Regular Article in Vitro Evaluation of Photosensitivity Risk Related to Genetic Polymorphisms of Human ABC Transporter ABCG2 and Inhibition by Drugs

Drug Metab. Pharmacokinet. 22 (6): 428–440 (2007).

Regular Article In Vitro Evaluation of Photosensitivity Risk Related to Genetic Polymorphisms of Human ABC Transporter ABCG2 and Inhibition by Drugs

Ai TAMURA1,*,YukoONISHI1,RanAN1, Shoko KOSHIBA1, Kanako WAKABAYASHI1,*, Kazuyuki HOSHIJIMA1,WaldemarPRIEBE2, Takashi YOSHIDA3,SatoshiKOMETANI3, Takayoshi MATSUBARA3,KentaMIKURIYA3 and Toshihisa ISHIKAWA1,** 1Department of Biomolecular Engineering, Graduate School of Bioscience and Biotechnology, Tokyo Institute of Technology, Yokohama, Japan 2Department of Experimental Therapeutics, The University of Texas M. D. Anderson Cancer Center, Houston, Texas, U.S.A. 3Drug Discovery Solution Department, Life Science Business Headquarters, Yokogawa Electric Corporation, Kanazawa, Japan *Japanese Society for the Promotion of Science (JSPS) Research Fellow

Full text of this paper is available at http://www.jstage.jst.go.jp/browse/dmpk

Summary: Since porphyrins are regarded as endogenous substrates for the ATP-binding cassette (ABC) transporter ABCG2, it is hypothesized that functional impairment owing to genetic polymorphisms or inhibition of ABCG2 by drugs may result in a disruption of cellular porphyrin homeostasis. In the present study, we expressed ABCG2 genetic variants, i.e., V12M, Q141K, S441N, and F489L, as well as the wild type (WT) in Flp-In-293 cells to examine the hypothesis. Cells expressing S441N and F489L variants exhibited high levels of both cellularly accumulated pheophorbide a and photosensitivity, when those cells were incubated with pheophorbide a and irradiated with visible light. To further elucidate the sig- niˆcance of ABCG2 in cellular porphyrin homeostasis, we observed cellular accumulation and compar- tmentation of porphyrin and pheophorbide a by means of a new ‰uorescence microscopy technology, and found that accumulation of porphyrin and pheophorbide a in the cytoplasm compartment was maintained at low levels in Flp-In-293 cells expressing ABCG2 WT, V12M, or Q141K. When ABCG2 was inhibited by imatinib or novobiocin, however, those cells became sensitive to light. Based on these results, it is strongly suggested that certain genetic polymorphisms and/or inhibition of ABCG2 by drugs can enhance the potential risk of photosensitivity.

Key words: ABCG2 (BCRP/MXR); genetic polymorphisms; photo-toxicity; porphyrin; pheophorbide a

ABCG2 is expressed in a wide variety of stem cells, Introduction and its potential role in the regulation of hematopoietic Human ABCG2, a member of the ABC transporter development is suggested.6) Expression of Abcg2, a mu- family,1–3) plays a pivotal role in mediating drug rine orthologue of human ABCG2, is a conserved fea- resistance of cancer cells and aŠecting the pharmacolog- ture of murine stem cells from diŠerent sources includ- ical behavior of a variety of drugs. In addition, it has re- ing the bone marrow, skeletal muscle, and embryonic cently been revealed that ABCG2 is responsible for the stem cells.6) Abcg2 mRNA is expressed at high levels in transport of endogenous metabolites, such as porphy- primitive murine hematopoietic stem cells and is sharply rins.4,5) downregulated with diŠerentiation. Enforced expression of the human ABCG2 cDNA directly conferred the The ˆrst and second authors contributed equally to this work. side population phenotype to bone marrow cells. Loss

Received; July 30, 2007, Accepted; September 21, 2007 **To whom correspondence should be addressed: Toshihisa ISHIKAWA, Professor, Department of Biomolecular Engineering, Graduate School of Bioscience and Biotechnology, Tokyo Institute of Technology, 4259-B-60 Nagatsuta, Midori-ku, Yokohama 226-8501, Japan. Tel. ＋81-45-924-5800, Fax. ＋81-45-924-5838, E-mail: tishikaw＠bio.titech.ac.jp

