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1.
FEBS Lett ; 581(24): 4571-6, 2007 Oct 02.
Article in English | MEDLINE | ID: mdl-17825299

ABSTRACT

Synthesis and maintenance of primary cilia are regulated by the von Hippel-Lindau (VHL) tumour suppressor protein. Recent studies indicate that this regulation is linked to microtubule-dependent functions of pVHL such as orienting microtubule growth and increasing plus-end microtubule stability, however little is known how this occurs. We have identified the kinesin-2 motor complex, known to regulate cilia, as a novel and endogenous pVHL binding partner. The interaction with kinesin-2 facilitates pVHL binding to microtubules. These data suggest that microtubule-dependent functions of pVHL are influenced by kinesin-2.


Subject(s)
Kinesins/metabolism , Microtubules/metabolism , Von Hippel-Lindau Tumor Suppressor Protein/metabolism , Alleles , Animals , Cell Line , Humans , Kinesins/classification , Kinesins/genetics , Mice , Mutation/genetics , Protein Binding , Von Hippel-Lindau Tumor Suppressor Protein/genetics
2.
Urology ; 66(2): 434-40, 2005 Aug.
Article in English | MEDLINE | ID: mdl-16098373

ABSTRACT

OBJECTIVES: To evaluate the efficacy of the selective cyclooxygenase-2 (COX-2) inhibitor NS-398 in treating experimental T24 bladder carcinoma, and to assess its effect on tumor cell proliferation and survival and tumor vascularization. COX-2 overexpression is frequently observed in bladder carcinoma and has been correlated with an increased disease stage and with reduced patient survival. METHODS: NS-398 was used to inhibit COX-2 selectively in human T24 bladder carcinoma cells. Lentiviral transduction of the firefly luciferase gene allowed us to assess the effect of NS-398 on orthotopic bladder carcinoma growth over time in a noninvasive manner. Immunohistochemistry on bladder tissue sections was performed to determine the effect of NS-398 treatment on tumor cell proliferation (Ki-67), apoptosis (cleaved caspase-3), and angiogenesis (von Willebrand factor; CD31). RESULTS: T24 cells expressed COX-2 and secreted prostaglandin E2 (PGE2). Selective COX-2 inhibition using NS-398 abrogated PGE2 secretion and inhibited cell proliferation in vitro in a dose-dependent manner, without affecting cell viability. In vivo administration of NS-398 reduced the outgrowth of experimental orthotopic T24 bladder carcinoma. This was accompanied by a significant reduction in Ki-67-positive tumor cells but not by a reduction in tumor cell viability or tumor vascularization. CONCLUSIONS: Selective COX-2 inhibition by NS-398 reduces the outgrowth of T24 human bladder carcinoma in an orthotopic mouse model. The therapeutic activity was most likely caused by inhibition of tumor cell proliferation, rather than by inhibition of angiogenesis or tumor cell survival.


Subject(s)
Carcinoma, Transitional Cell/prevention & control , Cyclooxygenase Inhibitors/therapeutic use , Neoplasms, Experimental/prevention & control , Nitrobenzenes/therapeutic use , Sulfonamides/therapeutic use , Urinary Bladder Neoplasms/prevention & control , Animals , Carcinoma, Transitional Cell/pathology , Cell Division/drug effects , Cyclooxygenase Inhibitors/pharmacology , Humans , Male , Mice , Mice, SCID , Neoplasms, Experimental/pathology , Nitrobenzenes/pharmacology , Sulfonamides/pharmacology , Tumor Cells, Cultured , Urinary Bladder Neoplasms/pathology
3.
J Biol Chem ; 280(23): 22205-11, 2005 Jun 10.
Article in English | MEDLINE | ID: mdl-15824109

ABSTRACT

The tumor suppressor function of the von Hippel-Lindau protein (pVHL) has previously been linked to its role in regulating hypoxia-inducible factor levels. However, VHL gene mutations suggest a hypoxia-inducible factor-independent function for the N-terminal acidic domain in tumor suppression. Here, we report that phosphorylation of the N-terminal acidic domain of pVHL by casein kinase-2 is essential for its tumor suppressor function. This post-translational modification did not affect the levels of hypoxia-inducible factor; however, it did change the binding of pVHL to another known binding partner, fibronectin. Cells expressing phospho-defective mutants caused improper fibronectin matrix deposition and demonstrated retarded tumor formation in mice. We propose that phosphorylation of the acidic domain plays a role in the regulation of proper fibronectin matrix deposition and that this may be relevant for the development of VHL-associated malignancies.


Subject(s)
Tumor Suppressor Proteins/physiology , Ubiquitin-Protein Ligases/physiology , Animals , Cell Line , Chromatography, Gel , DNA-Binding Proteins/metabolism , Dose-Response Relationship, Drug , Electrophoresis, Polyacrylamide Gel , Enzyme-Linked Immunosorbent Assay , Fibronectins/metabolism , Humans , Hypoxia/metabolism , Hypoxia-Inducible Factor 1 , Hypoxia-Inducible Factor 1, alpha Subunit , Immunoblotting , Immunoprecipitation , Mice , Mice, SCID , Mutation , Neoplasm Transplantation , Nuclear Proteins/metabolism , Phosphorylation , Plasmids/metabolism , Protein Binding , Protein Processing, Post-Translational , Protein Structure, Tertiary , Time Factors , Transcription Factors/metabolism , Transfection , Tumor Suppressor Proteins/chemistry , Tumor Suppressor Proteins/metabolism , Ubiquitin/metabolism , Ubiquitin-Protein Ligases/chemistry , Ubiquitin-Protein Ligases/metabolism , Von Hippel-Lindau Tumor Suppressor Protein
4.
FEBS Lett ; 556(1-3): 137-42, 2004 Jan 02.
Article in English | MEDLINE | ID: mdl-14706840

ABSTRACT

The von Hippel-Lindau (VHL) tumor suppressor gene regulates the extracellular matrix by controlling fibronectin deposition. To identify novel VHL target genes, we subjected mRNA from VHL-deficient RCC cells (786-0-pRC) and a transfectant re-expressing wildtype VHL (786-0-VHL) to differential expression profiling. Among the differentially expressed genes, we detected that fibronectin is upregulated in the presence of VHL, while it is not affected by hypoxia. Thus regulation of fibronectin deposition by VHL occurs at the transcriptional level, irrespective of oxygen levels.


Subject(s)
Fibronectins/metabolism , Gene Expression Regulation, Neoplastic/physiology , Genes, Tumor Suppressor , Tumor Suppressor Proteins/genetics , Tumor Suppressor Proteins/metabolism , Ubiquitin-Protein Ligases/genetics , Ubiquitin-Protein Ligases/metabolism , Carcinoma, Renal Cell/genetics , Carcinoma, Renal Cell/metabolism , Cell Hypoxia/physiology , Ceruloplasmin/biosynthesis , Ceruloplasmin/genetics , Fibronectins/genetics , Gene Expression Profiling , Humans , Insulin-Like Growth Factor Binding Protein 3/biosynthesis , Insulin-Like Growth Factor Binding Protein 3/genetics , RNA, Messenger/biosynthesis , RNA, Messenger/genetics , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Transcription, Genetic/genetics , Transfection , Tumor Cells, Cultured , Up-Regulation , Vascular Endothelial Growth Factors/biosynthesis , Vascular Endothelial Growth Factors/genetics , Von Hippel-Lindau Tumor Suppressor Protein
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