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ABCG2/BCRP Expression Modulates D-Luciferin–Based Bioluminescence Imaging

¹ Russell H. Morgan Department of Radiology, ² Department of Environmental Health Sciences, The Johns Hopkins Bloomberg School of Public Health, ³ The Kennedy Krieger Institute, ⁴ Department of Neurology, and ⁵ Pharmacology and Molecular Sciences, The Johns Hopkins School of Medicine, Baltimore, Maryland

Requests for reprints: Martin G. Pomper, Johns Hopkins Medical Institutions, 1550 Orleans Street, 492 CRB II, Baltimore, MD 21231. Phone: 410-955-2789; Fax: 443-817-0990; E-mail: mpomper{at}jhmi.edu.

	Abstract

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Bioluminescence imaging (BLI) is becoming indispensable to the study of transgene expression during development and, in many in vivo models of disease such as cancer, for high throughput drug screening in vitro. Because reaction of D-luciferin with firefly luciferase (fLuc) produces photons of sufficiently long wavelength to permit imaging in intact animals, use of this substrate and enzyme pair has become the method of choice for performing BLI in vivo. We now show that expression of the ATP-binding cassette (ABC) family transporter ABCG2/BCRP affects BLI signal output from the substrate D-luciferin. In vitro studies show that D-luciferin is a substrate for ABCG2/BCRP but not for the MDR1 P-glycoprotein (ABCB1/Pgp), multidrug resistance protein 1 (MRP1/ABCC1), or multidrug resistance protein 2 (MRP2/ABCC2). D-Luciferin uptake within cells is shown to be modulated by ABC transporter inhibitors, including the potent and selective ABCG2/BCRP inhibitor fumitremorgin C. Images of xenografts engineered to express transgenic ABCG2/BCRP, as well as xenografts derived from the human prostate cancer cell line 22Rv1 that naturally express ABCG2/BCRP, show that ABCG2/BCRP expression and function within regions of interest substantially influence D-luciferin–dependent bioluminescent output in vivo. These findings highlight the need to consider ABCG2/BCRP effects during D-luciferin–based BLI and suggest novel high throughput methods for identifying new ABCG2/BCRP inhibitors. [Cancer Res 2007;67(19):9389–97]

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion References

 
Bioluminescence imaging (BLI) is the most widely used method to measure transgene expression in vivo (1–4). Although originally developed as a way to detect bacterial pathogens in living hosts, the technique has been extended to monitoring tumor growth and therapeutic response, measuring protein-protein interactions, observing the trafficking and proliferation of immune and stem cells, and as a method to study gene expression patterns (5). The pharmaceutical industry has begun incorporating BLI into its drug development programs routinely (6). The method relies upon the administration of D-luciferin, or another suitable substrate, to cultured cells or to an animal harboring cells engineered to express luciferase. The most commonly used enzyme-substrate pair is firefly luciferase (fLuc) and D-luciferin. Upon oxidation by fLuc, D-luciferin emits photons of a sufficiently long wavelength that they are capable of traversing living tissue and can be detected by a cooled CCD camera to generate an image. Its application to high throughput screening and imaging of cell functions in intact animals makes the technique particularly versatile and attractive. BLI is exquisitely sensitive and specific due, in part, to the lack of background noise with which fluorescence imaging is often fraught, enabling the quantification of small changes in light output magnitude and the visualization of as few as 1,000 cells deep within a living mouse (3).

However, unlike fluorescence imaging, BLI is a complex process that requires the presence of ATP and oxygen and generates a signal that can only purport to be directly related to the amount of luciferase present when the enzyme is limiting and substrate is in excess. The readout from BLI is necessarily downstream from a series of physiologic and cellular events. First, substrate must reach the cytoplasm of cells, a process that may be attenuated if the luciferase substrate serves also as a substrate for a membrane-bound pump, such as those produced by the ATP-binding cassette (ABC) family. Thus, experimental agents that unknowingly modulate substrate access to luciferase-expressing cells might alter BLI output independent of changes in luciferase activity. For example, coelenterazine is a substrate for the MDR1 P-glycoprotein pump (ABCB1/Pgp), as well as for Renilla luciferase (rLuc), necessitating an understanding of how experimental conditions influence ABCB1/Pgp activity in cells undergoing rLuc imaging (7).

