We report that in puma latest shoes glioblastoma, EGFR/EGFRvIII paradoxically co-expresses with p53-upregulated modulator of apoptosis (PUMA), a proapoptotic member of the Bcl-2 family of proteins primarily located on the mitochondria. EGFR/EGFRvIII binds to PUMA constitutively and under apoptotic stress, and subsequently sequesters PUMA in the cytoplasm. The EGFR-PUMA interaction is independent of EGFR activation and is sustained under EGFR inhibition. A Bcl-2/Bcl-xL inhibitor that mimics PUMA activity sensitizes EGFR/EGFRvIII-expressing glioblastoma cells to Iressa. Collectively, we uncovered a novel kinase-independent function of EGFR/EGFRvIII that leads to tumor drug resistance.

Conversely, the proapoptotic members of the Bcl-2 family of proteins function by antagonizing the anti-apoptotic proteins and include the multi-BH3 domain proteins (Bax, Bak, and Bok) and the BH3-only proteins (Bid, Bim, Bmf and PUMA). Although, these proapoptotic proteins appear to be functionally redundant, their expression has been shown to be tumor-specific [ 23 ] and puma leather sneakers one of the main ones, PUMA, has recently been shown to mediate EGFR inhibitor-induced apoptosis in head and neck cancer [ 24 ]. Although, a direct regulatory role of EGFR/EGFRvIII in apoptosis has not been reported, it has been recently shown that ErbB4, a member of the puma lifestyle shoes EGFR family of receptor tyrosine kinases, interacts with the anti-apoptotic Bcl-2 protein to promotes apoptosis [ 25 ].

Therapeutic activation of the apoptotic pathway has emerged as an attractive treatment strategy for a number of cancers, including, GBM [ 26 ; 27 ]. In GBMs, however, to date, a systemic analysis of the expression profile of the proapoptotic members of Bcl-2 family of proteins has not been reported and the relationship between these proapoptotic proteins and EGFR/EGFRvIII remains unclear in GBMs. In this study, we investigated the interaction between PUMA and EGFR and its potential puma mexico role in EGFR-targeted mono and combinational therapies of GBMs. Our results show that, paradoxically, both wild-type EGFR and EGFRvIII co-express with proapoptotic protein PUMA in GBM cells, in vitro and in vivo .

All chemicals were purchased from Sigma (St. Louis, MO) unless otherwise stated. Rabbit polyclonal anti-EGFR antibody used in western blotting was purchased from Santa Cruz Biotech. (sc-03; Santa Cruz, CA). The EGFR and EGFRvIII expression vectors were previously generated in our laboratory [ 9 ] and both proteins were expressed as Myc-tagged fusion proteins. Anti-Myc mouse monoclonal antibody was purchased from Roche (Indianapolis, IN). Anti-lamin B mouse monoclonal antibody was from Calbiochem (San Diego, CA). ²-actin and ±-tubulin antibodies were obtained from Sigma.

This was performed, as we described previously [ 9 ]. For EGFR/EGFRvIII, two tissue arrays (Imgenex; IMT-01240 and IMT-01241) were immunostained. A tissue array (IMT-01255) was previously stained for EGFR/EGFRvIII [ 9 ]. The anti-EGFR mouse monoclonal antibody used in IHC recognizes the C-terminus of both EGFR and EGFRvIII. For PUMA, we immunostained all three tissue arrays that are consisted of 12 normal brain tissues puma official site and 101 primary gliomas. The tissue sections were deparaffinized, dehydrated, and subjected to antigen retrieval in an EDTA-containing buffer in an oven.

Endogenous peroxidase activity was blocked with 0.3% hydrogen peroxide and the slides incubated with 10% normal goat serum for 30 min and then with anti-EGFR mouse monoclonal antibody (1:50; Novocastra RTU-EGFR-384) and anti-PUMA rabbit polyclonal antibody (1:100; Cell Signaling) at 4°C overnight. Following washes with PBS, the slides were incubated with biotinylated secondary antibodies and then with avidin-biotin-horseradish peroxidase complex. Detection was performed using 0.125% aminoethylcarbazole chromogen. After counterstaining with Mayer's hematoxylin (Sigma), the sides were mounted.