Inhibition of radiation-induced nuclear factor-κB 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 radio-sensitization. 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-κB (NF-κB), a radiation-responsive transcription factor associated with cell survival. In U251 human glioma cells, radiation-induced NF-κB was significantly attenuated by prior transduction of the anti-Ras scFv adenovirus. This effect appeared to involve an inhibition of IκB kinase activity and IκBa phosphorylation. Inhibitors to the Ras effectors mitogen-activated protein kinase kinase, phosphatidylinositol 3-kinase, and p38, however, did not reduce radiation-induced NF-κB. Whereas AV1Y28 inhibited NF-κB activation by hydrogen peroxide and ferricyanide, it had no effect of tumor necrosis factor-α-induced NF-κB activation. These results are consistent with a novel Ras-dependent, oxidant-specific signaling pathway mediating the activation of NF-κB. In additional cell lines radiosensitized by AV1Y28, radiation -induced NF-κB activation was also inhibited by the anti-Ras scFv, whereas in cell lines not radiosensitized, radiation did not activate NF-κB. This correlation suggested that AV1Y28-mediated radiosensitization involved the inhibition of radiation-induced NF-κB activation. However, inhibition of NF-κB activation via the expression of a dominant-negative form of IκBa 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-κB activation, this process does not appear to play a direct role in its radiosensitizing actions.