We found two lines with EGFR EC variations. Both mutations resulted in amino acid substitutions at 289, the most common site of extracellular EGFR missense mutations in human GBMs. Alanine was substituted by valine in SF268 cells and by aspartic acid in SKMG3 cells. We examined whether depletion of the EGFR protein was sufficient to cause cell death in these p53 ubiquitination lines. Acute infection of SF268 and SKMG3 cells with retroviral shRNA constructs targeting two different aspects of the mRNA led to loss in EGFR protein expression within 72 hours of infection and effective cell demise induction after 5 days. EGFR knock-down in human astrocytes and two GBM cell lines without EGFR mutation didn’t cause cell death. Of notice, SKMG3 cells don’t express the cyst suppressor protein Gene expression Phosphatase and Tensin homolog, confirming our earlier findings that PTEN inactivation isn’t adequate to relieve EGFR mutant cancer cells from their reliance upon EGFR for success. We performed similar experiments with shRNA constructs targeting the EGF receptor family member HER2 transfer oncogenic indicators in a few cellular contexts and because HER2 can heterodimerize with EGFR. HER2 knockdown didn’t induce an important number of cell death as measured by the trypan blue dye exclusion assay and immunoblotting for the cleaved Caspase3 substrate Poly polymerase. HER2 destruction also did not influence EGFR phosphorylation at tyrosine 1068, suggesting that basal EGFR phosphorylation in SF268 and SKMG3 cells is not the result of trans phosphorylation by the kinase. Many prosurvival Cathepsin Inhibitor 225120-65-0 characteristics of EGFR have been related to kinase separate qualities of the receptor protein. We treated them with the 2nd era EGFR kinase inhibitor HKI 272, to examine whether EGFR kinase activity is required for the survival of SKMG3 and SF268 cells. This drug irreversibly inhibits EGFR because covalent interactions are formed by it with cysteines within the ATP cleft of the kinase domain. HKI 272 induced cell death in SKMG3 and SF268 cells, but not in EGFR wildtype GBM, lung cancer cells, or human astrocytes. We repeated our experiments with CI 1033, to give our observations with HKI 272 to a second EGFR kinase inhibitor. Like HKI 272, CI 1033 inhibits phosphorylation of wildtype EGFR in intact cells with similar potency and is definitely an irreversible, ATP site competitive inhibitor of ErbB receptors as HKI 272. To your surprise, CI 1033 did not induce cell death in both SF268 or SKMG3 cells. Immunoblots of total cell lysates from SKMG3 cells treated with either inhibitor showed that CI 1033 inhibited EGFR phosphorylation less effectively than HKI 272. We wondered if the differential effect of CI 1033 on EGFR and HKI 272 was special to GBM cells with EGFR EC strains. We therefore also compared the activity of both compounds in HCC827 lung cancer cells which harbor a deletion within the EGFR kinase domain.