title: Adenovirus-mediated overexpression of dominant negative epidermal growth factor receptor-CD533 as a gene therapeutic approach radiosensitizes human carcinoma and malignant glioma cells.
abstract: OBJECTIVE
  Epidermal growth factor receptor (EGFR) and other members of the ErbB family of receptor tyrosine kinases (RTK) mediate autocrine growth regulation in a wide spectrum of human tumor cells. We have previously demonstrated that in stably transfected mammary carcinoma cells a dominant negative (DN) mutant of EGFR, EGFR-CD533 is a potent inhibitor of EGFR and its cytoprotective signaling after exposure to ionizing radiation. In the present study, we further investigate the capacity of a genetic approach, using replication-incompetent adenovirus (Ad)-mediated transfer of EGFR-CD533 (Ad-EGFR-CD533), to enhance the radiosensitivity in vitro of four cell lines representative of three major cancer phenotypes.
  
  
  METHODS
  The cell lines MDA-MB-231 and T-47D mammary carcinoma, 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 relative radiosensitivities of tumor cells were assessed by standard colony formation assays after infection with control vector (Ad-LacZ) or Ad-EGFR-CD533.
  
  
  RESULTS
  The expression profiles demonstrated varying levels of EGFR, ErbB2, ErbB3, and ErbB4 expression. The overexpression of EGFR-CD533 after infection with Ad-EGFR-CD533 completely inhibited the radiation-induced stimulation of EGFR Tyr-P relative to the immediate 2.4- to 3.1-fold increases in EGFR Tyr-P in control infected cells (Ad-LacZ). Ad-EGFR-CD533-infected cells demonstrated significant (p < 0.001) radiosensitization over a range of radiation doses (1-8 Gy), yielding dose-enhancement ratios (DER) between 1.4 and 1.7. This radiosensitization was maintained under conditions of repeated 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 carcinoma and glioma cells to single and repeated radiation exposures irrespective of their ErbB expression levels. Therefore, transduction of human tumor cells with EGFR-CD533 holds promise as a gene therapeutic approach for the radiosensitization of neoplastic cells that are growth-regulated by EGFR or other ErbB receptors.
authors.affiliation: Department of Radiation Oncology, Medical College of Virginia, Virginia Commonwealth University, Richmond 23298-0058, USA.
authors.forename: G
authors.initials: G
authors.lastname: Lammering
authors.forename: P S
authors.initials: PS
authors.lastname: Lin
authors.forename: J N
authors.initials: JN
authors.lastname: Contessa
authors.forename: J L
authors.initials: JL
authors.lastname: Hampton
authors.forename: K
authors.initials: K
authors.lastname: Valerie
authors.forename: R K
authors.initials: RK
authors.lastname: Schmidt-Ullrich
mesh_terms: D000256:Adenoviridae / Q000235:genetics; D001943:Breast Neoplasms / Q000235:genetics / Q000378:metabolism* / Q000628:therapy; D002294:Carcinoma, Squamous Cell / Q000235:genetics / Q000378:metabolism* / Q000628:therapy; D066246:ErbB Receptors / Q000235:genetics / Q000378:metabolism*; D015972:Gene Expression Regulation, Neoplastic; D005799:Genes, Dominant; D015316:Genetic Therapy / Q000379:methods*; D005910:Glioma / Q000235:genetics / Q000378:metabolism* / Q000628:therapy; D006801:Humans; D010766:Phosphorylation; D011836:Radiation Tolerance; D018719:Erb-b2 Receptor Tyrosine Kinases / Q000235:genetics / Q000378:metabolism*; D020893:Receptor, ErbB-3 / Q000235:genetics / Q000378:metabolism*; D066247:Receptor, ErbB-4; D014407:Tumor Cells, Cultured / Q000528:radiation effects; D014410:Tumor Stem Cell Assay
chemical_list: D066246:ErbB Receptors; D018719:Erb-b2 Receptor Tyrosine Kinases; D020893:Receptor, ErbB-3; D066247:Receptor, ErbB-4; C587689:ERBB4 protein, human