thor manuscript, available in PMC 2008 March 25. NIH PA Author Manuscript NIH PA Author Manuscript NIH PA Author Manuscript PE38. Consistent with the ability of the EGFRvIII to undergo activation induced downregulation, PS-341 Velcade we found that treatment with AG 1478 caused an approximately 1000 fold increase in 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. 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 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 CCT128930 885499-61-6 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 implications for the treatment of malignancies. Therapies, such as immunotoxins, that exploit the down regulation of the EGFRvIII or therapies aimed at enhancing the activation induced degradation of this mutant offer a promising approach to the treatment of EGFRvIII expressing tumors. However, the use of TK inhibitors in conjunction with these therapies may decrease their efficacy.
Materials and methods Materials Dulbecco,s modified Eagle,s medium, fetal bovine serum, penicillin, streptomycin sulfate, and Zeocin were obtained from Invitrogen. Dulbecco,s phosphate buffered saline and G 418 sulfate were purchased from Mediatech Inc.. AG 1478, ALLN, cycloheximide, MG 132, lactacystin, and folimycin were acquired from EMD Biosciences Inc.. Leupeptin hemisulfate was bought from MP Biomedicals. Chloroquine, ammonium chloride, and DMSO were obtained from Sigma Aldrich Corp.. Recombinant human EGF was purchased from BD Biosciences, Inc.. A recombinant immunotoxin generated from an EGFRvIII specific single chain Fv domain fused to domains I and II of the Pseudomonas exotoxin PE38 was provided by Dr Ira Pastan. Tissue culture plastic ware and other laboratory consumables were purchased from commercial sources.
Expression constructs The expression plasmids for full length WT and HA epitope tagged Cbl, Cbl b, and Cbl c along with HA epitope tagged full length RING finger mutant Cbl b, C2/3 Cbl b, N1/2 Cbl b, and the control vector have been described previously. The cDNA for the EGFRvIII was a gift from Dr Gordon N Gill and was cloned into pSVZeo. Site directed mutagenesis of EGFRvIII was performed using the Quick Change Kit. All of the constructs were confirmed by DNA sequencing. The GFP expression plasmid was obtained from Invitrogen. The HA epitope tagged ubiquitin expression plasmid was provided by Dr Dirk Bohmann. Cell culture, transfections, and foci assays CHO, HEK 293T, and NIH 3T3 cells were maintained in culture in DMEM supplemented with 10% FBS, 100 U/ml penicillin, and 100 g/ml streptomycin sulfate. NR 6 cells were maintained in DMEM supplemented with 5% FBS, 100 U/ml penicillin, and 100 g/ml Davies et al. Page 9 Oncogene. Author manuscript, available in PMC 2008 March 25. NIH PA