428 Genetic Polymorphisms of ABCG2 and Photosensitivity Risk 429 of the Abcg2 gene led to a reduction in the number of of photosensitivity. In this context, our present study side population cells in the bone marrow and skeletal may provide a new approach to studying drug-induced muscle.7) Abcg2 null hematopoietic cells were sig- phototoxicity in vitro. niˆcantly more sensitive than normal hematopoietic Materials and Methods cells to mitoxantrone in drug-treated transplanted mice. Mice lacking Abcg2 displayed a previously unknown Chemicals and biological reagents: The following type of protoporphyria, where erythrocyte levels of pro- compounds and therapeutic drugs were purchased from toporphyrin IX were increased about 10-fold.8) It has re- the commercial sources indicated in parentheses: ATP, cently been demonstrated that under hypoxic condi- hematoporphyrin, and protoporphyrin IX (SIGMA- tions, heme molecules and/or porphyrins are detrimen- ALDRICH, St. Louis, MO, USA); pheophorbide a tal to Abcg2－/－ progenitor cells.9) Abcg2 speciˆcally (Frontier Scientiˆc, Inc., Utah, USA); L-glutamine binds heme, and cells lacking Abcg2 accumulate por- (Wako Pure Chemical Industries, Ltd., Osaka, Japan); phyrins. In addition, Abcg2 expression is upregulated creatine kinase, creatine phosphate, ethyleneglycol- by hypoxia, where the hypoxia-inducible transcription bis(2-aminoethyl)-N,N,N?,N?-tetracetic acid (EGTA), factor complex HIF-1 is involved. These ˆndings sug- ethylenediaminetetraacetic acid (EDTA), tris(hydrox- gest that Abcg2 confers a strong survival advantage on ymethyl)aminomethane (Tris), 2-[4-(2-Hydroxyethyl)-1- stem cells under hypoxic conditions. Furthermore, piperazinyl]ethanesulfonic acid (HEPES), and high-glu- erythroid cells engineered to express Abcg2 had sig- cose Dulbecco's modiêd Eagle's medium (D-MEM) niˆcantly lower intracellular levels of protoporphyrin (Nacalai Tesque, Inc., Kyoto, Japan); fetal calf serum IX, suggesting that the protoporphyrin IX level is (FCS) (Dainippon Pharmaceuticals, Osaka, Japan); and modulated by human ABCG2.10,11) antibiotic-antimycotic cocktail solution and hygromy- Single nucleotide polymorphisms (SNP) of ABC cin B (Invitrogen, Carlsbad, CA, USA). Imatinib transporters have been reported to play a signiˆcant role (Gleevec or STI571), 4-(4-methylpiperazin-1-ylmethyl)- in patients' responses to medication and/or the risk of N-[4 -methyl-3 -(4-pyridin-3-ylpyrimidin-2 -ylamino) diseases.12) Sequencing of the ABCG2 gene from human phenyl]-benzamide monomethanesulfonate, was chemi- samples has revealed over 80 diŠerent, naturally occur- cally synthesized as reported previously.25) All other ring sequence variations.13–22) The analysis and valida- chemicals used were of analytical grade. tion of those SNPs occurring in the ABCG2 gene are of SNP data on nonsynonymous polymorphisms of hu- clinical importance. Based on the functional validation, man ABCG2 gene: SNP data on the polymorphisms we have recently classiêd the non-synonymous SNPs of ABCG2 were obtained from the NCBI dbSNP data- and acquired mutations of ABCG2 into four groups.23) base and recent publications.13–22) To clarify the possible physiological or pathological Preparation of plasmids carrying ABCG2 variant relevance of the ABCG2 polymorphism, we attempted cDNA: Wild-type (WT) ABCG2 cDNA inserted into to functionally characterize genetic variants. By using the pcDNA5/FRT plasmid26) wasusedasthetemplate, plasma membrane vesicles and a high-speed screening and nonsynonymous SNP variants were generated by system, we precisely evaluated functional changes asso- using the QuikChange} Site-Directed Mutagenesis Kit ciated with genetic polymorphisms in vitro.24) Since por- (Stratagene, La Jolla, CA, USA), as described previous- phyrins are considered to be endogenous substrates for ly.4) The mutations were conˆrmed by sequencing the in- ABCG2, we have investigated the transport of porphy- serted cDNA. rins with a total of 18 variant forms of human ABCG2 Expression of ABCG2 and its variants in Flp-In-293 in the plasma membrane vesicle system.4) As a result, we cells: Flp-InTM-293 cells (Invitrogen, Carlsbad, CA, found that the variants Q126stop, F208S, S248P, USA) were maintained in high-glucose Dulbecco's mo- E334stop, S441N, and F489L are defective or impaired diêd Eagle's medium (D-MEM) supplemented with in the transport of porphyrins. From our data, it is sug- 10z (v/v) heat-inactivated FCS, 2 mM L-glutamine, gested that those genetic polymorphisms in the ABCG2 penicillin (100 U/mL), and streptomycin (100 mg/mL) gene may be related to the risk of certain diseases result- at 379C in a humidiêd atmosphere of 5z CO2 in air. ing from disruption of porphyrin homeostasis. The number of viable cells was determined from counts In the present study, we aimed to evaluate the impact made in a hemocytometer with Trypan Blue dye exclu- of genetic polymorphisms of ABCG2 as well as the inhi- sion. bition by drugs of ABCG2 function on photo-sensitivi- Flp-In-293 cells were transfected with the ABCG2- ty. By using Flp-In-293 cells and plasma membrane vesi- pcDNA5/FRT vector, the Flp recombinase expression cles prepared from Sf9 insect cells, we established new plasmid pOG44, and LipofectAmineTM-2000 (Invitro- methods for photosensitivity assays. In this paper, we gen, Carlsbad, CA, USA) according to the manufac- provide evidence that certain genetic polymorphisms turer's instructions. Single colonies resistant to and inhibition of ABCG2 by drugs can enhance the risk hygromycin B (Invitrogen, Carlsbad, CA, USA) were 430 Ai TAMURA, et al. picked and subcultured. Selection of positive colonies for 20 min. Thereafter, cell membranes were permeabi- was performed by immunoblotting, as described below. lized by incubation with 0.02z Triton X-100 in PBS at The resulting cells are described as Flp-In-293/ABCG2 room temperature for 5 min. To block free aldehyde cells throughout this manuscript. Mock cells (Flp-In- groups of the formaldehyde, cells were treated with gly- 293/Mock) were prepared by transfecting Flp-In-293 cine (10 mg/mL) in PBS at room temperature for 10 cells with pcDNA5/FRT and pOG44 vectors in the same min, which was followed by a further incubation with manner as described above. 