Aims: To determine if immunohistochemistry (IHC) could be used to monitor nuclear factor-κB (NF-κB) activity in oesophageal adenocarcinoma and pre-malignant (Barrett’s) oesophageal tissues, relative to normal oesophageal mucosa. The pro-inflammatory cytokine interleukin-8 (IL-8), a transcriptional target of NF-κB, was also studied to better understand NF-κB functionality; its RNA and protein levels were assessed in oesophageal tissues.
Methods: IHC was employed using an antibody against the nuclear localisation sequence (NLS) of the p65 subunit as well as an antibody against IL-8. To assess NF-κB function, changes in gene expression of NF-κB controlled genes (IL-8 and I-κB) were also assessed in the histological sequence using real-time PCR. More global expression changes were also studied using membrane arrays.
Results: IHC was effective at monitoring overall NF-κB activity and IL-8 abundance. This method also allowed NF-κB activity and IL-8 abundance to be pinpointed in specific cell types. There were significant increases in nuclear NF-κB activity and IL-8 abundance across the histological series. Gene expression analysis also showed consistent up-regulation of IL-8, confirming the IHC data and showing enhanced transcriptional NF-κB activity. I-κB (another NF-κB target) showed down-regulation in dysplastic and adenocarcinoma tissues. Down-regulation of I-κB gene expression may partly explain increased NF-κB activity.
Conclusion: IHC, using antibodies against the NLS of p65, may be useful in monitoring overall NF-κB activity in oesophageal tissues. As IHC is amenable to high-throughput screening (whereas traditional electrophoretic mobility shift assay methods are not), this may lead to the development of a better screening tool for early cancer risk.
- Barrett’s oesophagus
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Funding: This work was partly funded by the Welsh Office for Research and Development (Grant WS01/1/009).
Competing interests: None.
- nuclear factor-κB
- nuclear localisation sequence