title: CD133 glycosylation is enhanced by hypoxia in cultured glioma stem cells.
abstract: The cancer stem cell (CSC) marker CD133 is widely expressed in gliomas and             employed mostly by use of the CD133/1 antibody which binds the extracellular glycosylated             AC133 epitope. CD133 recognition may, however, be affected by its glycosylation             pattern and oxygen tension. The present study investigates the effect of oxygen             deprivation on CD133 expression and glycosylation status employing a high AC133-expressing             glioblastoma multiforme (GBM) cell line, IN699. IN699 cells were cultured under             normoxic (21% O2) and hypoxic (3% O2) conditions. CD133 expression was analysed             by western blotting (WB), qRT-PCR, immunocytochemistry (ICC) and flow cytometry             using the glycosylation-specific antibody CD133/1 and ab19898 which binds the             unglycosylated intra-cellular residues of CD133. By flow cytometry, ab19898 detected             94.1% and 96.2% CD133+ cells under normoxia and hypoxia, respectively. Hypoxia             significantly increased the percentage of CD133+ cells from 69% to 92% using CD133/1             (p<0.005). Moreover, a significantly higher geomean fluorescence intensity             (GMI) was demonstrated by ab19898 (p<0.005) in CD133+ cells. WB and qRT-PCR             results were consistent with flow cytometry data. Furthermore, over a period of             72-h incubation under normoxic and hypoxic conditions after autoMACS sorting,             an average of 31.8% and 42.2%, respectively, of CD133-negative IN699 cells became             positive using CD133/1. Our data show that a) previously reported CD133- cells             may have been misidentified using the glycosylation-specific CD133/1 as constitutive             expression of CD133 was detected by the intracellular antibody ab19898; b) hypoxia             promotes glycosylation status of CD133, indicating possible involvement of glycosylated             CD133 in the process of anti-hypoxia-mediated apoptosis.
authors: Cellular and Molecular Neuro-Oncology Research Group, Institute                     of Biomedical and Biomolecular Science, School of Pharmacy and Biomedical                     Sciences, University of Portsmouth, Portsmouth, UK.
authors: Kristina S
authors: KS
authors: Lehnus
authors: Laura K
authors: LK
authors: Donovan
authors: Xiaowei
authors: X
authors: Huang
authors: Ninghui
authors: N
authors: Zhao
authors: Tracy J
authors: TJ
authors: Warr
authors: Geoffrey J
authors: GJ
authors: Pilkington
authors: Qian
authors: Q
authors: An
mesh_terms: D000071916:AC133 Antigen; D000911:Antibodies, Monoclonal / Q000276:immunology; D015703:Antigens, CD / Q000276:immunology / Q000378:metabolism*; D017209:Apoptosis; D001932:Brain Neoplasms / Q000378:metabolism*; D015687:Cell Hypoxia; D045744:Cell Line, Tumor; D005910:Glioma / Q000378:metabolism*; D006023:Glycoproteins / Q000276:immunology / Q000378:metabolism*; D006031:Glycosylation; D006801:Humans; D014411:Neoplastic Stem Cells / Q000378:metabolism*; D010455:Peptides / Q000276:immunology / Q000378:metabolism*
chemical_list: D000071916:AC133 Antigen; D000911:Antibodies, Monoclonal; D015703:Antigens, CD; D006023:Glycoproteins; C000606023:PROM1 protein, human; D010455:Peptides