0.5z (w/v) albumin in PBS at room temperature for 1 Detection of mRNA by RT-PCR: Total RNA was h. To detect the ABCG2 protein, cells were treated with extracted from cultured cells with NucleoSpin} RNA II the BXP-21 antibody (1:1000 dilution; SIGNET, Ded- (MACHEREY-NAGEL GmbH & Co. KG, Duren, Ger- ham, MA, USA) as the ˆrst antibody and subsequently many) according to the manufacturer's protocol. cDNA with the Alexa Fluor 488-conjugated anti-mouse IgG was prepared from the extracted RNA in a reverse tran- antibody (1:1000 dilution; Invitrogen, Carlsbad, CA, scriptase reaction with SuperScript II RT (Invitrogen, USA). In the same preparations, nuclear DNA was Carlsbad, CA, USA) and random hexamers according stained with propidium iodide (4 mg/mL) in PBS con- to the manufacturer's instructions. The mRNA levels of taining 0.5z (w/v) albumin. The immuno‰uorescence ABCG2 were determined by polymerase chain reaction of Flp-In-293 cells was detected with a confocal laser- (PCR)inaniCyclerTM thermal cycler (BIO-RAD, Her- scanning ‰uorescence microscope IX70/FLUOVIEW cules, CA, USA) with the following speciˆc primer sets: (Olympus, Tokyo, Japan). 5?-GATCTCTCACCCTGGGGCTTGTGGA,5?-TGTG Cellular accumulation of pheophorbide a: The in- CAACAGTGTGATG GCAAGGGA. The PCR reac- tracellular pheophorbide a accumulation was deter- tion consisted of hot-start incubation at 949Cfor2min mined by ‰uorescence spectroscopy. Cells were seeded and 30 cycles of 949C for 30 sec, 599C for 30 sec, and 72 in 6-well plates (1×106 cell/well) and cultured at 379C 9C for 30 sec. After the PCR, products were separated for 24 hours. Pheophorbide a was added to the culture by agarose gel electrophoresis and detected with ethydi- medium at increasing concentrations and then incuba- um bromide under UV light. tion was continued in the dark for 4 hours. Thereafter, Gel electrophoresis and detection of ABCG2 protein cells were rinsed with PBS and subsequently treated by immunoblotting: The ABCG2 protein expressed in with the lysis buŠer containing 50 mM Tris/HCl (pH Flp-In-293 cells was detected by immunoblotting with 7.4), 1z (w/v) Triton X-100, 1 mM DTT, and a pro- BXP-21 (SIGNET, Dedham, MA, USA), a speciˆc an- tease inhibitor cocktail (Nacalai Tesque, Kyoto, Japan). tibody to human ABCG2. Cells were rinsed with ice- A portion (100 mL) of the cell lysate was mixed with cold phosphate-buŠered saline (PBS) and subsequently 1900 mL of 10 mM NaOH solution. Pheophorbide a in treated with the lysis buŠer containing 50 mM Tris/HCl the resulting solution was quantitatively determined by (pH 7.4), 1z (w/v) Triton X-100, 1 mM DTT, and a measuring its ‰uorescence in a Hitachi Fluorescence protease inhibitor cocktail (Nacalai Tesque, Kyoto, Spectrophotometer Type F2000. An excitation wave- Japan). The samples (10 mg of protein) were subjected length of 400 nm and an emission wavelength of 667 nm to SDS-PAGE in the presence of 2-mercaptoethanol were used to measure the ‰uorescence intensity. (see Results for more details). Brie‰y, proteins were Pheophorbide a concentrations were determined by separated by electrophoresis on 7.5z polyacrylamide comparing ‰uorescence measurements with a calibra- gels and then electroblotted onto Hy-bond ECL tion curve. nitrocellulose membranes (Amersham, Buckingham- Cellular photosensitivity assay with 96-well plates: shire, UK). Immunoblotting was performed by using The eŠect of pheophorbide a on the photo-sensitivity of BXP-21 (1:500 dilution) as the ˆrst antibody and an an- mock or ABCG2-expressing Flp-In-293 cells was meas- ti-mouse IgG-horseradish peroxidase (HRP)-conjugate ured as described by Robey et al.27) Brie‰y, cells were (1:3000 dilution; Cell Signaling Technology, Beverly, seeded in 96-well plates (15,000 cell/well) and cultured MA, USA) as the secondary antibody. HRP-dependent at 379C for 24 hours. Pheophorbide a was added to the luminescence was developed by using Western Lighting culture medium at various concentrations and then in- Chemiluminescent Reagent Plus (PerkinElmer Life cubation was continued in the dark for 4 hours. Subse- Sciences, Boston, MA, USA) and detected in a Lumino quently, the culture medium was replaced with fresh Imaging Analyzer FAS-1000 (TOYOBO, Osaka, medium. Cells were then exposed to light for 90 min in Japan). the cell culture chamber, where 96-well plates were Immuno‰uorescence microscopy: ABCG2-express- placed on a light viewer (Hakuba Model 5700). Cells ing Flp-In-293 cells were seeded onto collagen type I- were again incubated in the dark for 24 hours, and cell coated cover glasses and incubated under the above- viability was measured by the MTT assay as described mentioned culture conditions for 24 h. Cells were ˆxed previously.28) Thereafter, 100 mLof10z (w/v) SDS in with 4z paraformaldehyde in PBS at room temperature PBS was added to the culture medium, and the mixture Genetic Polymorphisms of ABCG2 and Photosensitivity Risk 431 was incubated at 379C overnight. The absorbance of Sf9 cells were further infected with the harvested virus formazan, a metabolite of MTT, in the resulting solu- and maintained at 279C for 72 hours. After the incubation was photometrically measured at a test wavelength tion, the culture medium was harvested by centrifuga- of 570 nm and a reference wavelength of 630 nm in a tion. This process was repeated two times. Multiskan JX system (Dainippon Pharmaceuticals Co., Sf9 cells (1×106 cells/mL) were infected with the am-

