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Banyu Tsukuba Research Institute in collaboration with Merck Research Laboratories, Ibaraki, 300-2611, Japan [H. Kom., H. Kot., Y. H., R. N., H. A., S. N.], and Clinical Development Institute, Banyu Pharmaceutical Co. Ltd., Tokyo 103-0026, Japan [M. M.]
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ABSTRACT |
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Introduction |
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TopABSTRACTIntroductionMaterials and MethodsResultsDiscussionREFERENCES |
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Two members of this family, P-gp (3 , 4) and MRP1 (5) , are well characterized and have been shown to be involved in resistance to multiple anticancer drugs, which is known as multidrug resistance. Both molecules transport a variety of anticancer drugs with unrelated structures and functions. In addition, increasing numbers of ABC proteins have recently been identified as associated with the drug resistance.
One such transporter, BCRP, is a half-transporter and has the highest homology to the Drosophila white gene family, which transports eye pigment precursor. This gene was isolated independently from human placenta (6) , from a cell line selected with doxorubicin and verapamil (7) , and from a cell line selected with mitoxantrone (8) , and the genes were designated as ABCP, BCRP, and MXR, respectively. A minute variation of amino acid sequences was observed in these clones. BCRP was reported to confer resistance to mitoxantrone (7) , and some cell lines with overexpression of this gene showed cross-resistance to camptothecin derivatives (9 , 10) . BCRP is localized in the plasma membrane (11 , 12) and thus is thought to be a novel type of drug efflux pump with a substrate spectrum different from those of P-gp and MRP1. However, the spectrum of cross-resistance varied, depending on the cell line and experimental system (7 , 9) , which suggests that complex mechanisms determine the substrate specificity.
Indolocarbazole compounds are a novel class of anticancer agents with a unique structure. These agents include the topo I inhibitor NB-506 (13) and J-107088 (14) and protein kinase inhibitors such as UCN-01 (15) . They show strong antitumor activity, and J-107088 and UCN-01 are currently under clinical evaluation. Moreover, study of NB-506 suggested that it is not a substrate of P-gp or MRP1 (13 , 16) , implying that the indolocarbazole topo I inhibitors are effective in multidrug-resistant cells.
On the other hand, there are unknown mechanisms controlling the intracellular concentration of indolocarbazole topo I inhibitors. Analysis of the cytotoxicity of NB-506 against various cell lines suggested that the tumor cell lines showed different levels of accumulation of NB-506 and that the accumulation levels were associated with the cellular sensitivity to the drug (13) . In addition, some NB-506-resistant cell lines showed reduced accumulation of NB-506 (17) . Therefore, we assumed that the putative transporter protein(s) is involved in the control of intracellular concentration of NB-506. To identify this molecule(s), we established several NB-506-resistant cell lines showing reduced accumulation of NB-506 and investigated changes in expression of thousands of genes by oligonucleotide microarray. In this way, we were able to identify BCRP as a transporter of these indolocarbazole drugs.
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Materials and Methods |
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Cell Culture and Isolation of the NB-506-resistant Cell Line.
The mouse fibroblast cell line LY, the human colon carcinoma cell line HCT116, and the human adenocarcinoma cell line HeLa were grown in DMEM supplemented with 10% FCS and 420 µg/ml of kanamycin. The human lung cancer cell line PC-13 was grown in RPMI 1640 supplemented with 10% FCS and 420 µg/ml of kanamycin. The NB-506-resistant LY cell lines were selected by continuous stepwise exposure to NB-506. The exposure of cells was started at 0.1 µM NB-506 for 2 weeks and then at 0.3 µM for 3 weeks. Finally, two clonal cell lines that grew in the presence of 0.3 µM NB-506 were isolated and designated as LY/NR1 and LY/NR2, respectively. The NB-506-resistant HCT116 and PC-13 cell lines were obtained by continuous exposure to 1.1 µM NB-506. These cell lines were further selected by culture with 20 µM NB-506, and the isolated cell lines were designated as HCT116/NR1 and PC-13/NR13, respectively.
Drug Sensitivity Test.
The cytotoxicity of anticancer drugs was determined as previously described (13)
using a sulforhodamine B dye-staining method (18)
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Drug Accumulation Assay.
For measurement of drug accumulation in the cells, the cells seeded at a density of 2 x 106 in 25-cm2 culture flasks were incubated in medium supplemented with 25 mM HEPES or HBSS buffer containing various concentrations of [14C]NB-506 at 37°C. For measurement of the accumulation in energy-depleted cells, the cells were pretreated with HBSS containing 1 mM DNP for 15 min, and then incorporation of NB-506 was performed for an additional 60 min in the presence of 1 mM DNP. After incorporation of the drug, the cells were washed with ice-cold PBS and treated with trypsin on ice. The suspended cells were counted, washed again with PBS, and solubilized in 0.2% Triton X-100. The lysates were centrifuged at 2000 x g for 10 min, the supernatants were collected, and remaining pellets were solubilized in 2 N NaOH to lyse the nuclei. The radioactivities of supernatant fractions and nuclear lysates were counted in a liquid scintillation counter. The values of the two fractions were added and normalized to the cell numbers to determine the amount of NB-506 accumulated per 107 cells.
