期刊
CANCER SCIENCE
卷 105, 期 7, 页码 870-874出版社
WILEY-BLACKWELL
DOI: 10.1111/cas.12420
关键词
Antineoplastic agents; dehydroxymethylepoxyquinomicin; DNA-binding proteins; fluorescence resonance energy transfer; NF-kappa B
类别
资金
- Ministry of Education, Culture, Sports, Science and Technology of Japan
- Grants-in-Aid for Scientific Research [221S0001, 23300363, 25112711, 25701005, 26310106, 26640091, 26290047] Funding Source: KAKEN
Nuclear factor-kappa B (NF-kappa B) is a key regulator of cancer progression and the inflammatory effects of disease. To identify inhibitors of DNA binding to NF-kappa B, we developed a new homogeneous method for detection of sequence-specific DNA-binding proteins. This method, which we refer to as DSE-FRET, is based on two phenomena: protein-dependent blocking of spontaneous DNA strand exchange (DSE) between partially double-stranded DNA probes, and fluorescence resonance energy transfer (FRET). If a probe labeled with a fluorophore and quencher is mixed with a non-labeled probe in the absence of a target protein, strand exchange occurs between the probes and results in fluorescence elevation. In contrast, blocking of strand exchange by a target protein results in lower fluorescence intensity. Recombinant human NF-kappa B (p50) suppressed the fluorescence elevation of a specific probe in a concentration-dependent manner, but had no effect on a non-specific probe. Competitors bearing a NF-kappa B binding site restored fluorescence, and the degree of restoration was inversely correlated with the number of nucleotide substitutions within the NF-kappa B binding site of the competitor. Evaluation of two NF-kappa B inhibitors, Evans Blue and dehydroxymethylepoxyquinomicin ([-]-DHMEQ), was carried out using p50 and p52 (another form of NF-kappa B), and IC50 values were obtained. The DSE-FRET technique also detected the differential effect of (-)-DHMEQ on p50 and p52 inhibition. These data indicate that DSE-FRET can be used for high throughput screening of anticancer drugs targeted to DNA-binding proteins.
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