Osaka, Japan). IC50 values were calculated from dose- pliêd recombinant baculoviruses and cultured in EX- response curves (i.e., cell survival vs. drug concentra- CELLTM 420 Insect serum-free medium at 279Cwith tion). gentle shaking. Three days after the infection, cells were Fluorescence microscopy with a new confocal scanner harvested by centrifugation. Cells were subsequently unit: Cells were seeded (1×105 cells) onto collagen washed with PBS, collected by centrifugation, and type IV-coated glass bottom culture dishes and incubat- stored at －809C until used. ed under the above-mentioned culture conditions for 24 Preparation of the plasma membrane vesicles from h. d-Aminolevlinic acid (ALA) or pheophorbide a was Sf9 cells: Plasma membrane vesicles were prepared added to the culture medium at 1 mM or 1 mM, respec- from ABCG2-expressing Sf9 cells as described previous- tively, and then incubation was continued in the dark ly.24) The frozen cell pellet was thawed quickly, diluted for 4 hours. Thereafter, the culture medium was 40-fold with a hypotonic buŠer (0.5 mM Tris/HEPES, replaced with fresh medium, and immediately the pH 7.4, 0.1 mM EGTA), and then homogenized with a ‰uorescence of protoporphyrin or pheophorbide a was Potter-Elvehjem homogenizer. After centrifugation at measured by using a newly developed confocal scanner 2,000×g for 10 min, the supernatant was further cen- unit (Yokogawa Electric Corporation, Kanazawa, trifuged at 100,000×g for 30 min. The resulting pellet Japan). The confocal scanner unit was equipped with a was suspended in 0.25 M sucrose containing 10 mM CCD camera (Cool SNAP HQ, Roper Industries, Inc., Tris/HEPES, pH 7.4. The crude membrane fraction Florida, USA) and an image intensiêr unit (C9016-01, was layered over 40z (w/v) sucrose solution and cen- Hamamatsu Photonics K.K., Hamamatsu, Japan) to trifuged at 100,000×g for 30 min. The turbid layer at capture digital images. The ‰uorescence of protopor- the interface was collected, suspended in 0.25 M sucrose phyrin or pheophorbide a was observed with an excita- containing 10 mM Tris/HEPES, pH 7.4, and cen- tion laser light at 405 nm and through a 600-nm long- trifuged at 100,000×g for 30 min. The membrane frac- pass ˆlter for detection. To visualize mitochondria, cells tion was collected and resuspended in a small volume were pre-incubated with 50 nM MitoTracker} Green (150 to 250 mL) of 0.25 M sucrose containing 10 mM FM (Invitrogen Co., Carlsbad, CA, USA) for 10 min Tris/HEPES, pH 7.4. After the protein concentration and then the medium was replaced with fresh incubation was measured with the BCA Protein Assay Kit medium prior to the 4-hour incubation. The MitoTrack- (PIERCE, Rockford, IL, USA), the membrane solution er was excited with excitation laser light at 488 nm, and was stored at －809C until used. its emission was detected through a band-pass (505–555 High-speed detection of ABCG2-mediated hemato- nm) ˆlter. porphyrin transport: The frozen stocked membrane Expression of ABCG2 in Sf9 cells: Competent solution was thawed quickly at 49C. Plasma membrane DH10Bac E. coli cells were transformed by the ABCG2- vesicles (20 mg of protein) were incubated with 20 mM pFanstBac1 plasmids. Then, the ABCG2 cDNA was hematoporphyrin in the presence or absence of 1 mM transposed into a bacmid, which is a baculovirus shuttle ATP in 30 mL of the standard incubation medium (0.25 vector carrying the baculovirus genome, in DH10Bac M sucrose and 10 mM Tris/HEPES, pH 7.4, 10 mM cells with the help of a helper plasmid. The recombinant creatine phosphate, 100 mg/mL creatine kinase, and 10 bacmid was isolated and puriêd. mM MgCl2)at379C for 10 min. After a speciêd incu- Insect Spodoptera frugiperda Sf9 cells were grown in bation period, the reaction mixture was mixed with 80 EX-CELLTM 420 Insect serum-free medium (JRH mL of ice-cold stop solution (10 mM EDTA, 0.25 M su- Biosciences, Inc., Lenexa, KZ, USA) supplemented crose and 10 mM Tris/HEPES, pH 7.4); and then 50 mL with 1z (v/v) heat-inactivated fetal calf serum (FCS), of the resulting solution was loaded onto a 96-well sepa- penicillin (100 U/mL), and streptomycin (100 mg/mL) ration plate (100 mL of bed volume) packed with Sepha- (Invitrogen Co., Carlsbad, CA, USA) with gentle shak- dex G-25 equilibrated with 0.25 M sucrose and 10 mM ing at 279C. Sf9 cells were then transfected with the Tris/HEPES, pH 7.4. The plate was immediately cen- ABCG2-recombinant bacmid in the presence of Cellfec- trifuged in a swing-type rotor at 1,600×g for 5 min, tin} reagent (Invitrogen Co., Carlsbad, CA, USA) ac- whereby the eluate was collected into a 96-well micro cording to the manufacturer's protocol. Ninety-six plate.Theeluateineachwellwasmixedwith350mLof hours after the transfection, the culture medium con- 10 mM NaOH solution to dissolve plasma membrane taining the recombinant baculovirus was harvested by vesicles. Hematoporphyrin in the resulting solution was centrifugation. To amplify recombinant baculovirus, quantitatively analyzed by measuring its ‰uorescence in 432 Ai TAMURA, et al.