Drug Efflux Assay.
For analyses of LY cell lines, the cells were preloaded with 50 µM [14C]NB-506 in the presence of DNP for 60 min. For analyses of BCRP-transfected PC-13 cells, the vector transfectant and the BCRP transfectants were preloaded with 10 and 75 µM [14C]J-107088, respectively, for 120 min. The cells were rapidly chilled on ice, washed with PBS after incorporation of the drugs, and incubated in the medium supplemented with 10% FCS for 15 min on ice to recover the cells from an energy-depleted condition. Then the cells were incubated in fresh medium supplemented with 10% FCS for the indicated times. After incubation, the cells were collected by trypsinization on ice, and the radioactivity remaining in the cells was measured as described above.
Oligonucleotide Microarray Analysis.
Total RNA was isolated using a RNeasy total RNA isolation kit (QIAGEN, Valencia, CA). Total RNA was biotin-labeled with T7 RNA polymerase (Enzo Diagnostics, Farmingdale, NY; Refs. 19, 20, 21
). Five oligonucleotide microarrays containing mouse probes for 
30,000 genes were used for hybridizations (Mu11KsubA, subB, Mu19KsubA, subB, and subC; Affymetrix, Santa Clara, CA). Hybridization and the array wash procedure were performed according to the Affymetrix Fluidics Station Mu11K and Mu19K protocols.
Microarray Data Analysis.
Each gene, either full-length or EST, is represented by 20 probes, 25 mer oligonucleotide, on the GeneChip. The expression levels of genes were measured by comparing the signal intensities of hybridization of these probe sets to the one-base, mismatch oligonucleotide probes by GeneChip software (average difference). Expression of the genes was scored as presence when the perfect match signal exceeded mismatch counterpart, and this parameter was called "absolute call." Experimental data were compared with those of parental cell lines as the baseline file in Comparison Analysis on the GeneChip software to evaluate the change. The data in this article represent the data sets that met the following criteria: (a) fold change value compare with the parental cell line >4.0; (b) genes with presence call on absolute call on NR2 lines; and (c) genes with given score of increase on differential call.
Northern Blots.
Total RNAs were extracted from each cell line with ISOGEN (Nippon Gene, Toyama, Japan), and mRNAs were isolated by oligodeoxythymidylate columns. Samples of 0.8 µg of poly(A)+ RNA or 8 µg of total RNA were separated on 1% agarose gel and transferred to nylon membranes. The membranes were probed with a 32P-labeled 425-bp fragment of mouse BCRP cDNA (1230–1654 of AF140218) or 269-bp fragment of human BCRP cDNA (161–429 of AF103796).
Cloning of BCRP cDNA from HeLa Cells.
The entire coding sequence of BCRP cDNA was generated by RT-PCR from total RNA of HeLa cells. The PCR amplification of the cDNA was performed with LA-Taq polymerase (Takara, Tokyo, Japan) using primers: 5'-CAAAAAGCTTAAGACCGAGCTCTATTAAGC and 5'-GAATTAAGGGGAAATTTAAGAAT, which were based on the AF103796 sequence with an added HindIII linker. These fragments were cloned into pcDNA3.1/V5-His-TOPO (Invitrogen, Carlsbad, CA).
Nucleotide Sequencing Analysis of Human BCRP.
The coding region of a cDNA clone was completely sequenced with a Dyedeoxy Terminator Cycle Sequencing Kit (Applied Biosystems, Foster City, CA). The sequence of the clone was deposited in GenBank under accession no. AB051855. For the analysis of nucleotide sequences of BCRP from human tissue, human BCRP was amplified by PCR with the primers 5'-CAAAAAGCTTAAGACCGAGCTCTATTAAGC and 5'-AGAGATCGATGCCCTGCTTTACCA from human placenta cDNA, which was a pool of samples from seven individuals, and human kidney cDNA, which was a pool of samples of eight individuals, in Multiple Tissue cDNA Panels (CLONTECH, Palo Alto, CA). The PCR fragments were directly sequenced using a Dyedeoxy Terminator Cycle Sequencing Kit (Applied Biosystems). We also performed direct sequencing analysis of PCR fragments derived from BCRP transcripts from HCT116/NR1 and PC-13/NR13 cells as described above.
Transfection Studies.
