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EGFR and EGFRvIII both interact with PUMA constitutively and under apoptotic stress, leading to cytoplasmic sequestration of PUMA in GBM cells. In contrast, PUMA is exclusively localized on the mitochondrial membranes, as expected, in EGFR-negative GBM cells. These results, together with additional data from functional studies provide evidence for a novel inverse functional link between the EGFR/EGFRvIII signaling and the proapoptotic pathway and this can be a mechanism that accounts for EGFR-associated drug resistance in GBM and, potentially, in other cancer types that also co-express EGFR and PUMA.

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 cheap new balance 1500 B mouse monoclonal antibody was from Calbiochem (San Diego, CA). ²-actin and ±-tubulin antibodies were obtained from Sigma.

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