Table 1. Genetic polymorphisms of human ABCG2 and pheophorbide a-photosensitivity

In vitro experiments SNP data

IC50 ( mM) Photosensitivity ratio (fold) Ethnic group N Allele frequency (z) Reference WT 3.0 1.0 — — — —

V12M 4.1 0.7 Caucasian 546 5.6 22, 13, 21, 20, 14 Japanese 259 17.6 18, 22, 20 African 181 6.3 22, 20

Q141K 2.9 1.0 Caucasian 717 11.0 22, 13, 21, 15, 20, 14 Japanese 354 30.6 18, 22, 20 African 1213 1.4 22, 15, 14 S441N 0.5 6.0 Japanese 100 0.5 20

F489L 1.7 1.8 Japanese 160 0.6 19, 20

Pheophorbide a-photosensitivity ratios and IC50 values were determined from the data shown in Fig. 2B.

a Fluoroskan Ascent FL (Thermo Labsystems, Hel- tected, suggesting that endogenous expression of sinki, Finland) (excitation at 405 nm; emission at 612 ABCG2 was negligibly low. This was in accordance with nm). the immunoblotting data (Fig. 1A). In contrast, strong green ‰uorescence was observed at the plasma mem- Results brane and within intracellular compartments in Flp-In- Expression of ABCG2 WT and SNP variants in 293 cells expressing ABCG2 WT as well as the SNP vari- Flp-In-293 cells: In the present study, we aimed to exa- ants of V12M, Q141K, and F489L. In the case of the mine the impact of hitherto reported major SNPs S441N variant, however, the variant protein was detect- (V12M, Q141K, S441N, or F489L) on the photo-sen- ed only within intracellular compartments in Flp-In-293 sitivity. Table 1 summarizes the allele frequencies of cells. those SNPs in diŠerent ethnic populations. Based on Cellular accumulation of pheophorbide a in Flp-In- SNP sequence data, we prepared each variant cDNA by 293 cells expressing ABCG2 WT and SNP variants: site-directed mutagenesis. Thereafter, we integrated one Since the chlorophyll metabolite pherophorbide a is one single copy of the resulting cDNA into FRT-tagged of substrates for ABCG2, we examined the cellular ac- genomic DNA by using the Flp-In method. The cDNA cumulation of this metabolite in Flp-In-293 cells ex- was found to be integrated into the telomeric region of pressing ABCG2 WT and those SNP variants to inves- the short arm on one of chromosomes 12 in Flp-In-293 tigate any functional diŠerences among them. Cells cells (data not shown; see ref. 23). As shown in Fig. 1A, were incubated with pherophorbide a at diŠerent con- ABCG2 mRNA levels detected by RT-PCR were almost centrations at 379C for 4 hours in the dark. Thereafter, equal among those SNP variants as well as ABCG2 WT. cells were harvested, and the amount of pheophorbide a Immunoblotting with the anti-ABCG2 antibody accumulated in those cells was determined by measuring BXP-21, however, revealed varying levels of ABCG2 both the intensity of pheophorbide a ‰uorescence and proteins. Namely, the protein expression level of the the total protein amount in each cell lysate sample. As S441N variant was markedly low (Fig. 1A). In addition, demonstrated in Fig. 2A, in the concentration range of the protein level of the Q141K SNP variant was moder- up to 2.5 mM, the accumulation of pheophorbide a in ately lower than that of ABCG2 WT. The expression of Flp-In-293/ABCG2 (S441N) was high, being similar to endogenous ABCG2 in Flp-In-293/Mock cells was un- that in Flp-In-293/Mock cells. In addition, the cellular der detectable levels. pherophorbide a accumulation in Flp-In-293/ABCG2 Figure 1B depicts the immuno‰uorescence images of (F489L) cells was about the same level as those in Flp- Flp-In-293 cells expressing ABCG2 WT and those SNP In-293/ABCG2 (S441N) and Flp-In-293/Mock cells at variants (i.e., V12M, Q141K, S441N, and F489L) as the concentration of 2.5 mM. In contrast, intracellular well as mock vector-transfected cells (Flp-In-293/ accumulations of pheophorbide a in both Flp-In- Mock). ABCG2 proteins were probed with the BXP-21 293/ABCG2 (V12M) and Flp-In-293/ABCG2 (Q141K) antibody and then labeled with green ‰uorescence dye cells were signiˆcantly lower, being similar to the levels (Alexa Fluor 488), whereas DNA in the nuclei was observed in Flp-In-293/ABCG2 (WT) cells. It is sug- stained with propydium iodide (red ‰uorescence). In gested that two variants, V12M and Q141K, actively ex- Flp-In-293/Mock cells, no green ‰uorescence was de- truded pheophorbide a out of cells as did ABCG2 WT, Genetic Polymorphisms of ABCG2 and Photosensitivity Risk 433

Fig. 1. Expression of human ABCG2 WT, V12M, Q141K, S441N, and F489L in Flp-In-293 cells. A, ABCG2 WT and SNP variants were expressed in Flp-In-293 cells as described previously (Tamura A, et al. J Exp Ther Oncol 2006; Cancer Sci 2007). The mRNA and protein levels of ABCG2 WT and SNP variants were detected by RT-PCR and immunoblotting, respectively (see details in Materials and Methods). B,cellular localization of ABCG2 WT and SNP variants was analyzed by immuno‰uorescence microscopy as described in Materials and Methods. The ABCG2 protein is demonstrated in green color and the nuclei in red. Horizontal bars indicate 20 mmofdistance.

whereas the S441N variant and F489L appeared unable of pheophorbide a in the incubation medium increased, to export pheophorbide a. the cells became more sensitive to the visible light. Im- Photosensitivity of Flp-In-293 cells expressing portantly, signiˆcant diŠerences were noticed in the ABCG2 WT and SNP variants: Figure 2B demon- photosensitivity among Flp-In-293/Mock cells as well as strates the cellular photosensitivity proˆles of Flp-In- the Flp-In-293 cells expressing ABCG2 WT and SNP 293 cells expressing ABCG2 WT, V12M, Q141K, variants. Flp-In-293/Mock and Flp-In-293/ABCG2 S441N, and F489L, as well as that of Flp-In-293/Mock (S441N) cells were very sensitive to light, whereas Flp- cells. In that experiment, cells were ˆrst incubated with In-293/ABCG2 (V12M), Flp-In-293/ABCG2 (Q141K), pheophorbide a at various concentrations in the dark and Flp-In-293/ABCG2 (WT) cells were signiˆcantly for 4 hours. Subsequently, the culture medium was more resistant. Flp-In-293/ABCG2 (F489L) cells ex- replaced with fresh medium, and then cells were exhibited a moderate photosensitivity in the range between posed to light for 90 min. During this period, the those of Flp-In-293/Mock and Flp-In-293/ABCG2 pheophorbide a accumulated in cells was excited by visi- (WT) cells. Table 1 summarizes the observed IC50 values 1 ble light to produce singlet oxygen ( O2), a reactive and the calculated photosensitivity ratios for those cells oxygen species, within the cells. When the concentration tested. These results were well correlated with the levels 434 Ai TAMURA, et al.

