ATP-binding cassette transporters modulate both coelenterazine- and D-luciferin-based bioluminescence imaging

Bioluminescence imaging (BLI) of luciferase reporters provides a cost-effective and sensitive means to image biological processes. However, transport of luciferase substrates across the cell membrane does affect BLI readout intensity from intact living cells. To investigate the effect of ATP-binding cassette (ABC) transporters on BLI readout, we generated click beetle (cLuc), firefly (fLuc), Renilla (rLuc), and Gaussia (gLuc) luciferase HEK-293 reporter cells that overexpressed different ABC transporters (ABCB1, ABCC1, and ABCG2). In vitro studies showed a significant BLI intensity decrease in intact cells compared to cell lysates, when ABCG2 was overexpressed in HEK-293/cLuc, fLuc, and rLuc cells. Selective ABC transporter inhibitors were also applied. Inhibition of ABCG2 activity increased the BLI intensity more than two-fold in HEK-293/cLuc, fLuc, and rLuc cells; inhibition of ABCB1 elevated the BLI intensity two-fold only in HEK-293/rLuc cells. BLI of xenografts derived from HEK-293/ABC transporter/luciferase reporter cells confirmed the results of inhibitor treatment in vivo. These findings demonstrate that coelenterazine-based rLuc-BLI intensity can be modulated by ABCB1 and ABCG2. ABCG2 modulates d-luciferin-based BLI in a luciferase type-independent manner. Little ABC transporter effect on gLuc-BLI intensity is observed because a large fraction of gLuc is secreted. The expression level of ABC transporters is one key factor affecting BLI intensity, and this may be particularly important in luciferase-based applications in stem cell research.     

Positive and negative regulation of the human heme oxygenase-1 gene expression in cultured cells.

To elucidate the regulation of the human heme oxygenase-1 (hHO-1) gene expression, we assessed approximately 4 kb of the 5'-flanking region of the hHO-1 gene for basal promoter activity and sequenced approximately 2 kb of the 5'-flanking region. A series of deletion mutants of the 5'-flanking region linked to the luciferase gene was constructed. Basal level expression of these constructs was tested in HepG2 human hepatoma cells and HeLa cervical cancer cells. By measuring luciferase activity, which was transiently expressed in the transfected cells, we found a positive regulatory region at position -1976 to -1655 bp. This region functions in HepG2 cells but not in HeLa cells. A negative regulatory region was also found at position -981 to -412 bp that functions in both HepG2 cells and HeLa cells.     

呼吸道黏蛋白5AC基因转录表达的顺式调控元件分析

目的：探讨呼吸道黏蛋白（mucin,MUC）5AC基因5'上游序列顺式调控元件在中性粒细胞弹力酶(neutrophil elastase , NE)诱导MUC5AC基因转录表达的调控机制。方法：应用DNA重组技术,构建含萤光素酶报告基因和MUC5AC启动子不同长度片段的嵌合质粒。采用定点突变技术,在嵌合质粒的基础上构建MUC5AC启动子区特殊蛋白（specificity protein）-1和核因子(nuclear factor, NF)-κB结合位点单独突变体,并测定NE刺激的转染细胞荧光素酶相对活性。结果：成功构建了4种含有不同长度MUC5AC基因启动子序列的荧光索酶报告基因质粒。含有启动子序列-1330bp、-689bp、-324bp的嵌合质粒荧光素酶相对光强度较对照组均显著增加,而含有启动子序列-64bp的嵌合质粒荧光素酶相对光强度与对照组相比差异无统计学意义。NE可诱导含有MUC5AC启动子区NF-кB结合位点单独突变体（pGL3E-MUC5AC-NF-кB-MU）荧光素酶相对光强度增加,而NE不能诱导Sp-l结合位点单独突变体（pGL3E-MUC5AC-SP-1-MU）荧光素酶表达增加。结论：MUC5AC 5'上游序列中-324～-64位点存在参与NE诱导MUC5AC基因表达的重要调控元件,位于此区域的顺式作用元件Sp-1位点在NE诱导MUC5AC基因表达机制中起重要作用,该位点可能作为靶向性基因治疗的关键调控元件。