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ÿþWe examined a panel of new balance australia eight GBM and two AA cell lines for expression of EGFR/EGFRvIII and proapoptotic members of the Bcl-2 family of proteins. As shown in Fig. 1a , the majority of these cell lines co-express high levels of EGFR and three proapoptotic proteins, namely, PUMA, Bax, and Bmf. In contrast, these cells express low levels of Bad, Bim and Bok. To determine whether EGFRvIII also co-expresses with proapoptotic proteins similar to EGFR, we examined three EGFRvIII-carrying GBM xenografts since EGFRvIII expression is not maintained in vitro [ 35 ].

EGFR and EGFRvIII both paradoxically co-express with proapoptotic proteins and specifically interact with PUMA in GBM. (A) Expression of EGFR and proapoptotic proteins in human malignant glioma cell new balance shoes lines. Total cell extracts isolated from eight GBM and two AA cell lines were subjected to western blotting to determine the levels of EGFR and Bcl-2 family of proapoptotic proteins, including, Bad, PUMA, Bax, Bim, Bmf and Bok.

A new balance 247 previous report [ 36 ] showed that EGFR can exist in the cytoplasm in a free non-membrane-bound form. In this context, activated cell-surface EGFR is endocytosed and trafficked to the ER where it associates with Sec61beta, a component of the Sec61 translocon, and is then retrotranslocated from the ER to the cytoplasm as a non-membrane-bound receptor [ 37 ]. Our results in Fig. S2 in Supplemental Data show that PUMA primarily interacts with the EGFR protein that is not associated new balance 574 with plasma membranes.

As shown in Fig. 2a , the extent of EGFR/EGFRvIII-PUMA co-expression is significantly higher in high-grade/malignant gliomas (20% in AAs and 34.1% in GBMs) than in low-grade gliomas (6.3-7.7%) and normal brain tissues (0%). Regression analysis further indicates that EGFR alone, PUMA alone and EGFR/PUMA co-expression correlate significantly with glioma grade ( Fig. 2a ). Two representative immunostained gliomas are shown in Fig.

The EGFR antibody used in these studies recognized both EGFR and EGFRvIII. Regression analysis showed that expression of EGFR correlates significantly and positively with PUMA (p=0.039, R=0.9) and that EGFR alone, PUMA alone and their co-expression correlated significantly and positively with glioma grade (*EGFR/PUMA, p=0.018, R=0.94; **EGFR, p=0.00005, R=1.0; ***PUMA, p=0.021, R=0.93). (B) Representative immunostained primary gliomas. Top panel: a primary GBM stained positively for both EGFR/EGFRvIII and PUMA. Bottom new balance outlet panel: a grade II glioma showing negative staining for both proteins. Consecutive tumor sections were used in these studies.

© Increased EGFRvIII expression confers resistance of GBM cells to apoptosis. U87MG-vector and U87MG-EGFRvIII stable transfectant cells were exposed to 100 ng/ml anisomycin for 48 hrs and subjected to the TUNEL assay to determine the extent of apoptosis. Generation and characterization of the isogenic cell lines were described previously [ 9 ]. (D) Western blotting indicates that U87MG-vector cells express a very low level of endogenous EGFR (higher molecular weight; 175 [Image: new balance outlet-680mil.jpg] kD) whereas U87MG-EGFRvIII cells contain significant EGFRvIII (140 kD) expression.
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