precursor of porphyrin biosynthesis, we tried to produce protoporphyrin IX within those cells by adding ALA into the incubation medium. Weak ‰uorescence of protoporphyrin was observed in both the cytosol space and mitochondria of Flp-In-293/Mock cells (Fig. 3) when the cells were irradiated with the excitation laser light at 405 nm. In the case of Flp-In-293/ABCG2 (WT) cells, on the other hand, the ‰uorescence intensity of protoporphyrin in the cytosol space was weaker than that observed in Flp-In-293/Mock cells. A marked diŠerence was obtained between Flp-In- 293/Mock and Flp-In-293/ABCG2 (WT) cells with respect to the cellular porphyrin distribution, however when those cells were incubated with pheophorbide a (Fig. 3). The strong ‰uorescence of pheophorbide a was observed in both the cytosol space and mitochondria within Flp-In-293/Mock cells, whereas Flp-In-293/ ABCG2 (WT) cells exhibited very weak levels of pheophorbide a ‰uorescence. These results suggest that ABCG2 is playing a signiˆcant role in the extrusion of porphyrins from the cells. Inhibition of ABCG2-mediated porphyrin transport by imatinib, novobiocin, and quercetin: By using 96- well gel ˆltration plates and plasma membrane vesicles prepared from Sf9 cells expressing human ABCG2 WT, we have developed a high-speed in vitro screening system to precisely evaluate the functional changes associated with ABCG2 inhibition by using a variety of test Fig. 2. Intracellular accumulation of pheophorbide a (A) and pho- compounds and drugs. For this purpose, plasma mem- tosensitivity of Flp-In-293 cells expressing ABCG2 WT and SNP vari- brane vesicles were prepared from ABCG2 (WT)-ex- ants (B). A, Flp-In-293 cells expressing human ABCG2 WT and SNP pressing Sf9 cells. Figure 4A shows the time courses for variants (V12M, Q141K, S441N, and F489L) were incubated with hematoporphyrin transport into the membrane vesicles pheophorbide a at diŠerent concentrations (0, 0.63, 1.25, and 2.5 mM) in the presence or absence of ATP. The activity of ATP- at 379C for 4 hours. Thereafter, cells were rinsed with PBS, and pheophorbide a accumulated in the cells was assayed by measuring the dependent hematoporphyrin transport was very low in ‰uorescence intensity (excitation 400 nm; emission 667 nm). The plasma membrane vesicles prepared from mock-infect- amount of pheophorbide a was deterimined by referring to the exter- ed Sf9 cells (data not shown). In addition to hematopor- nal standards of pheophorbide a. B, to determine the photosensitivity, phyrin, we tested protoporphyrin, coproporphyrin, Flp-In-293 cells expressing human ABCG2 WT and SNP variants were uroporphyrin, and pheophorbide a; however, the best incubated with pheophorbide a at diŠerent concentrations in 96-well plates under dark conditions for 4 hours. Thereafter the incubation signal vs. noise ratio in ATP-dependent transport was medium was replaced with fresh medium, and cells were exposed to obtained with hematoporpohyrin. light for 90 min, as described in Materials and Methods. Cell viability As demonstrated in Fig. 4B, imatinib, novobiocin, was measured by the MTT assay. and quercetin strongly inhibited ABCG2-mediated hematoporphyrin transport in the membrane vesicle

transport system with IC50 values of 5.0, 4.6, and 1.2 of pheophorbide a accumulation (Fig. 2A). mM, respectively. Quercetin was found to be the stron- Cellular distribution of protoporphyrin and pheo- gest inhibitor among these test compounds in this mem- phorbide a in Flp-In-293 cells expressing ABCG2 WT: brane vesicle transport experiment. To elucidate the role of ABCG2 WT in cellular Photosensitization of Flp-In-293 cells by inhibiting homeostatis and extrusion of porphyrin, we incubated ABCG2 WT with imatinib, novobiocin, and quercetin: Flp-In-293/Mock and Flp-In-293/ABCG2 (WT) with To examine the potential link between ABCG2 inhibi- ALA and then observed the intracellular distribution of tion and photosensitivity, we incubated Flp-In-293 cells protoporphyrin with the confocal scanner unit devel- expressing ABCG2 WT with varying concentrations of oped by Yokogawa Electric Corporation (Kanazawa, pheophorbide a in the presence of quercetin, novobio- Japan). Mitochondria were visualized with MitoTracker cin, and imatinib. Except for the addition of these test as described in Materials and Methods. Since ALA is a compounds into the incubation medium, the experi- Genetic Polymorphisms of ABCG2 and Photosensitivity Risk 435

Fig. 3. Detection of intracellular distribution of protoporphyrin and pheophorbide a in Flp-In-293 cells by confocal ‰uorescence microscopy. Flp-In-293/Mock and Flp-In-293/ABCG2(WT) cells were incubated with 1 mM ALA or 1 mM pheophorbide a under dark conditions for 4 hours. Thereafter, the culture medium was replaced with fresh medium, and immediately the ‰uorescence of protoporphyrin or pheophorbide a was measured by using the confocal scanner unit. Protoporphyrin or pheophorbide a (Phe a) was excited with laser light at 405 nm, and their ‰uorescence was detected with a 600-nm long-pass ˆlter. On the other hand, the ‰uorescence of MitoTracker (Mito) was observed with the excitation laser light at 488 nm and through a band-pass (505–555 nm) ˆlter. ments were performed in the same manner as shown in accumulation of pheophorbide a in Flp-In-293/ Fig. 2B. Figure 5 demonstrates that 100 mM novobiocin ABCG2(WT) cells: To further investigate whether the and 5 mM imatinib signiˆcantly enhanced the pheophor- enhanced photosensitivity was due to increased intracel- bide a-induced photosensitivity in Flp-In-293 cells ex- lular levels of pheophorbide a, we observed the intracel- pressing ABCG2 WT. Both novobiocin and imatinib lular accumulation and distribution of pheophorbide a per se had no eŠect on the photosensitivity of those by using a ‰uorescence microscope equipped with the cells. Although quercetin strongly inhibited the confocal scanner unit. Figure 6 depicts ‰uorescence im- ABCG2-mediated hematophorphyrin transport in plas- ages of Flp-In-293/ABCG2 (WT) cells that were incu- ma membrane vesicles (Fig. 4), its eŠect on the pho- batedwith1mM pheophorbide a in the absence or tosensitization in Flp-In-293/ABCG2 (WT) cells was presence of 5 mMimatinibor100mM novobiocin for 4 minimal. hours. In the absence of those ABCG2 inhibitors, in- EŠect of imatinib and novobiocin on the intracellular tracellular accumulation of pheophorbide a was very 436 Ai TAMURA, et al.

