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« em: Junho 01, 2020, 07:35:20 »

EGFR and EGFRvIII both paradoxically co-express with puma australia proapoptotic proteins and specifically interact with PUMA in GBM. (A) Expression of EGFR and proapoptotic proteins in human malignant glioma cell 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.

Collectively, these results indicate that EGFR and EGFRvIII both paradoxically co-express with proapoptotic proteins and specifically interact with PUMA in human GBM.Using a cohort of 101 gliomas and 12 normal brain tissues, we examined the levels of EGFR/EGFRvIII and PUMA via IHC. The results indicate a significant positive puma shoes correlation between expression levels of EGFR/EGFRvIII and PUMA (p=0.039).

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 puma suede 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.

To address whether PUMA is essential for the induction of apoptosis in GBM, PUMA-specific siRNA was used to transcriptionally down-regulate PUMA expression and the response of U87MG GBM cells, which express very low levels of EGFR, to anisomycin, a potent puma womens shoes inducer of apoptosis [ 38 ], was examined. As shown in Fig. 3a , the TUNEL assay showed that PUMA knockdown renders U87MG cells resistant to apoptosis (2.5%), induced by 48 hr anisomycin treatments. In contrast, U87MG cells transfected with the control siRNA undergo significant anisomycin-induced apoptosis (45.5%). In the assay, the yellow merged signals represent nuclear fragmented DNA.

As expected, untreated cells showed no evidence of apoptosis (data not shown). The western blotting ( Fig. 3b ) indicated that PUMA expression was specifically and efficiently down-regulated by the PUMA-specific siRNA.Effects of PUMA expression down-regulation and increased EGFRvIII expression on the induction of apoptosis in GBM cells. Extent of apoptosis is computed as: (# of apoptotic nuclei) / (total # of nuclei). In both panels, 250-300 puma mens shoes cells were examined in each experiment and three independent experiments were conducted to derive means and standard deviations.

(C) 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 kD) whereas U87MG-EGFRvIII cells contain significant EGFRvIII (140 kD) expression.
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