CD133 glycosylation is enhanced by hypoxia in cultured glioma stem cells

The cancer stem cell (CSC) marker CD133 is widely expressed in gliomas andemployed mostly by use of the CD133/1 antibody which binds the extracellular glycosylatedAC133 epitope. CD133 recognition may, however, be affected by its glycosylationpattern and oxygen tension. The present study investigates the effect of oxygendeprivation on CD133 expression and glycosylation status employing a high AC133-expressingglioblastoma multiforme (GBM) cell line, IN699. IN699 cells were cultured undernormoxic (21% O2) and hypoxic (3% O2) conditions. CD133 expression was analysedby western blotting (WB), qRT-PCR, immunocytochemistry (ICC) and flow cytometryusing the glycosylation-specific antibody CD133/1 and ab19898 which binds theunglycosylated intra-cellular residues of CD133. By flow cytometry, ab19898 detected94.1% and 96.2% CD133+ cells under normoxia and hypoxia, respectively. Hypoxiasignificantly 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-PCRresults were consistent with flow cytometry data. Furthermore, over a period of72-h incubation under normoxic and hypoxic conditions after autoMACS sorting,an average of 31.8% and 42.2%, respectively, of CD133-negative IN699 cells becamepositive using CD133/1. Our data show that a) previously reported CD133- cellsmay have been misidentified using the glycosylation-specific CD133/1 as constitutiveexpression of CD133 was detected by the intracellular antibody ab19898; b) hypoxiapromotes glycosylation status of CD133, indicating possible involvement of glycosylatedCD133 in the process of anti-hypoxia-mediated apoptosis.