Our recent efforts to apply fLuc to study several new inhibitors of the tumor promoting hedgehog (Hh) signaling pathway led us to uncover that BLI using D-luciferin as a substrate is influenced substantially by cell expression of the ABC transporter family member ABCG2/BCRP. We show that changes in ABCG2/BCRP expression and function alter D-luciferin–dependent BLI output independent of luciferase expression modulation in intact cells and in vivo. These findings reveal new cautions and potential applications for BLI in living cells and tissues.

	Materials and Methods

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Reagents. D-Luciferin sodium salt was obtained from Gold Biotechnology, Inc. Verapamil, indomethacin, doxorubicin, and colchicine were purchased from Sigma Chemical Company. Fumitremorgin C was a kind gift of Dr. Susan Bates (National Cancer Institute). All compounds were prepared in DMSO for in vitro experiments, and fumitremorgin C was dissolved in ethanol/cremophor EL/saline (1:1:6) as described for in vivo studies. Expression plasmids for Gli1 and Gli3 were kindly provided by Dr. Phillip Beachy (Johns Hopkins University; refs. 8, 9). Dr. Robert Robey (National Cancer Institute) generously provided the mutant ABCG2/BCRP-expressing cells (10). HhAntag-691 was kindly provided by Genentech, Inc. (11, 12).

Construction of reporter plasmids. fLuc-mRFP-HSV1–truncated sr39tk (fLuc-mrfp-ttk) driven by the cytomegalovirus promoter (CMV-tri) was a generous gift of Dr. S. Sam Gambhir (Stanford University) and was prepared as previously described (13). A unique ligation strategy involving three DNA fragments was used to generate the plasmid with the fLuc-mrfp-ttk gene under the control of eight tandem minimal Gli-binding sites (Gli-tri). The fLuc-mrfp-ttk gene fragment (3.38 kb) was obtained from CMV-tri by cutting with NheI and NotI. The 8x-Gli promoter (0.6 kb) was derived from pGl3b/8XGli-lc-luc, kindly provided by Dr. Phillip Beachy (Johns Hopkins University), cut with KpnI and NheI (14), and the backbone (4.6 kb) carrying a G418 selection marker was from pGlow-TOPO-p53-1F by cutting with NotI and KpnI. The DNA fragments were purified with QIAGEN gel extraction kit (Qiagen, Inc.) and then ligated. Gli-tri plasmids were prepared from transformants from the ligation product and confirmed by restriction digestion and sequencing.

Cell culture. The human prostate cancer cell line 22Rv1 was cultured in RPMI 1640 (Invitrogen) supplemented with 10% fetal bovine serum (FBS), 30 mg/L Na₂CO₃, 45 mg/L glucose, 0.1 mmol/L HEPES, and 0.01 mmol/L sodium pyruvate. Madin-Darby canine kidney cells (MDCK) were cultured in DMEM medium (Invitrogen) supplemented with 10% FBS; HEK-293 cells were cultured in MEM (Invitrogen) supplemented with 10% FBS. All cultures were maintained at 37°C in a humidified 5% CO₂/95% air incubator.

Transfection. Transient transfection was done with Fugene6 transfection reagent (Roche Pharmaceuticals) according to the manufacturer's instructions. 22Rv1 cells stably transfected with CMV-tri or Gli-tri were selected in 1.5 mg/mL G418.

Reverse transcription–PCR. RNA was isolated from cells using RNeasy Mini kit (Qiagen, Inc.), and oligonucleotides used for PCR amplification were as follows:

ABCB1/Pgp: forward 5'-GCCTGGCAGCTGGAAGACAAATACACAAAATT-3'.

backward 5'-CAGACAGCAGCTGACAGTCCAAGAACAGGACT-3'.

