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DNA Damage and Cellular Stress Responses

Epidermal Growth Factor Receptor vIII Expression in U87 Glioblastoma Cells Alters Their Proteasome Composition, Function, and Response to Irradiation

¹ Department of Radiation Oncology, Experimental Division, David Geffen School of Medicine at UCLA, University of California Los Angeles, Los Angeles, California and ² Human Oncology and Pathogenesis Program, Memorial Sloan-Kettering Cancer Center, New York, New York

Requests for reprints: William H. McBride, Department of Radiation Oncology, Roy E. Coats Research Laboratories, Room B3-109, David Geffen School of Medicine at University of California at Los Angeles, 10833 Le Conte Avenue, Los Angeles, CA 90095-1714. Phone: 310-794-7051; Fax: 310-206-1249. E-mail: wmcbride{at}mednet.ucla.edu

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 (PA28 and β) 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. (Mol Cancer Res 2008;6(3):426–34)