Inhibition of radiation-induced nuclear factor-kappaB activation by an anti-Ras single-chain antibody fragment: lack of involvement in radiosensitization.
We have shown previously that the transduction of a number of human tumor cell lines with an adenovirus (AV1Y28) expressing a single-chain antibody fragment (scFv) directed against Ras proteins results in radiosensitization. Because Ras is involved in the regulation of a number of transcription factors, we have determined the effects of this adenovirus on the activation of nuclear factor-kappaB (NF-kappaB), a radiation-responsive transcription factor associated with cell survival. In U251 human glioma cells, radiation-induced NF-kappaB was significantly attenuated by prior transduction of the anti-Ras scFv adenovirus. This effect appeared to involve an inhibition of IkappaB kinase activity and IkappaBalpha phosphorylation. Inhibitors to the Ras effectors mitogen-activated protein kinase kinase, phosphatidylinositol 3-kinase, and p38, however, did not reduce radiation-induced NF-kappaB. Whereas AV1Y28 inhibited NF-kappaB activation by hydrogen peroxide and ferricyanide, it had no effect of tumor necrosis factor-alpha-induced NF-kappaB activation. These results are consistent with a novel Ras-dependent, oxidant-specific signaling pathway mediating the activation of NF-kappaB. In additional cell lines radiosensitized by AV1Y28, radiation-induced NF-kappaB activation was also inhibited by the anti-Ras scFv, whereas in cell lines not radiosensitized, radiation did not activate NF-kappaB. This correlation suggested that AV1Y28-mediated radiosensitization involved the inhibition of radiation-induced NF-kappaB activation. However, inhibition of NF-kappaB activation via the expression of a dominant-negative form of IkappaBalpha in U251 cells had no effect on radiation-induced cell killing and did not influence AV1Y28-mediated radiosensitization. Therefore, whereas AV1Y28 inhibits radiation-induced NF-kappaB activation, this process does not appear to play a direct role in its radiosensitizing actions.
Russell
Jeffery S
JS
Molecular Radiation Therapeutics Branch, Radiation Oncology Science Program, EPN/6015A, National Cancer Institute/NIH, 6130 Executive Boulevard, Bethesda, MD 20892, USA.
Raju
Uma
U
Gumin
Glenice J
GJ
Lang
Fredrick F
FF
Wilson
Deborah R
DR
Huet
Thierry
T
Tofilon
Philip J
PJ
D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism; D005347:Fibroblasts / Q000502:physiology / Q000528:radiation effects; D005909:Glioblastoma / Q000235:genetics / Q000532:radiotherapy; D006801:Humans; D020672:I-kappa B Proteins; D007128:Immunoglobulin Fragments / Q000235:genetics / Q000502:physiology*; D009154:Mutation; D000072000:NF-KappaB Inhibitor alpha; D016328:NF-kappa B / Q000037:antagonists & inhibitors* / Q000502:physiology / Q000528:radiation effects; D010766:Phosphorylation; D011836:Radiation Tolerance / Q000276:immunology / Q000502:physiology*; D014162:Transfection; D014407:Tumor Cells, Cultured; D018631:ras Proteins / Q000235:genetics / Q000276:immunology*
D004268:DNA-Binding Proteins; D020672:I-kappa B Proteins; D007128:Immunoglobulin Fragments; D016328:NF-kappa B; C000606204:NFKBIA protein, human; D000072000:NF-KappaB Inhibitor alpha; D018631:ras Proteins