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DSE-FRET: A new anticancer drug screening assay for DNA binding proteins.

Miyagi T, Shiotani B, Miyoshi R, Yamamoto T, Oka T, Umezawa K, Ochiya T, Takano M, Tahara H - Cancer Sci. (2014)

Bottom Line: Evaluation of two NF-κB inhibitors, Evans Blue and dehydroxymethylepoxyquinomicin ([-]-DHMEQ), was carried out using p50 and p52 (another form of NF-κB), 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.

View Article: PubMed Central - PubMed

Affiliation: Department of Cellular and Molecular Biology, Graduate School of Biomedical & Health Science, Hiroshima University, Hiroshima, Japan; Japanese Red Cross Kanto-koshinetsu Block Blood Center, Tokyo, Japan.

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Dose-dependent suppression of DNA strand exchange by p50. (a) Five microliters of 40 nM NF-D1 was mixed with 5 μL of 0 nM (open circles), 400 nM (open squares), 800 nM (closed squares), 1600 nM (open triangles), or 3200 nM (closed triangles) p50 and incubated for 30 min. Then, 40 μL of 10 nM NF-D2 was added and fluorescence was measured; thus, the final concentrations of p50 were 0, 40, 80, 160, and 320 nM, respectively. Closed circles with a dotted line represent NF-D1 alone. (b) Fluorescence at 60 min for a positive control completely exchanged product (NF-01F + NF-02, named NF-01F02), an exchanged product (NF-D1 + NF-D2), and an unexchanged product (NF-D1 alone) with 0 or 320 nM p50. Error bars represent ±3 SD (n = 4).
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fig02: Dose-dependent suppression of DNA strand exchange by p50. (a) Five microliters of 40 nM NF-D1 was mixed with 5 μL of 0 nM (open circles), 400 nM (open squares), 800 nM (closed squares), 1600 nM (open triangles), or 3200 nM (closed triangles) p50 and incubated for 30 min. Then, 40 μL of 10 nM NF-D2 was added and fluorescence was measured; thus, the final concentrations of p50 were 0, 40, 80, 160, and 320 nM, respectively. Closed circles with a dotted line represent NF-D1 alone. (b) Fluorescence at 60 min for a positive control completely exchanged product (NF-01F + NF-02, named NF-01F02), an exchanged product (NF-D1 + NF-D2), and an unexchanged product (NF-D1 alone) with 0 or 320 nM p50. Error bars represent ±3 SD (n = 4).

Mentions: To illustrate the method, we attempted to detect the NF-κB (p50) interaction with DNA. Nuclear factor-κB plays a pivotal role in the coordinated transcription of multiple inflammatory genes and is a probable drug target.(14–16) Two probes, NF-D1 and NF-D2, were prepared to test quantitative detection of p50 binding to DNA. Their double-stranded regions are identical and include an NF-κB binding sequence, d(GGGACTTTCC). These probes interact with each other through their single-stranded tails and are then involved in a strand exchange reaction. Each strand of NF-D1 was labeled with 6FAM and DABCYL at the double-stranded terminus. NF-D1, various concentrations of recombinant p50, and NF-D2 were mixed in a half-area 96-well microplate and changes in fluorescence were measured. Time courses of these changes are shown in Figure 2. The fluorescence signal of NF-D1 increased rapidly within 30 min after addition of NF-D2 and was fivefold higher than that of NF-D1 alone at 60 min in the absence of p50. Fluorescence elevation was suppressed in a p50 concentration-dependent manner by half at 40 nM p50 and almost completely at 320 nM p50.


DSE-FRET: A new anticancer drug screening assay for DNA binding proteins.

Miyagi T, Shiotani B, Miyoshi R, Yamamoto T, Oka T, Umezawa K, Ochiya T, Takano M, Tahara H - Cancer Sci. (2014)

Dose-dependent suppression of DNA strand exchange by p50. (a) Five microliters of 40 nM NF-D1 was mixed with 5 μL of 0 nM (open circles), 400 nM (open squares), 800 nM (closed squares), 1600 nM (open triangles), or 3200 nM (closed triangles) p50 and incubated for 30 min. Then, 40 μL of 10 nM NF-D2 was added and fluorescence was measured; thus, the final concentrations of p50 were 0, 40, 80, 160, and 320 nM, respectively. Closed circles with a dotted line represent NF-D1 alone. (b) Fluorescence at 60 min for a positive control completely exchanged product (NF-01F + NF-02, named NF-01F02), an exchanged product (NF-D1 + NF-D2), and an unexchanged product (NF-D1 alone) with 0 or 320 nM p50. Error bars represent ±3 SD (n = 4).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4317927&req=5

fig02: Dose-dependent suppression of DNA strand exchange by p50. (a) Five microliters of 40 nM NF-D1 was mixed with 5 μL of 0 nM (open circles), 400 nM (open squares), 800 nM (closed squares), 1600 nM (open triangles), or 3200 nM (closed triangles) p50 and incubated for 30 min. Then, 40 μL of 10 nM NF-D2 was added and fluorescence was measured; thus, the final concentrations of p50 were 0, 40, 80, 160, and 320 nM, respectively. Closed circles with a dotted line represent NF-D1 alone. (b) Fluorescence at 60 min for a positive control completely exchanged product (NF-01F + NF-02, named NF-01F02), an exchanged product (NF-D1 + NF-D2), and an unexchanged product (NF-D1 alone) with 0 or 320 nM p50. Error bars represent ±3 SD (n = 4).
Mentions: To illustrate the method, we attempted to detect the NF-κB (p50) interaction with DNA. Nuclear factor-κB plays a pivotal role in the coordinated transcription of multiple inflammatory genes and is a probable drug target.(14–16) Two probes, NF-D1 and NF-D2, were prepared to test quantitative detection of p50 binding to DNA. Their double-stranded regions are identical and include an NF-κB binding sequence, d(GGGACTTTCC). These probes interact with each other through their single-stranded tails and are then involved in a strand exchange reaction. Each strand of NF-D1 was labeled with 6FAM and DABCYL at the double-stranded terminus. NF-D1, various concentrations of recombinant p50, and NF-D2 were mixed in a half-area 96-well microplate and changes in fluorescence were measured. Time courses of these changes are shown in Figure 2. The fluorescence signal of NF-D1 increased rapidly within 30 min after addition of NF-D2 and was fivefold higher than that of NF-D1 alone at 60 min in the absence of p50. Fluorescence elevation was suppressed in a p50 concentration-dependent manner by half at 40 nM p50 and almost completely at 320 nM p50.

Bottom Line: Evaluation of two NF-κB inhibitors, Evans Blue and dehydroxymethylepoxyquinomicin ([-]-DHMEQ), was carried out using p50 and p52 (another form of NF-κB), 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.

View Article: PubMed Central - PubMed

Affiliation: Department of Cellular and Molecular Biology, Graduate School of Biomedical & Health Science, Hiroshima University, Hiroshima, Japan; Japanese Red Cross Kanto-koshinetsu Block Blood Center, Tokyo, Japan.

Show MeSH
Related in: MedlinePlus