.

ABCC1/MRP1: forward 5'-CACACTGAATGGCATCACCTTC-3'.

backward 5'-CCTTCTCGCCAATCTCTGTCC-3'.

.

ABCC2/MRP2: forward 5'-CCAATCTACTCTCACTTCAGCGAGA-3'.

backward 5'-AGATCCAGCTCAGGTCGGTACC-3'.

.

ABCC3/MRP3: forward 5'-TCTATGCAGCCACATCACGG-3'.

backward 5'-GTCACCTGCAAGGAGTAGGACAC-3'.

.

ABCG2/BCRP: forward 5'-TTTCAGCCGTGGAACTCTTT-3'.

backward 5'-TGAGTCCTGGGCAGAAGTTT-3'.

.

fLuc assay. Cell extracts were prepared from 22Rv1 cells using passive lysis buffer (Promega) according to the manufacturer's instructions, and the luciferase assay was done using the Luciferase Assay System (Promega).

BLI of cell lines. 22Rv1 stable transfectants were plated into 24-well plates at the density of 3 x 10⁵ per well. MDCK and HEK-293 cell lines were transiently transfected with CMV-tri for 2 days and then plated into 24-well plates at the same density. The following day, before imaging, each well was changed into fresh medium containing drugs at indicated concentrations. D-Luciferin was then added to a final concentration of 150 µg/mL according to the manufacturer's (Xenogen Corp.) recommendation, and imaging commenced immediately. BLI was done with the IVIS 200 small animal imaging system (Xenogen Corp.). Acquisition times varied depending on signal intensity.

In vitro [³H]penciclovir uptake assay. 22Rv1/CMV-tri cells were plated into 12-well plates at the density of 1 x 10⁶ per well. The following day, cells were changed into fresh medium containing 1.0 µCi/mL [³H]penciclovir (Moravek Biochemicals and Radiochemicals) with or without 5 µmol/L fumitremorgin C, and incubated for 0, 1, and 3 h at 37°C. After incubation, cells were washed with cold PBS four times, lysed in 250 µL 1x passive lysis buffer (Promega), and then counted in a liquid scintillation counter (Model LS6500, Beckman Coulter, Inc.).

In vivo BLI. Animal protocols were approved by the Johns Hopkins University Animal Care and Use Committee. HEK-293 cells with or without ABCG2/BCRP overexpression were transiently transfected with CMV-tri for 2 days, then 1 x 10⁶ cells of each were implanted s.c. into 6-week-old female nude mice. Animals were anesthetized with 2.5% isoflurane during imaging. BLI was done with the IVIS 200 small animal imaging system (Xenogen Corp.). Within 1 to 4 days, mice were injected i.p. with D-luciferin (150 mg/kg) and imaged every few minutes for >1 h. Fumitremorgin C was injected by tail vein (25 mg/kg) at one point during the imaging time course, and imaging continued thereafter. 22Rv1/CMV-tri stable cells were implanted s.c. into mice (5 x 10⁶ cells per site). When the tumors became palpable, osmotic pumps (Alzet Model 1007D, Durect Corp.) with a 0.5 µL/h infusion rate loaded with D-luciferin (50 mg/mL) were implanted s.c. into the contralateral side of the mice. The next day mice were subjected to BLI, then fumitremorgin C or vehicle was injected by tail vein (25 mg/kg), and imaging continued thereafter.

