ABSTRACT
Glioblastoma multiforme (GBM) is among the most aggressive tumor types and is essentially an incurable malignancy characterized by resistance to chemo-, radio-, and immunotherapy. GBM is maintained by a hierarchical cell organization that includes stem-like, precursor, and differentiated cells. Recurrence and maintenance of the tumor is attributed to a small population of undifferentiated tumor-initiating cells, defined as glioblastoma stem-like cells (GSLCs). This cellular hierarchy offers a potential treatment to induce differentiation of GSLCs away from tumor initiation to a more benign phenotype or to a cell type more amenable to standard therapies. Bone morphogenetic proteins (BMPs), members of the TGF-ß superfamily, have numerous biological activities including control of growth and differentiation. In vitro, a BMP7 variant (BMP7v) decreased primary human GSLC proliferation, endothelial cord formation, and stem cell marker expression while enhancing neuronal and astrocyte differentiation marker expression. In subcutaneous and orthotopic GSLC xenografts, which closely reproduce the human disease, BMP7v decreased tumor growth and stem cell marker expression, while enhancing astrocyte and neuronal differentiation compared with control mice. In addition, BMP7v reduced brain invasion, angiogenesis, and associated mortality in the orthotopic model. Inducing differentiation of GSLCs and inhibiting angiogenesis with BMP7v provides a potentially powerful and novel approach to the treatment of GBM.
Subject(s)
Bone Morphogenetic Protein 7/pharmacology , Neoplastic Stem Cells/metabolism , Animals , Bone Morphogenetic Protein 7/genetics , Bone Morphogenetic Protein 7/therapeutic use , Brain Neoplasms/drug therapy , Brain Neoplasms/metabolism , Brain Neoplasms/pathology , Cell Differentiation/drug effects , Cell Line, Tumor , Cell Proliferation/drug effects , Cytokines/metabolism , Glioblastoma/drug therapy , Glioblastoma/metabolism , Glioblastoma/pathology , HCT116 Cells , Humans , Mice , Neoplastic Stem Cells/drug effects , Neovascularization, Pathologic , Transplantation, HeterologousABSTRACT
The serine/threonine kinase Akt has been implicated in the control of cell survival and metabolism. Here we report the disruption of the most ubiquitously expressed member of the akt family of genes, akt1, in the mouse. Akt1(-/-) mice are viable but smaller when compared to wild-type littermates. In addition, the life span of Akt1(-/-) mice, upon exposure to genotoxic stress, is shorter. However, Akt1(-/-) mice do not display a diabetic phenotype. Increased spontaneous apoptosis in testes, and attenuation of spermatogenesis is observed in Akt1(-/-) male mice. Increased spontaneous apoptosis is also observed in the thymi of Akt1(-/-) mice, and Akt1(-/-) thymocytes are more sensitive to apoptosis induced by gamma-irradiation and dexamethasone. Finally, Akt1(-/-) mouse embryo fibroblasts (MEFs) are more susceptible to apoptosis induced by TNF, anti-Fas, UV irradiation, and serum withdrawal.
Subject(s)
Apoptosis , Arabidopsis Proteins , Plant Proteins/genetics , Plant Proteins/physiology , Potassium Channels/genetics , Potassium Channels/physiology , Alleles , Animals , Blotting, Western , Body Weight/genetics , Cells, Cultured , Crosses, Genetic , Culture Media, Serum-Free/pharmacology , Dexamethasone/pharmacology , Diabetes Mellitus, Experimental/genetics , Fibroblasts/metabolism , Gamma Rays , Genotype , Glucocorticoids/pharmacology , Homozygote , In Situ Nick-End Labeling , Male , Mice , Mice, Knockout , Models, Genetic , Mutagenesis, Site-Directed , Mutation , Phenotype , Spermatogenesis/genetics , T-Lymphocytes/metabolism , Testis/pathology , Thymus Gland/metabolism , Time Factors , Tumor Necrosis Factor-alpha/pharmacology , fas Receptor/metabolismABSTRACT
The structure of a ubiquitin-like protein, small ubiquitin-related modifier-1 (SUMO-1), was earlier determined using homonuclear nuclear magnetic resonance (NMR) spectroscopy, since the spectral quality of the protein was not suitable for heteronuclear NMR data collection. In this study, a slightly different construct of the SUMO-1 gene was used for protein over-expression. The protein purified from this construct showed high spectral qualities, therefore, multi-dimensional heteronuclear NMR data for a dynamic study and structural determination were acquired. The structure of SUMO-1 obtained in this study differs in several respects from the structure obtained from homonuclear NMR data. Furthermore, structural differences were observed between the new SUMO-1 and ubiquitin structures. These differences may be important for SUMO-1-specific recognition in cells. Additionally, relaxation parameters indicate that SUMO-1 undergoes highly anisotropic tumbling in solution and that the long amino (N)-terminal sequence of SUMO-1 is highly dynamic with increasing flexibility towards the end.