PC-13 cells were transfected with the expression vector pcDNA3.1/V5-His-TOPO containing the full-length BCRP cDNA or vector alone using Effecten Transfection Reagent (QIAGEN). After selection by culture in medium containing 0.2 mg/ml of G418, the emerging colonies were isolated with cloning rings. Clones with high-level expressions of BCRP were selected by Northern blot analysis.
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Results |
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TopABSTRACTIntroductionMaterials and MethodsResultsDiscussionREFERENCES |
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. The human resistant cell line, HCT116/NR1 displayed 2400- and 230-fold resistances to NB-506 and J-107088, respectively, and PC13/NR13 cells displayed more than 4300- and 140-fold resistances to NB-506 and J-107088, respectively, compared with the parental cells (Table 1)
. These cell lines showed low cross-resistances to topotecan and mitoxantrone.
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. The accumulation of NB-506 was considerably reduced in both resistant cell lines, and the accumulations in LY/NR1 and LY/NR2 were 50 and 14%, respectively, of that in parental cells. Thus, an increase in relative resistance appeared to be associated with a decrease in drug accumulation. The accumulation of J-107088 was also reduced in these two resistant cells in a manner similar to that of NB-506 (data not shown). Additional analysis suggested that reduced drug accumulation in the resistant cells was attributable to enhancement of energy-dependent efflux (Fig. 1B)
. First, NB-506 incorporation in the energy-depleted condition achieved by incubation with DNP reversed the accumulation of NB-506 in LY/NR1 and LY/NR2 to almost the same level as that in parental cells. Then, when the cells with incorporated NB-506 were incubated in drug-free media, 70 and 20% of the drug were effluxed within 15 min from LY/NR2 cells and LY/NR2 cells, respectively, whereas 100% of intracellular NB-506 remained in the parental cells. Therefore, the putative efflux pump for NB-506 seemed to be activated in the LY/NR2 cells. Reduced accumulation and enhanced efflux of NB-506 were also detected in the human resistant cell lines HCT116/NR1 and PC-13/NR13 (data not shown).
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. Among the 34,020 genes surveyed, 12 showed more than 4-fold higher expression in LY/NR2 cells, and only 2 of these showed more than 10-fold higher expression. The gene whose expression was most prominently elevated in LY/NR2 cells was BCRP, an ABC transporter involved in mitoxantrone resistance. The expression of this gene was 31-fold higher in the LY/NR2-resistant cells and was strikingly high as determined by the average difference change. Furthermore, BCRP was expressed 6.0-fold higher than in LY/NR1 cells, which showed an intermediate level of resistance and drug accumulation. Thus, the expression of BCRP was associated with the indolocarbazole resistance of NB-506-resistant LY cells.
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Overexpression of BCRP mRNA in the Resistant Cell Lines.
To confirm the results obtained by microarray analysis, we performed Northern blot analysis of the BCRP gene in the LY parental and resistant cells. As seen in Fig. 2
, BCRP showed a correlation of expression with resistance, as detected in the microarray. We also examined the expression in the human resistant cell lines HCT116/NR1 and PC-13/NR13 and found that human BCRP was selectively overexpressed in both NB-506-resistant cell lines compared with their parental cells. The fact that BCRP was overexpressed in all of the resistant cell lines strongly suggested that this gene is involved in NB-506 and J-107088 resistance.
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. Differences in codon 482 were observed in clones isolated in different laboratories, and the clone previously isolated from placenta by Allikmets et al. (6)
also encoded Arg at this position (Fig. 3B)
. To investigate which amino acids are found at codon 482 in BCRP of normal human tissues, we performed direct sequencing analysis of the BCRP transcript from human kidney and placenta. We found that codon 482 was AGG, encoding Arg, in BCRP from a human kidney cDNA sample, which was a pool of samples from eight individuals (Fig. 3A)
and from a human placenta cDNA (Fig. 3B)
. These results suggested that the BCRP transcript carrying Arg at codon 482 was expressed in human tissues. We also analyzed the nucleotide sequences of codon 482 of the BCRP transcripts expressed in the NB-506-resistant cell lines HCT116/NR1 and PC-13/NR13. As detected in human tissues, codon 482 of BCRP expressed in the resistant cells encoded Arg (Fig. 3B)
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, clones 2-2 and 2-3 showed high expression of BCRP, whereas a vector transfectant did not express it. The expression levels of BCRP in clones 2-2 and 2-3 were about 
and 
, respectively, of that in NB-506-selected resistant cell lines, PC13/NR13. The drug sensitivities of these clones were compared with that of a vector transfectant (Fig. 4A)
. Clones 2-2 and 2-3 showed 22- and 9-fold higher resistance to NB-506, respectively, than the vector-transfected cells. Similarly, 2-2 and 2-3 cells showed about 10-fold higher resistance to J-107088. On the other hand, these clones were not resistant to camptothecin, topotecan, or etoposide. Unexpectedly, both cell lines overexpressing BCRP were not resistant to mitoxantrone. Clones 2-2 and 2-3 showed 0.42- and 0.60-fold higher resistance, respectively, compared with the vector transfectant. We also tested the sensitivities to doxorubicin and paclitaxel. The sensitivities of clones 2-2 and 2-3 were similar to those of vector transfectant (data not shown).