Data analysis. LivingImage (Xenogen Corp.) and IGOR (Wavemetrics) image analysis software were used to superimpose and analyze the corresponding gray scale photographs and false color BLI images. Signal intensities of regions of interests were defined manually. Light intensities of the regions of interests were expressed as total flux (photons per second). Background total flux was subtracted from that produced by tumors in the case of the 22Rv1 tumor imaging. Data are presented as mean ± SE, where indicated.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
Discordance of fLuc reporter function in live cells and cell extracts. Toward our goal of imaging the pharmacologic modulation of Hh pathway activity in vivo, we constructed a triple modality imaging reporter consisting of the trifusion reporter gene that produces fLuc, red fluorescent protein (RFP), and a mutated form of herpes simplex virus 1 thymidine kinase (sr39ttk) under transcriptional control of the Gli promoter (13). 22Rv1 prostate cancer cells stably transfected with the Gli trifusion construct were established by selection with G418 (designated 22Rv1/Gli-tri). The effects of well-defined modulators of Gli promoter activity validated Gli trifusion reporter function in these cells (14, 15). When transiently transfected with a plasmid expressing Gli1, a positive regulator of Gli promoter activity, 22Rv1/Gli-tri cells showed an 18-fold increase in fLuc reporter expression by fLuc assay of cell extracts (Fig. 1A ). Alternatively, expressing Gli3, a negative regulator of the Gli promoter function, substantially inhibited fLuc expression in Gli1-transfected cells (Fig. 1A). These results confirmed physiologic patterns of Gli trifusion reporter expression regulation in the 22Rv1/Gli-tri cell line.

View larger version (23K): [in this window] [in a new window] [Download PPT slide]   Figure 1. BLI signal in 22Rv1 prostate cancer cells is enhanced by treatment with HhAntag-691, a known Hh pathway inhibitor. A, 22Rv1 cells stably transfected with the Gli trifusion reporter (22Rv1/Gli-tri) were transiently transfected with expression vectors coding for either Gli1, a Gli transcription enhancer, with Gli3, a Gli transcription suppressor, or both. Cell extracts assayed for fLuc expression reveal that reporter expression is Hh pathway dependent, i.e., stimulated by Gli1 and suppressed by Gli3. Columns, mean (n = 3); bars, SE. B, 22Rv1/Gli-tri cells were treated with the Hh pathway inhibitor HhAntag-691. Luciferase assays done on cell extracts show that HhAntag-691 inhibits Gli-tri expression in a concentration-dependent manner. 22Rv1/Gli-tri cells were treated with increasing concentrations of HhAntag-691 for 2 d before the luciferase assay. Points, mean (n = 3); bars, SE. C, BLI of intact 22Rv1/Gli-tri cells treated with HhAntag-691 as in (B) shows a concentration-dependent increase in signal intensity. Top, representative plate of cells imaged; bottom: quantification of BLI. The reading in each well at 2 d was normalized to the reading of the same well before addition of HhAntag-691. Points, mean (n = 4); bars, SE. D, 22Rv1/Gli-tri cell extracts were mixed with different concentrations of HhAntag-691 before quantification of fLuc activity. The presence of HhAntag-691 in cell extracts does not interfere with the fLuc signal. Points, mean (n = 3); bars, SE.

ABC transporter inhibitors alter D-luciferin–dependent BLI. We examined the effects of several ABC transporter inhibitors on D-luciferin–dependent BLI in intact 22Rv1/CMV-tri cells. These included verapamil and doxorubicin for ABCB1/Pgp inhibition, indomethacin for ABCC [multidrug resistance protein (MRP)] inhibition, and fumitremorgin C for ABCG2/BCRP inhibition (18–21). Bioluminescence images were obtained immediately and every few minutes for up to 2 h after the addition of D-luciferin to obtain the kinetics of the inhibitor effects. Verapamil, indomethacin, and fumitremorgin C enhanced the fLuc-mediated BLI signal in 22Rv1/CMV-tri cells similar to the effect of HhAntag-691 in the 22Rv1/Gli-tri and 22Rv1/CMV-tri cells (Fig. 2 ). Doxorubicin had no effect. Enhancement of BLI signal by HhAntag-691, verapamil, indomethacin, and fumitremorgin C occurred in a dose- and time-dependent manner. Verapamil and fumitremorgin C showed the strongest enhancement (fumitremorgin C > verapamil), up to more than double the BLI signal at the highest concentrations tested (50 µmol/L for verapamil and 5 µmol/L for fumitremorgin C) compared with control. These results were consistent with D-luciferin being a substrate for an ABC transporter that pumps D-luciferin out of cells under baseline conditions.