Subject(s)
Models, Molecular , Ubiquitins/chemistry , Escherichia coli/genetics , Humans , Magnetic Resonance Spectroscopy/methods , Protein Conformation , Protein Folding , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Recombinant Proteins/isolation & purification , SUMO-1 Protein , Ubiquitins/genetics , Ubiquitins/isolation & purificationABSTRACT
The winged helix-turn-helix motif was initially identified in the mammalian hepatocyte-enriched transcription factor HNF-3 and the Drosophila forkhead homeotic protein. Proteins containing the winged helix motif have been shown to play important roles in tissue-specific developmental regulation. In this report, by using a genomic binding site selection method, we demonstrate that the winged helix protein YFKH-1 from the yeast Saccharomyces cerevisiae recognizes conserved sequence in yeast centromeres. Thus, our data suggest that the winged helix proteins of the yeast may be involved in centromeric functions of the yeast.
Subject(s)
Centromere/metabolism , DNA-Binding Proteins/genetics , Fungal Proteins/genetics , Saccharomyces cerevisiae Proteins , Saccharomyces cerevisiae/genetics , Amino Acid Sequence , Binding Sites/genetics , Conserved Sequence/genetics , DNA, Fungal/metabolism , DNA-Binding Proteins/metabolism , Fungal Proteins/metabolism , Molecular Sequence Data , Plasmids/genetics , Plasmids/isolation & purification , Protein Structure, Tertiary/genetics , Saccharomyces cerevisiae/metabolism , Sequence Homology, Amino AcidABSTRACT
The dissociation constants and the half-lives of the DNA complexes of winged helix protein Genesis and its mutants were investigated. Our data show that substitutions of non-DNA contact residues in the two highly dynamic wing sequences dramatically change the off rates of the complexes. Thus, our data indicate that the off rate of a DNA complex is encoded in the amino acid sequence of the DNA-binding protein and/or should be a unique property for a protein-DNA complex. Our data also indicate that the local structural transition of a DNA-binding protein in its DNA recognition process is likely to go through a transition state, which can control the on and off rates of a complex.
Subject(s)
Amino Acid Substitution , DNA-Binding Proteins/metabolism , DNA/metabolism , Repressor Proteins/metabolism , Amino Acid Sequence , Animals , Binding, Competitive , DNA-Binding Proteins/chemistry , DNA-Binding Proteins/genetics , Forkhead Transcription Factors , Half-Life , Helix-Turn-Helix Motifs , Kinetics , Molecular Sequence Data , Nuclear Magnetic Resonance, Biomolecular , Proline/genetics , Proline/metabolism , Protein Folding , Protein Structure, Secondary , Rats , Repressor Proteins/chemistry , Repressor Proteins/genetics , ThermodynamicsABSTRACT
The hepatocyte nuclear factor 3 (HNF-3)/fork head (fkh) family contains a large number of transcription factors which recognize divergent DNA sequences via a winged-helix binding motif. In this report we present studies on the DNA binding properties of winged-helix HNF-3/fkh homologues 1 (HFH-1) and 2 (HFH-2) which recognize a shared DNA binding site with different affinities. To explore how HFH-1 and HFH-2 proteins recognize this DNA binding sequence, the binding affinities of these two HFH proteins toward a series of DNA sites containing a single strand trimer loop insertion at different positions were studied. This insertion induces a bend of approximately 80 degrees in the DNA binding site (prebending). HFH-1 and HFH-2 were shown to recognize DNA sites prebent at many nucleotide positions on both strands of the DNA sequence. Both HFH-1 and HFH-2 were more sensitive to mismatch insertions on the sense strand of the DNA binding site, especially within the AAAATAAC sequence. Our data suggest that the recognition helix (helix 3) recognizes the AAAATAAC sequence and that the helix 3/DNA interaction results in bending of the DNA which narrows the major groove in the AAAATAAC sequence. Furthermore, the binding affinities of HFH-1 and HFH-2 toward DNA binding sites with base-pair reversion in the AAAATAAC sequence was also investigated. Different patterns of response from HFH-1 and HFH-2 to both prebent and base-pair reverted binding sites was observed. Our results demonstrate that even two highly conserved members of the winged-helix family may contact the same DNA sequence differently.
