NF-kappaB is a family of important transcription factors involved in many cellular functions, such as cell survival, proliferation, and stress responses. Many studies indicate that NF-kappaB is a stress-sensitive transcription factor and its activation is regulated by reactive oxygen species. In previous studies, we and others demonstrated that this transcription factor can be activated by transient oxidative stress. However, the effects of sustained oxidative stress on NF-kappaB activation are not clear. The objective of this study was to determine the effects of sustained oxidative stress on NF-kappaB activation and to elucidate the signaling events affected by sustained oxidative stress. Human lens epithelial cells (HLEC) that were subjected to 4 h of continuous influx of hydrogen peroxide were used to investigate the effects of sustained oxidative stress on NF-kappaB activation. The data showed that, unlike transient oxidative stress, sustained exposure of HLEC to physiologically relevant levels of H(2)O(2) (50-100 microM for 4 h) did not induce the degradation of I-kappaB and activation of NF-kappaB, but attenuated TNFalpha-induced degradation of I-kappaB and activation of NF-kappaB. Sustained exposure of HLEC to these levels of H(2)O(2) also inactivated proteasome activity by 50-80%. Consistent with the role of the proteasome in degradation of I-kappaB and activation of NF-kappaB, treatment of HLEC with proteasome inhibitors also attenuated TNFalpha-induced I-kappaB degradation and NF-kappaB activation. The data also indicate that activation of NF-kappaB is essential for the cells to recover from oxidative stress. Inhibiting NF-kappaB activation during recovery from transient oxidative stress significantly reduced the cell viability. Together, these data indicate that sustained oxidative stress may inactivate the proteasome and subsequently inhibit NF-kappaB activation by impeding the degradation of I-kappaB. The oxidative inactivation of the proteasome and subsequent impairment of NF-kappaB activation may contribute to the death of lens epithelial cells, a common feature associated with cataract.