View larger version (39K): [in this window] [in a new window] [Download PPT slide]   Figure 2. ABC transporter inhibitors enhance the BLI signal in 22Rv1 cells stably transfected with the CMV trifusion reporter. A, representative plates of cells showing that verapamil (VP) enhances the bioluminescence of 22Rv1/CMV-tri cells at various times after administration of D-luciferin (150 µg/mL). B, quantitative BLI of 22Rv1/CMV-tri cells in the presence of different transporter inhibitors over 2 h after the addition of D-luciferin (150 µg/mL). Inhibitors were used at the indicated concentrations. Verapamil, indomeficin (Ind), and fumitremorgin C (FTC), but not doxorubicin (Dox), enhance the BLI signal. Columns, mean (n = 3); bars, SE.

D-Luciferin is a specific ABCG2/BCRP substrate. We examined which of the ABC transporters were responsible for the diminished cellular uptake of D-luciferin. Reverse transcription–PCR was used to identify the ABC transporters expressed by 22Rv1 cells. Transcripts for multiple ABC transporters, including ABCB1/Pgp, MRP1, MRP2, MRP3, and ABCG2/BCRP, were identified (Supplementary Fig. S3 online). We examined the effects of ABC transporter inhibitors on quantitative fLuc-mediated BLI in cell lines engineered to stably express specific ABC transporters. Cells engineered to express wild-type (wt) ABCG2/BCRP or mutant ABCG2/BCRP transporters T10 (R482T) and G2 (R482G) were transiently transfected with the CMV trifusion reporter and then seeded into 24-well plates for imaging in the presence or absence of transporter inhibitors (10). Mutation at amino acid 482 alters the substrate specificity of ABCG2/BCRP dramatically. For example, HEK-293 cells transfected with the mutant ABCG2/BCRP T10 were 71-fold less sensitive to the Pgp substrate doxorubicin but those transfected with the wt transporter were only 3-fold less sensitive (10). Fumitremorgin C enhanced the BLI signal 3-fold within cells expressing transgenic wt ABCG2/BCRP within 10 min of adding D-luciferin. Fumitremorgin C had no effect on control-transfected cells that did not express the ABCG2/BCRP transgene (Fig. 3 ). Verapamil and indomethacin, which most potently inhibit ABCB1/Pgp and MRP (ABCC), respectively, generated only mild signal enhancement in the ABCG2/BCRP overexpressing cells. Verapamil, indomethacin, and fumitremorgin C only minimally enhanced BLI in cells engineered to express the mutant ABCG2/BCRP transporters T10 and G2 (Fig. 3). In contrast to cells overexpressing wt ABCG2/BCRP, fumitremorgin C had no effect on the BLI output in cells overexpressing ABCB1/Pgp, MRP1, or MRP2. To show that the ABC transporters that were overexpressed were functional, we did a calcein AM dye uptake assay (Supplementary Fig. S4 online; ref. 23). Similarly, verapamil, indomethacin, and doxorubicin did not alter light output in cells overexpressing these ABC transporters (Fig. 4 ; doxorubicin data not shown). These multiple, complementary findings show that D-luciferin is a specific substrate for ABCG2/BCRP and suggest that pharmacologic transporter inhibition enhances BLI by increasing the cytoplasmic concentrations of this luciferase substrate.