Fig. 5. Photosentitization of Flp-In-293/ABCG2(WT) cells by inhibiting ABCG2 WT with imatinib, novobiocin or quercetin. Flp-In- 293 cells expressing ABCG2 WT were incubated with pheophorbide a inthepresenceof5mM imatinib, 100 mM novobiocin, or 100 mM quercetin at 379C for 4 hours. Thereafter, the incubation medium was replaced with fresh medium, and cells were exposed to light for 90 min, in the same manner as described in Fig. 2B. Cell viability was measured by the MTT assay.

Discussion Critical role of ABCG2 in intracellular porphyrin biosynthesis and homeostasis: Hemes play a critical role in diverse biological processes, such as respiration and oxidative metabolism. Heme molecules are synthe- Fig. 4. Inhibition of ABCG2-mediated hematoporphyrin transport sized from glycine and succinyl CoA via an eight-step by quercetin, novobiocin, and imatinib. ABCG2-expressing plasma pathway that is spatially shared between mitochondria membrane vesicles (20 mg of protein) were incubated with 20 mM and cytoplasm compartments (Fig. 7). The transport of hematoporphyrin in the presence of imatinib, novobiocin, or querce- porphyrins across cellular membranes is critical in the tin (ˆnal concentration: 0, 0.3, 1, 3, 10, or 30 mM) in the standard in- metabolic compartment and cellular homeostasis of cubation medium (0.25 M sucrose and 10 mM Tris/HEPES, pH 7.4, 1 mM ATP, 10 mM creatine phosphate, 100 mg/mL creatine kinase, 10 porphyrins. Recently, the involvement of ABCB6 has mM MgCl2)at379C for 10 min. Hematoporphyrin transported into been implicated in the transport of coproporphyrinogen membrane vesicles was detected as described in Materials and from the cytosol compartment to mitocondria.29) This Methods. transport is considered to be one of the critical steps in porphyrin biosynthesis. On the other hand, an im- portant role of ABCG2 in the regulation of cellular por- low, being under detectable levels. However, incubation phyrin levels was suggested in Abcg2-knockout mice, with 5 mMimatinibor100mM novobiocin clearly en- where erythrocyte levels of protoporphyrin IX were in- hanced the intracellular accumulation of pheophorbide creased 10-fold.8) In addition, these Abcg2-knockout a, as represented by the red ‰uorescence in Fig. 6.Inad- mice developed a skin phototoxicity that was associated dition, it is noteworthy that major part of intracellular with an increase in dietary chlorophyll that produced an pheophorbide a was localized in both the cytosolic com- accumulation of pheophorbide a.8) partment and mitochondria. Based on these results, it is Porphyria is recognized as a peculiar group of genetic suggested that inhibition of ABCG2 function with im- disorders resulting from hereditary or acquired deˆcien- atinib or novobiocin resulted in the accumulation of cies in the biosynthetic pathway of hemes.30,31) Depend- pheophorbide a in cytosolic and mitochondrial com- ing on the site of heme-biosynthetic enzyme defect, por- partments and thereby enhanced the photosensitivity of phyrias are classiˆed as being erythropoietic or hepatic. cells. There are nine basic clinical forms of porphyria related to a deˆciency in each of the involved enzymes. Genetic Polymorphisms of ABCG2 and Photosensitivity Risk 437

Fig. 6. EŠect of imatinib and novobiocin on the intracellular distribution of pheophorbide a in Flp-In-293/ABCG2(WT) cells. Flp-In-293 cells expressing ABCG2 WT were incubated with pheophorbide a in the presence of 5 mM imatinib or 100 mM novobiocin at 379C for 4 hours. There- after, the incubation medium was replaced with fresh medium, and immediately the ‰uorescence of pheophorbide a was measured with the excitation laser light at 405 nm by using the confocal scanner unit with a 600-nm long-pass ˆlter. To visualize mitochondria, the MitoTracker ‰uorescence was recorded with the excitation laser light at 488 nm, where its emission was detected through a band-pass (505–555 nm) ˆlter, as described in Materials and Methods.

However, we anticipate that genetic polymorphisms of Clinical SNP detection combined with functional evalu- ABCG2 and its inhibition by certain drugs may be relat- ations of ABCG2 would provide clear insights into the ed to photosensitivity or the risk of porphyria. molecular mechanisms underlying individual diŠerences Impact of genetic polymorphisms of ABCG2 on pho- in the photosensitivity of patients. tosensitivity: It is only recently that the physiological Potential risk of drug-induced photosensitization in importance of ABCG2 has been recognized in terms of the inhibition of ABCG2 function: Photosensitive porphyrin homeostasis and photosensitivity. Indeed, we skin reactions occur when human skin reacts abnormal- reported that the variants Q126stop, F208S, S248P, ly to ultraviolet radiation or visible light. The phenotype E334stop, and S441N are defective in the transport of of photosensitivity includes phototoxicity and pho- hematoporphyrin.4) The F489L variant showed im- toallergy. Phototoxic disorders occur in high incidence, paired transport activity. As demonstrated in the whereas photoallergic reactions are much less frequent present study, as well as in our previous one,4) Flp-In- in the human population. Several hundred substances, 293 cells expressing the F208S, S248P, S441N, and including chemicals and drugs, may invoke phototoxic F489L variants were sensitive to light when cells were and photoallergic reactions. Especially, drug photosen- treated with pheophorbide a. Thus, it is likely that hu- sitization is a major problem since these abnormal reac- mans with these alleles may be more susceptible to tions seriously limit or exclude the usage of drugs, such porphyrin-induced phototoxicity. as ‰uoroquinolones.32–34) The phototoxicity risk associ- While the protein expression level of Q141K was low- ated with the use of drugs may depend on both environ- er than that of WT in Flp-In-293 cells (Fig. 1A), its level mental and genetic factors. If the photosensitizing eŠect is considered to be su‹cient to extrude pheophorbide a of a chemical or drug is known before the patient's ex- from Flp-In-293/ABCG2(Q141K) cells. Since our in- posure, appropriate clinical management may help to vitro studies suggest that genetic polymorphisms of control the photosensitivity reactions. ABCG2 may be related to photosensitivity, clinical It is speculated that drugs that interact with ABCG2 research should be carried out to prove our concept. inhibit the porphyrin transport activity, and then cause 438 Ai TAMURA, et al.

