Gamma-Secretase Represents a Therapeutic Target for the Treatment of Invasive Glioma Mediated by the p75 Neurotrophin Receptor
Figure 1
p75NTR Is Proteolytically Processed in Patient Specimens and Invasive Glioma Cells
(A) Western blot analysis detected p75NTR-positive fragments migrating at 24 and 19 kDa in patient specimens expressing p75NTR (eight of nine GBMs, two of five Grade III glioma) but was undetectable in tissue from normal human brain. U87 cells transfected with p75NTR and grown in the presence of the proteasome inhibitor epoxomicin (1 μM) were used as a positive control (C). Western blots probed for beta-actin were used as a loading control. Molecular weight markers are indicated on the left.
(B) The highly invasive glioma cell lines U87R and U251R isolated by serial in vivo selection were treated with the proteasome inhibitor epoxomicin (Epo, 1 μM) and/or the γ-secretase inhibitor, Compound X (CompX, 2 μM) for 4 h. Western blots for p75NTR were probed with an antibody specific to the cytoplasmic domain of p75NTR which detects full-length (75 kDa), CTF (indicated by the less than symbol [<]; 25 kDa), and ICD (indicated by the asterisk [*]; 19 kDa) peptides. The ICD is derived from the cleavage of the CTF, which is shown to visibly accumulate in the presence of a γ-secretase inhibitor.
(C) p75NTR proteolytic processing is a global event in human glioma cells. pcDNA3.1 encoding human p75NTR or the empty pcDNA3.1 vector were stably transfected into U87, U251, U118, and U343. Cells expressing p75NTR (U87p75, U251p75, U118p75, and U343p75) were treated as described above. Western blot analysis showed that all glioma cell lines tested cleaved the full-length p75NTR to generate first the 24-kDa CTF and then the 19-kDa ICD.