View larger version (39K): [in this window] [in a new window] [Download PPT slide]   Figure 3. D-Luciferin is a substrate of ABCG2/BCRP. A, HEK-293 cells stably transfected with wt ABCG2/BCRP, empty vector, or mutant ABCG2/BCRP (T10 or G2) were transiently transfected with CMV-tri and imaged in the presence of different ABC transporter inhibitors as indicated. A, shown are bioluminescent images of cell culture plates and a corresponding schematic of inhibitors and their concentrations. B, quantification of the imaging experiment shown in (A). Fumitremorgin C, a potent and specific inhibitor of ABCG2/BCRP, enhances the BLI signal in cells expressing transgenic wt ABCG2/BCRP and has no effect on control cells. The effect of fumitremorgin C is minimal in cells expressing the T10 and G2 ABCG2/BCRP mutants. Verapamil and indomethacin show mild signal enhancement, whereas no effect was seen with doxorubicin or colchicine (Col). Imaging was done within 2 min of adding D-luciferin to cultures. All readings were normalized to the condition with no drug added with this baseline reading set at 1 arbitrary unit. Fumitremorgin C increased the BLI signal 3-fold at concentrations of 1 to 5 µmol/L in cells expressing wt ABCG2/BCRP. Columns, mean (n = 3); bars, SE.

View larger version (32K): [in this window] [in a new window] [Download PPT slide]   Figure 4. D-Luciferin is not a substrate of Pgp, MRP1, or MRP2. BLI was done on MDCK cells stably transfected with Pgp (A), MRP1 (B), or MRP2 (C) and transiently transfected with CMV-tri as described in Fig. 3. Doses of ABC transporter inhibitor are indicated. Neither fumitremorgin C, verapamil, indomethacin, nor doxorubicin altered the signal output from cells expressing these ABC transporters. Columns, mean (n = 3); bars, SE.

View larger version (44K): [in this window] [in a new window] [Download PPT slide]   Figure 5. ABCG2/BCRP-modulated bioluminescence in living mice. HEK-293 cells stably transfected with ABCG2/BCRP or empty vector (control) and transiently transfected with CMV-tri were implanted to the flanks (left and right, respectively) of immunocompromised (nude) mice. A, a representative mouse showing BLI acquired 30 min after i.p. administration of D-luciferin, immediately before administration of fumitremorgin C. B, the same mouse as in (A) imaged 12 min after i.v. administration of fumitremorgin C. C, time course of BLI signal from control and transgenic ABCG2/BCRP-expressing tumors. The BLI signal from the BCRP-transfected tumor was 70% less than the control tumor before fumitremorgin C injection and then increased 4-fold after fumitremorgin C injection to levels comparable to the control tumor. Data are representative of results from four separate animals.

View larger version (54K): [in this window] [in a new window] [Download PPT slide]   Figure 6. The specific ABCG2/BCRP inhibitor fumitremorgin C enhances BLI signal from 22Rv1 tumors in vivo. 22Rv1 cells stably transfected with CMV-tri were implanted s.c. into the right flank of mice and allowed to form small tumors. Osmotic minipumps implanted s.c. into the left side of the mice were used to deliver D-luciferin continuously (25 µg/h), and imaging was performed before and after a single i.v. injection of fumitremorgin C or vehicle. A, representative images of the same two mice, before (left) and 30 min after fumitremorgin C or vehicle injection (right). B, time course of quantitative BLI before and after fumitremorgin C or vehicle injection. BLI signal increased gradually by 2-fold after fumitremorgin C administration in contrast to signal from the mouse treated with vehicle, which remained relatively unchanged. Arrows indicate fumitremorgin C and vehicle injection times. Data are representative of results from four separate animals.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

Our findings are consistent with those of Pichler et al. who previously found that D-luciferin is not a substrate for ABCB1/Pgp (7). However, they reported in that study that coelenterazine, another important although less commonly used luciferase substrate (for rLuc), is a substrate for ABCB1/Pgp. But rLuc and coelenterazine are not used for imaging in vivo due to low quantum yield, instability of coelenterazine in plasma, and poor tissue penetration of emitted photons (27, 28). fLuc is the standard in vivo molecular imaging reporter, and an industry is rapidly developing around its use with D-luciferin as a substrate. In fact, D-luciferin is the nearly exclusive substrate for in vitro cell-based assays using BLI as well.