Fig. 7. Schematic illustration of porphyrin biosynthesis and transport mediated by ABCG2 and ABCB6. Protoporphyrin IX is synthesized from succinyl CoA and glycine via d-aminolevulinic acid (ALA) in mitochondrial and cytoplasmic compartments. ABCB6 is involved in transport of coproporphyrinogen from the cytoplasm into mitochondria. Protoporphyrin IX is converted to heme by the ferrochelatase enzyme in mitochondria. Protoporphyrin IX can be transported out of cells via ABCG2 to maintain the intracellular porphyrin homeostasis. However, photosensitivity that is due to intracellular porphyrin accumulation can occur when ABCG2 is inhibited by a drug or its metabolites. unpredictable toxicity induced by cellular porphyrin ac- ABCG2-mediated porphyrin transport (Fig. 4B). This cumulation. In the present study, we have examined discrepancy may be due to the rapid metabolism of that hypothesis of the drug-induced phototoxicity, quercetin within living cells or its low penetration across which is associated with the inhibition of ABCG2. As the plasma membrane of Flp-In-293 cells. demonstrated in Fig. 4B and Fig. 5, imatinib and In humans, pheophorbide a-induced phototoxicity novobiocin strongly inhibited ABCG2-mediated por- has been reported after the ingestion of chlorella phyrin transport and signiˆcantly enhanced the cellular tablets,38) a dried algae preparation taken as a natural photosensitivity. It fact, side-eŠects related with pho- health supplement. Several pickled vegetables also con- tosensitization were reported in patients treated with im- tain substantial amounts of pheophorbide a and have atinib mesylate.35,36) The reported cutaneous side-eŠects been shown to cause phototoxicity in rats.38) In the hu- include skin rash, hypopigmentation, and depigmenta- man population, there are many incompletely under- tion, and those adverse eŠects were observed only in stood incidences of idiosyncratic food and drug hyper- long-term-treated patients, suggesting that cumulative sensitivities, including phototoxicity.39–41) Individuals dosages of imatinib represent the unique determinant carrying an allele with impaired porphyrin transport ac- for cutaneous photosensitization. In addition, ocular tivity may have a higher risk of drug-induced photosen- side-eŠects (e.g., periorbital edema and epiphora) were sitivity. In this context, it would be of great interest to also reported.37) The mechanism of those side-eŠects is investigate whether hyper-photosensitivities are related not known at present, but our data suggests a possible to deˆciencies in the function of human ABCG2. involvement of ABCG2 inhibition. In the case of quer- Concluding remarks: The present study provides cetin, on the other hand, its photosensitization potency evidence that ABCG2 plays a pivotal role in photosen- was weak (Fig. 5) in spite of its strong inhibition of sitivity. In addition, we have developed assay methods Genetic Polymorphisms of ABCG2 and Photosensitivity Risk 439 for the inhibition of porphyrin transport and cell-based The ABC transporter Bcrp1/ABCG2 is expressed in a photosensitivity. Such technologies may oŠer a great wide variety of stem cells and is a molecular determinant advantage for predicting photosensitive reactions before of the side-population phenotype. Nat. Med., 7: drugs are introduced in therapy or products made avail- 1028–1034 (2001). able on the market. High-speed screening methods for 7) Zhou, S., Morris, J. J., Barnes, Y., Lan, L., Schuetz, J. detection of drug-induced photosensitivity in vitro as D. and Sorrentino, B. P.: Bcrp1 gene expression is re- quired for normal numbers of side population stem cells well as pharmacogenomic testing for ABCG2 genotypes in mice, and confers relative protection to mitoxantrone of patients would provide a better assessment of the risk in hematopoietic cells in vivo. Proc. Natl. Acad. Sci. of photosensitivity. The relevant studies are ongoing in USA, 99: 12339–12344 (2002). our laboratory, and those results will be reported else- 8) Jonker, J. W., Buitelaar, M., Wagenaar, E., Van Der where. Valk, M. A., ScheŠer, G. L., Scheper, R. J., Plosch, T., Kuipers,F.,Elferink,R.P.,Rosing,H.,Beijnen,J.H. Acknowledgments: The authors thank Prof. Ichiro and Schinkel, A. H.: The breast cancer resistance protein Okura, Assistant Prof. Kenji Tabata, and Ms. Kaori protects against a major chlorophyll–derived dietary Takahashi (Graduate School of Bioscience and phototoxin and protoporphyria. Proc.Natl.Acad.Sci. Biotechnology, Tokyo Institute of Technology) as well USA, 99: 15649–15654 (2002). 9) Krishnamurthy, P., Ross, D. D., Nakanishi, T., Bailey- as Dr. Shun-ichiro Ogura (Shizuoka Cancer Center) for Dell, K., Zhou, S., Mercer, K. E., Sarkadi, B., Sorrenti- their kind help and advice in performing ‰uorescence no, B. P. and Schuetz, J. D.: The stem cell marker microscopy experiments. This study was supported, in Bcrp/ABCG2 enhances hypoxic cell survival through in- part, by the NEDO International Joint Research Grant teractions with heme. J. Biol. Chem., 279: 24218–24225 program ``International standardization of functional (2004). analysis technology for genetic polymorphisms of drug 10) Latunde-Dada, G. O., Simpson, R. J. and McKie, A. 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