethylarginine dimethylaminohydrolase plays a pivotal role in tumour growth and the development of the tumour vasculature by regulating the concentration of nitric oxide and altering vascular endothelial cell growth factor production.
Subject(s)
Amidohydrolases/metabolism , Brain Neoplasms/blood supply , Brain Neoplasms/pathology , Endothelial Growth Factors/metabolism , Endothelium, Vascular/metabolism , Glioma/blood supply , Glioma/pathology , Lymphokines/metabolism , Neovascularization, Pathologic , Nitric Oxide/metabolism , Amidohydrolases/genetics , Amidohydrolases/pharmacology , Animals , Astrocytoma/metabolism , Astrocytoma/pathology , Blotting, Northern , Blotting, Western , Brain Neoplasms/metabolism , Cell Division/physiology , Cell Movement , Cells, Cultured , Cyclic GMP/metabolism , DNA Primers/chemistry , Enzyme-Linked Immunosorbent Assay , Female , Glioblastoma/metabolism , Glioblastoma/pathology , Glioma/metabolism , Humans , Mice , Mice, Nude , Neoplasm Transplantation , Neoplasms, Experimental/blood supply , Neoplasms, Experimental/metabolism , Neoplasms, Experimental/pathology , Neovascularization, Pathologic/metabolism , Rats , Reverse Transcriptase Polymerase Chain Reaction , Transfection , Vascular Endothelial Growth Factor A , Vascular Endothelial Growth FactorsABSTRACT
The archaeal H and eukaryotic RPB5 RNA polymerase subunits are highly homologous and are likely to play a fundamental role in transcription that extends from archaea to humans. We report the structure of subunit H, in solution, from the archaeon Methanococcus jannaschii using multidimensional nuclear magnetic resonance. The structure reveals a novel fold containing a four-stranded mixed beta sheet that is flanked on one side by three short helices. The dominant feature is beta-ribbon motif, which presents a hydrophobic, basic surface, and defines a general RNA polymerase architectural scaffold.