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1.
BMC Cancer ; 16: 399, 2016 07 07.
Article in English | MEDLINE | ID: mdl-27388476

ABSTRACT

BACKGROUND: Increased numbers and improperly positioned centrosomes, aneuploidy or polyploidy, and chromosomal instability are frequently observed characteristics of cancer cells. While some aspects of these events and the checkpoint mechanisms are well studied, not all players have yet been identified. As the role of proteases other than the proteasome in tumorigenesis is an insufficiently addressed question, we investigated the epigenetic control of the widely conserved protease HTRA1 and the phenotypes of deregulation. METHODS: Mouse embryonal fibroblasts and HCT116 and SW480 cells were used to study the mechanism of epigenetic silencing of HTRA1. In addition, using cell biological and genetic methods, the phenotypes of downregulation of HTRA1 expression were investigated. RESULTS: HTRA1 is epigenetically silenced in HCT116 colon carcinoma cells via the epigenetic adaptor protein MBD2. On the cellular level, HTRA1 depletion causes multiple phenotypes including acceleration of cell growth, centrosome amplification and polyploidy in SW480 colon adenocarcinoma cells as well as in primary mouse embryonic fibroblasts (MEFs). CONCLUSIONS: Downregulation of HTRA1 causes a number of phenotypes that are hallmarks of cancer cells suggesting that the methylation state of the HtrA1 promoter may be used as a biomarker for tumour cells or cells at risk of transformation.


Subject(s)
Colonic Neoplasms/genetics , DNA Methylation , DNA-Binding Proteins/metabolism , Fibroblasts/cytology , Serine Endopeptidases/genetics , Animals , Cell Line, Tumor , Cell Proliferation , Cells, Cultured , Centrosome/metabolism , Colonic Neoplasms/pathology , Down-Regulation , Epigenesis, Genetic , Fibroblasts/metabolism , Gene Expression Regulation, Neoplastic , HCT116 Cells , High-Temperature Requirement A Serine Peptidase 1 , Humans , Mice , Neoplasm Transplantation , Polyploidy , Promoter Regions, Genetic
2.
Eur J Mass Spectrom (Chichester) ; 11(5): 547-56, 2005.
Article in English | MEDLINE | ID: mdl-16322661

ABSTRACT

Alzheimer's disease (AD) is the most common cause for human age-related dementia, characterised by formation of diffuse plaques in brain that are directly involved in AD pathogenesis. The major component of AD plaques is beta-amyloid, a 40 to 42 amino acid polypeptide derived from the amyloid precursor protein (APP) by proteolytic degradation involving the specific proteases, beta-and gamma-secretase acting at the N- and C- terminal cleavage site, respectively. In this study we have prepared polypeptides comprising the carboxy-terminal and transmembrane sequences of APP, by bacterial expression and chemical synthesis, as substrates for studying the C-terminal processing of APP and its interaction with the gamma-secretase complex. Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR-MS) was used as a major tool for structure analysis. Immunisation of transgenic mouse models of AD with Abeta42 has been recently shown to be effective to inhibit and disaggregate Abeta-fibrils, and to reduce AD-related neuropathology and memory impairments. However, the mechanism underlying these therapeutic effects has been as yet unclear. Using proteolytic epitope excision from immune complexes in combination with FT-ICR-MS, we identified the epitope recognised by the therapeutically active antibody as the N-terminal Abeta(4-10) sequence; this soluble, nontoxic epitope opens new lead structures for AD vaccine development. A monoclonal antibody (Jonas; JmAb) directed against the cytosolic APP domain was used in studies of APP biochemistry and metabolism. Here we report the identification of the epitope recognised by the JmAb, using the combination of epitope excision and peptide mapping by FT-ICR-MS. The epitope was determined to be located at the C-terminal APP(740-747) sequence; it was confirmed by ELISA binding assays and authentic synthetic peptides and will be an efficient tool in the development of new specific vaccines. These results demonstrate high-resolution FT-ICR-MS as a powerful method for characterising biochemical pathways and molecular recognition structures of APP.


Subject(s)
Amyloid beta-Protein Precursor/chemistry , Mass Spectrometry/methods , Alzheimer Disease/genetics , Alzheimer Disease/immunology , Alzheimer Disease/metabolism , Amino Acid Sequence , Amyloid beta-Protein Precursor/genetics , Amyloid beta-Protein Precursor/immunology , Animals , Cyclotrons , Epitope Mapping , Epitopes/chemistry , Epitopes/genetics , Fourier Analysis , Humans , Ions , Mice , Mice, Transgenic , Molecular Sequence Data , Molecular Structure , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Recombinant Proteins/immunology
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