Adenovirus-mediated overexpression of dominant negative epidermal growth factor receptor-CD533 as a gene therapeutic approach radiosensitizes human carcinoma and malignant glioma cells

Purpose: Epidermal growth factor receptor (EGFR) and other members of the E rbB family of receptor tyrosine kinases (RTK) mediate autocrine growth regu lation in a wide spectrum of human tumor cells. We have previously demonstr ated that in stably transfected mammary carcinoma cells a dominant negative (DN) mutant of EGFR, EGFR-CD533 is a potent inhibitor of EGFR and its cyto protective signaling after exposure to ionizing radiation. In the present s tudy, we further investigate the capacity of a genetic approach, using repl ication-incompetent adenovirus (Ad)-mediated transfer of EGFR-CD533 (Ad-EGF R-CD533), to enhance the radiosensitivity in vitro of four cell lines repre sentative of three major cancer phenotypes. Methods and Materials: The cell lines MDA-MB-231 and T-47D mammary carcinom a, A-431 squamous carcinoma, and U-373 MG malignant glioma cells were used. The ErbB expression profiles and the EGFR tyrosine phosphorylation (Tyr-P) levels following irradiation were quantified by Western blotting. The rela tive radiosensitivities of tumor cells were assessed by standard colony for mation assays after infection with control vector (Ad-LacZ) or Ad-EGFR-CD53 3. Results: The expression profiles demonstrated varying levels of EGFR, ErbB2 , ErbB3, and ErbB4 expression. The overexpression of EGFR-CD533 after infec tion with Ad-EGFR-CD533 completely inhibited the radiation-induced stimulat ion of EGFR Tyr-P relative to the immediate 2.4- to 3.1-fold increases in E GFR Tyr-P in control infected cells (Ad-LacZ). Ad-EGFR-CD533-infected cells demonstrated significant (p < 0.001) radiosensitization over a range of ra diation doses (1-8 Gy), yielding dose-enhancement ratios (DER) between 1.4 and 1.7. This radiosensitization was maintained under conditions of repeate d radiation exposures, using 3 X 2 Gy, yielding DERs of 1.6 and 1.7 for MDA -MB-231 and U-373 cells, respectively. Conclusions: Overexpression of EGFR-CD533 significantly sensitizes human ca rcinoma and glioma cells to single and repeated radiation exposures irrespe ctive of their ErbB expression levels. Therefore, transduction of human tum or cells with EGFR-CD533 holds promise as a gene therapeutic approach for t he radiosensitization of neoplastic cells that are growth-regulated by EGFR or other ErbB receptors. (C) 2001 Elsevier Science Inc.