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

Abstract

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.

Figure 1

Generation of HEK-293 reporter cells with ABC-transporter overexpression (HEK-293/ABC-transporter reporter cells). A, structure of the dual-reporter system in a SFG backbone. Luciferase and GFP were constitutively expressed. GFP was used as a selection marker. B, validation of ABC-transporter overexpression in stably-transfected HEK-293/ABC-transporter cells by Western blotting. β-actin was used as a loading control. C, FACS analysis of the stably-transduced HEK-293/ABC-transporter reporter cells. Mock cells, empty vector stably-transfected HEK-293 cells, were used as a negative control; ABCG2(WT), wildtype ABCG2; ABCG2(482G) and ABCG2(482T), two ABCG2 mutants with point mutations at amino acid 482 (R→G, ABCG2(482G) and R→T, ABCG2(482T). cLuc, fLuc, rLuc and gLuc: HEK-293/ABC-transporter cells transduced with cLuc-IRES2-EGFP, fLuc-IRES2-EGFP, rLuc-IRES2-EGFP, and gLuc-IRES2-EGFP, respectively.

Figure 2

Relative BLI intensity of HEK-293 reporter cells with and without ABC transporter overexpression. Reporter cells were seeded into duplicate 96-well-plates for BLI intensity measurements in intact cells and in cell lysates. Relative BLI intensity was determined by calculating the BLI intensity ratio from intact cells divided by that from cell lysates, and then normalized to the value obtained from mock cells. Mock refers to empty vector stably-transfected HEK-293 cells without ABC-transporter overexpression; the cell-to-lysate BLI intensity ratio of the mock reporter cells (prior-to normalization) was: cLuc 0.095±0.003; fLuc 0.085±0.005; rLuc 0.059±0.002; gLuc 0.919±0.015. Error bar, SE; *, p<0.05.

Figure 3

Normalized BLI intensity of HEK-293 reporter cells with and without ABC transporter overexpression treated by selective ABC transporter inhibitors. Relative BLI intensity was determined by calculating the BLI intensity intact cells/cell lysates ratio, and then normalized to the value obtained from corresponding vehicle-treated cells. Reversin 121, ABCB1 inhibitor; MK-571, ABCC1 inhibitor; FTC, ABCG2 inhibitor. Error bar, SE; *, p<0.05.

Figure 4

Effects of specific ABC transporter inhibitors on BLI intensity from xenografts derived from HEK-293 reporter cells with and without ABC transporter overexpression. Reporter cells were subcutaneously implanted into the nude mice. After inhibitor or vehicle injection, sequential imaging was performed. Representative images of the same animal are shown. The pseudocolor bar shows BLI intensity (photons/cm²/sec/seradian). The white arrows point to xenografts with enhanced BLI intensity after inhibitor treatment. Reversin 121, ABCB1 inhibitor; FTC, ABCG2 inhibitor.

Figure 5

Time course of the normalized BLI signal from HEK-293 reporter-xenografts with and without ABC transporter overexpression treated with specific ABC transporter inhibitors. The BLI signal immediately after inhibitor or vehicle injection was defined as 1. Data are presented as mean normalized BLI signal from five xenografts in separate animals. *, the data with significant differences (p<0.05) compared with the normalized luciferase activity profiles of mock cells. Standard errors range from 0.012 to 0.125. Reversin 121, ABCB1 inhibitor; MK-571, ABCC1 inhibitor; FTC, ABCG2 inhibitor.

Figure 6

A, relative BLI intensity of mES-J1 and NIH/3T3 reporter cells treated by selective ABC transporter inhibitors. Relative BLI intensity is the ratio of BLI intensity from intact cells divided by that from cell lysates. Control, untreated cells; Reversin 121, ABCB1 inhibitor; MK-571, ABCC1 inhibitor; FTC, ABCG2 inhibitor. *, relative BLI intensity from the treated cells has significant difference compared with untreated cells, p<0.01. Error bar, SE. B, endogenous expression of ABC-transporters in mES-J1 and NIH/3T3 cells by semi-quantitative RT-PCR. β-actin was used as a loading control.