title: Epidermal growth factor receptor vIII expression in U87 glioblastoma cells alters their proteasome composition, function, and response to irradiation.
abstract: Little is known about the factors that influence the proteasome structures in cells and their activity, although this could be highly relevant to cancer therapy. We have previously shown that, within minutes, irradiation inhibits substrate degradation by the 26S proteasome in most cell types. Here, we report an exception in U87 glioblastoma cells transduced to express the epidermal growth factor receptor vIII (EGFRvIII) mutant (U87EGFRvIII), which does not respond to irradiation with 26S proteasome inhibition. This was assessed using either a fluorogenic substrate or a reporter gene, the ornithine decarboxylase degron fused to ZsGreen (cODCZsGreen), which targets the protein to the 26S proteasome. To elucidate whether this was due to alterations in proteasome composition, we used quantitative reverse transcription-PCR to quantify the constitutive (X, Y, Z) and inducible 20S subunits (Lmp7, Lmp2, Mecl1), and 11S (PA28alpha and beta) and 19S components (PSMC1 and PSMD4). U87 and U87EGFRvIII significantly differed in expression of proteasome subunits, and in particular immunosubunits. Interestingly, 2 Gy irradiation of U87 increased subunit expression levels by 16% to 324% at 6 hours, with a coincident 30% decrease in levels of the proteasome substrate c-myc, whereas they changed little in U87EGFRvIII. Responses similar to 2 Gy were seen in U87 treated with a proteasome inhibitor, NPI0052, suggesting that proteasome inhibition induced replacement of subunits independent of the means of inhibition. Our data clearly indicate that the composition and function of the 26S proteasome can be changed by expression of the EGFRvIII. How this relates to the increased radioresistance associated with this cell line remains to be established.
authors.affiliation: Department of Radiation Oncology, David Geffen School of Medicine at University of California at Los Angeles, Los Angeles, CA 90095-1714, USA.
authors.forename: Kwanghee
authors.initials: K
authors.lastname: Kim
authors.forename: James M
authors.initials: JM
authors.lastname: Brush
authors.forename: Philip A
authors.initials: PA
authors.lastname: Watson
authors.forename: Nicholas A
authors.initials: NA
authors.lastname: Cacalano
authors.forename: Keisuke S
authors.initials: KS
authors.lastname: Iwamoto
authors.forename: William H
authors.initials: WH
authors.lastname: McBride
mesh_terms: D001932:Brain Neoplasms / Q000235:genetics; D045744:Cell Line, Tumor; D017931:DNA Primers; D066246:ErbB Receptors / Q000235:genetics*; D005434:Flow Cytometry; D015972:Gene Expression Regulation, Neoplastic / Q000528:radiation effects; D005909:Glioblastoma / Q000235:genetics; D006801:Humans; D018613:Microscopy, Confocal; D046988:Proteasome Endopeptidase Complex / Q000528:radiation effects; D061988:Proteasome Inhibitors; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis; D012190:Retroviridae; D020133:Reverse Transcriptase Polymerase Chain Reaction
chemical_list: D017931:DNA Primers; D061988:Proteasome Inhibitors; D011993:Recombinant Fusion Proteins; C410723:epidermal growth factor receptor VIII; D066246:ErbB Receptors; D046988:Proteasome Endopeptidase Complex; C061553:ATP dependent 26S protease