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the interaction between the EGFRvIII and the Cbl proteins was below the level of sensitivity of the immunoprecipitation and immunoblotting procedure used by Schmidt et al The constitutive TK activity of the EGFRvIII results in the malignant transformation of cells . In this study, we found that the EGFRvIII is regulated by the Cbl proteins in an identical manner to the WT Ubiquitin conjugation inhibitor EGFR. This is unsurprising given that the activity and phosphorylation pattern of the dimerized EGFRvIII is similar to that of the WT EGFR following EGF stimulation . Indeed, we were able to detect phosphorylation of the Cbl TKBbinding site on the EGFRvIII using a specific antibody . In addition, Reist et al. reported that the EGFRvIII is internalized rapidly from the surface of fibroblasts transfected with the EGFR vIII, suggesting that it is downregulated.
Conversely, in a study using glioblastoma cells transfected with either the WT EGFR or the EGFRvIII, Huang et al. reported that, while the EGF stimulated WT EGFR is rapidly endocytosed, the EGFRvIII is internalized at a similar rate to that of the unstimulated WT EGFR. This suggests that the EGFRvIII is not downregulated. However, only a small proportion of the Ubiquitin conjugation inhibitor total EGFRvIII protein is active when compared to the ligand bound EGFR . It is likely that, compared to the spontaneous endocytosis of the overexpressed WT EGFR, the enhanced internalization of the small amount of active EGFRvIII does not significantly affect the overall rate of endocytosis. Our work indicates that active EGFRvIII is degraded by a Cbl protein dependent mechanism.
However, cancer cells with amplification of the EGFRvIII constitutively Docetaxel synthesize new inactive EGFRvIII protein. Experiments using the EGFR inhibitor AG 1478 demonstrate that the Cbl proteins do not mediate ubiquitination or degradation of inactive EGFRvIII . The amplification and overexpression of the EGFRvIII creates a large pool of inactive receptor, a small fraction of which spontaneously activates to replenish the pool of downregulated active EGFRvIII. Thus, at steady state equilibrium, there always will be active EGFRvIII and this results in the transformation of cells. The overexpression of Cbl b inhibits the transformation of fibroblasts by the EGFRvIII by enhancing the degradation of the active EGFRvIII. Conversely, the mutation of the Cbl binding site in the EGFRvIII increases its capacity to transform by preventing degradation of the active EGFRvIII.
The anti EGFRvIII immunotoxin, MR1 HSP 1 PE38, kills glioblastoma cells that ectopically express the EGFRvIII . In this study, we used an MTS dye reduction assay to test the ability of this immunotoxin to kill a Swiss 3T3 derived cell line that does not express the WT EGFR . Although MR1 1 PE38 did not effect the growth of NR 6 cells, it caused a concentration dependent death of EGFRvIIIexpressing NR 6m cells . This finding confirmed the previous report that MR1 1 PE38 specifically kills EGFRvIII expressing cells. The IC50 of MR1 1 PE38 in this study is similar to previously reported values . To function, immunotoxins must be internalized upon binding to their receptors ; indeed anti EGFRvIII monoclonal antibodies including MR1 1 PE38 are rapidly internalized by EGFRvIII expressing cells .
These internalized Docetaxel antibodies become localized to vesicles in the perinuclear Golgi region and are rapidly catabolized, suggesting that the internalized EGFRvIII:monoclonal antibody complex is trafficked to the lysosome. The Cbl proteins are critical regulators of the trafficking of the WT EGFR to the lysosome and this study has established that they regulate the constitutively active EGFRvIII. Furthermore, the inhibition of the TK activity of the EGFRvIII prevents its downregulation by the Cbl proteins and decreases the amount of EGFRvIII located in intracellular vesicles . Therefore, we tested whether inhibition of the EGFR vIII TK affects the efficacy of MR1 1 PE38.
Consistent with the ability of the EGFRvIII to undergo activation induced downregulation, we found that treatment with AG 1478 caused an approximately 1000 fold increase in Conjugating enzyme inhibitor the IC50 of MR1 1 PE38 . Thus, the inhibition of the TK activity of the EGFRvIII appears to antagonize MR1 1 PE38 in vitro. Like the WT EGFR, the EGFRvIII also can be spontaneously endocytosed in an activation independent manner. Docetaxel Thus, MR1 1 PE38 is still capable of killing cells in the presence of AG1478, albeit with an IC50 1000 fold higher than untreated cells. This finding suggests that TK inhibitors and immunotoxins may Docetaxel be antagonistic if used together for the treatment of EGFRvIII expressing tumors. This study has demonstrated that the EGFRvIII undergoes activation induced downregulation by the Cbl proteins. This suggests that the ability of the EGFRvIII to transform cells is not a consequence of unattenuated signaling from this mutant, but is due rather to the spontaneous activity of this TK. The ability of the EGFRvIII to be regulated by the Cbl proteins has implication