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Submitted to: Analytical Biochemistry
Publication Type: Peer Reviewed Journal Publication Acceptance Date: 12/21/2001 Publication Date: 2/2/2002 Citation: Wang, T.T. 2002. Beta-naphthoflavone, an inducer of xenobiotic metabolizing enzymes,inhibits luciferase activity. Analytical Biochemistry. 304:122-126. Interpretive Summary: It is a common practice to use reporter genes, which code for enzymes such as luciferases, to indirectly determine regulation of a gene in cell culture model systems. Firefly luciferase is an insect enzyme often used for such purposes. Determination of firefly luciferase activity allows one to indirectly assess gene regulation in cells of mammalian origin. The present study describes a novel observation that may affect choice of reporter gene in one's experimental design. We found that beta- naphthoflavone, a synthetic flavone commonly used in studies of drug detoxification enzymes, exerts an inhibitory effect on firefly luciferase. This effect appeared to be specific for firefly luciferase. This direct effect on firefly luciferase activity resulted in contradictory conclusions for gene regulation. Hence this study should serve as an important warning for scientists using reporter genes to conduct gene regulation research. Moreover, these results also suggest that care should be taken when working with compounds that may share structural similarity with beta-naphthoflavone. Technical Abstract: The present study describes a novel observation that may affect choice of reporter gene in one's experimental design. To study transcriptional regulation of NAD(P)H:quinone oxidoreductase 1 (EC 1.6.99.2) in mammary epithelial cells, a 342 bp promoter region of the gene coding for this enzyme was inserted into the firefly luciferase containing plasmid pTATALUC+. The resulting plasmid pQRTATA was used to assess transcriptional regulation in the human breast cancer cells MCF-7. Unexpectedly, we found that treatment of pQRTATA-transfected human breast cancer cells MCF-7 with 10 mM beta-naphthoflavone, a known inducer of quinone oxidoreductase activity, resulted in significantly lower relative luciferase value than that of the controls. This suggests an inhibition of the quinone oxidoreductase promoter activity by beta-naphthoflavone, which is contrary to the known effects of beta-naphthoflavone. This contradictory finding appeared to result from a direct inhibitory effect of beta-naphthoflavone on the firefly luciferase. Beta-naphthoflavone, but not alpha- naphthoflavone, inhibited the firefly luciferase activity with a KI of 0.1 mM. The effects of beta-naphthoflavone also appeared to be enzyme specific as renilla luciferase activity was not affected. |
