Title

Regulation of proteasome activity and substrate recognition in colon cancer cells

Date of Completion

January 2005

Keywords

Biology, Molecular|Biology, Cell|Health Sciences, Pharmacology|Health Sciences, Oncology

Degree

Ph.D.

Abstract

The proteasome is a multicatalytic protease complex that plays a central role in regulating fundamental cellular processes. NF-κB is a transcription factor whose activity is critically dependent on the proteasome, and plays a central role in regulating cell proliferation, apoptosis, and inflammatory responses. Experiments were performed to obtain insight into the role the proteasome plays in regulating NF-κB in colon cancer cells. ^ NF-κB is activated by the signal-induced proteasome degradation of the IκBα inhibitory protein, which allows NF-κB to transmigrate into the nucleus and activate target genes. However, IκBα levels are only transiently lowered since one gene activated by NF-κB is IκBα. We found that in addition to the transcriptional up-regulation of IκB, NF-κB stimuli also increased the stability of newly synthesized IκBα. This stabilization pathway involved the p38 MAP kinase, which suppressed the destabilizing effects of the C-terminal domain of IκBα. We propose that this p38-mediated pathway serves to replenish IκBα levels after stimulation, as well as limit sequential rounds of NF-κB activation. ^ We also obtained evidence that the level of proteasome subunit expression can impact NF-κB regulation. Histone deacetylase (HDAC) inhibitors induce cell cycle arrest and differentiation of colon cancer cells. We found that HDAC inhibitor-induced differentiation decreased proteasome activity and reprogrammed the NF-κB response. Specifically, TNF-α activation was suppressed, while IL-1β activation was largely unaffected. The selective impact of HDAC inhibitors on TNF-α-induced NF-κB activation appeared to relate to the fact that TNF-α-induced activation was mediated by the proteasome, whereas NF-κB activation by IL-1β was proteasome independent. The drop in proteasome activity was the result of reduced expression of the catalytic β-type subunits of the proteasome. These findings indicate that cellular differentiation status can significantly impact proteasome activity and NF-κB responses. This conclusion was further supported by the fact that proteasome activity and subunit expression naturally declined upon senescence of normal human cells. Our studies also revealed that HDACs play a central role in regulating senescence in normal cells. ^ These results highlight a number important biological features of proteasome and NF-κB regulation in colon cancer cells. These findings may be exploitable for the development of novel cancer treatments and anti-inflammatory agents. ^