Together, our findings and those of Pichler at al. indicate that ABCB1/Pgp and ABCG2/BCRP activity must be considered when luciferase imaging using coelenterazine or D-luciferin as substrates, respectively, is applied to intact cells or tissues. Failure to do so could result in inadvertently attributing relative differences in bioluminescence output to differences in luciferase reporter expression when differences in substrate transport might more appropriately be implicated. Some unexpected results in the literature may also be explained, in part, by the influence of ABCG2/BCRP on BLI. For example, the difference in the kinetics of photon emission from orthotopic compared with heterotopic brain tumor xenografts may be due to the presence of ABCG2/BCRP at the blood-brain barrier rather than low levels of fLuc expression in target cells within the central nervous system, as suggested by Burgos et al. (29, 30). Perhaps an even more salient example can be found in Zhang et al. (31). In that study, a transgenic (for fLuc) reporter mouse model was established to study transcriptional regulation of the human CYP3A4 gene by placing fLuc under the control of the CYP3A4 promoter. fLuc-luciferin BLI was used to compare relative levels of CYP3A4 expression in different organs and organ-specific CYP3A4 induction by drugs. Based on our findings and the fact that ABCG2/BCRP expression is not uniform throughout the mouse but differs considerably between organs and is gender dependent (32), the organ-specific expression based on the reported BLI may be inaccurate. Seven CYP3A4 inducers were then tested in this in vivo model, and the induction responses based on BLI were reported. Several of the compounds tested, including the compound that provided the greatest BLI output, could be ABCG2/BCRP inhibitors or substrates. Many CYP3A4 inducers concurrently serve as modulators of MDR pumps (33, 34). Specifically, one of the active compounds in the paper, nifedipine, is an ABCG2/BCRP inhibitor (35).

Because ABCG2/BCRP is widely expressed and its known substrates are structurally diverse, ABCG2/BCRP has become an important target for pharmacologic manipulation in efforts to overcome drug resistance in anticancer therapy and to enhance delivery of agents to the central nervous system (30, 36–39). The emerging importance of this relatively recently described ABC transporter is likely to intensify efforts to develop new, nontoxic inhibitors with therapeutic potential, in some cases via the application of high-throughput screens (40). That D-luciferin is a substrate for ABCG2/BCRP could facilitate the screening for new ABCG2/BCRP inhibitors. For example, BLI "reporter mice" have been generated and could provide an organ-specific, in vivo readout of ABCG2/BCRP modulation by various compounds (41). A high-throughput cellular assay for compounds that modulate ABCG2/BCRP can be readily developed based on BLI techniques similar to those used by us in this report. A BLI-based drug screening method would have distinct advantages over current fluorescent assays. Our findings also predict that BLI with fLuc could be applied to screen rapidly for ABCG2/BCRP functional mutations. Whereas D-luciferin proved to be a substrate of wt ABCG2/BCRP, single amino acid changes (R482T and R482G) within the transporter change the BLI output enhancement in response to various inhibitors. This suggests that BLI could provide a very sensitive readout on minor, i.e., single amino acid, changes that alter transporter function.

In summary, we have shown that D-luciferin is a substrate for ABCG2/BCRP and that changes in the activity of this transporter can substantially alter cell-based or in vivo BLI end points independent of other physiologic processes or molecular pathways specifically under investigation. Thus, ABCG2/BCRP expression and function must be considered in studies that rely on BLI with D-luciferin as a substrate in intact cells or tissues. Our findings also establish the feasibility of developing BLI-based high-throughput screens to identify novel modulators of ABCG2/BCRP for future testing in cancer and other disorders.

	Acknowledgments

 
Grant support: NIH grants CA92871 (M. Pomper), NS32148 (J. Laterra), NS43987 (J. Laterra), and The Dana Foundation for financial support.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

We thank Robert W. Robey for helpful discussions.

	Footnotes

 
Note: Supplementary data for this article are available at Cancer Research Online (http://cancerres.aacrjournals.org/).

J. Laterra and M.G. Pomper contributed equally to this work.

Received 3/12/07. Revised 6/29/07. Accepted 7/ 6/07.

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