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. The 60% of the intracellular J-107088 was transported out after a 30-min incubation in BCRP-transfected clone 2-2, whereas only 20% of the drug was transported out in vector-transfected cells. These results indicated the BCRP-mediated transport of J-107088. |
Discussion |
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TopABSTRACTIntroductionMaterials and MethodsResultsDiscussionREFERENCES |
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To identify the putative transporter by gene expression analysis, we prepared three highly resistant cell lines with reduced accumulation and enhanced efflux. Of these cell lines, two mouse cell lines were selected for subsequent gene expression analysis using oligonucleotide microarrays, because the degrees of resistance of the mouse highly resistant LY/NR2 cells and moderately resistant LY/NR1 cells were correlated with their degrees of activation of efflux. Therefore, it was expected that they would reflect the expression levels of the putative efflux pump. We found 31- and 6-fold elevations of BCRP expression in the highly and moderately resistant cells, respectively. The 31-fold increase of BCRP expression in the LY/NR2 cells was so high that it was easily distinguished from other up-regulated genes.
The involvement of this gene in NB-506 and J-107088 resistance was confirmed by its selective overexpression in human resistant cell lines and by analysis of BCRP transfected clones. The HCT116/NR1 and PC-13/NR13 cell lines showed high efflux of these compounds like mouse resistant cells (data not shown). Thus, the mechanism of the resistance is probably efflux of these compounds mediated by human BCRP.
The two resistant cell lines showed little or no cross-resistance to the topo I poison camptothecin and to substrates of P-gp or MRP1, such as doxorubicin, etoposide, paclitaxel, and vincristine (3 , 5) , suggesting little contribution of these known resistance factors to indolocarbazole resistance.
Analyses of BCRP-transfected PC-13 stable cell lines confirmed the involvement of BCRP in the resistance. The transfectant clone 2-2 showed 22- and 17-fold resistance to NB-506 and J-107088, respectively, and enhanced efflux activity of J-107088. These results strongly suggested that BCRP transports these indolocarbazole compounds and confers resistance by decreasing the intracellular concentrations of these compounds.
Our results demonstrated that BCRP expression did not confer resistance to mitoxantrone. This is in contrast with previous findings, because BCRP was reported to confer resistance to mitoxantrone when it was introduced into breast cancer cell lines (7) and because its overexpression was detected in several mitoxantrone-resistant cell lines (7 , 8 , 22 , 23) . Although the NB-506-resistant cell lines HCT116/NR1 and PC-13/NR13 showed 9.6- and 4.9-fold mitoxantrone-resistance, respectively, the degree of resistance was limited compared with those to NB-506 and J-107088. Furthermore, the relative resistance of PC-13 transfectant 2-2 cells to mitoxantrone was 0.42 despite their considerable expression of BCRP.
Several explanations are possible for this difference: (a) The difference in substrate specificity might be explained by the small difference in the amino acid sequences in BCRP. Our isolated BCRP differed from one isolated from MCF-7/AdVp by a single amino acid at codon 482. As previously reported, codon 482 varies in clones isolated in different laboratories (Fig. 3B)
. BCRP isolated from MCF-7/AdVp cells, which was previously shown to confer resistance to mitoxantrone, carries Thr at codon 482 (7)
. On the other hand, our BCRP clone carries Arg at codon 482, as detected in a clone from normal placenta (6)
. Codon 482 is located in the putative transmembrane 3-region (6)
, and analysis of MDR1 suggested that transmembrane regions of ABC transporters are likely to participate in substrate recognition (3)
. Therefore, it is possible that amino acid substitution at this position alters the substrate specificity of BCRP. Although these substitutions may reflect the occurrence of natural polymorphism, they may also be mutations occurring in the course of drug selection of cultured cells. Such mutations were reported in the case of MDR1 and were shown to affect substrate specificity (24)
. To clarify this point, we performed sequence analysis of the BCRP transcript in human tissues and demonstrated that the BCRP expressed in human tissue carries Arg at codon 482. Therefore, the characteristics of BCRP obtained in our analysis were not those of mutant protein and were likely to reflect the physiological function of this protein. (b) The different cross-resistance pattern is explained by putative cellular factors affecting the transport activity mediated by BCRP. Because the cell line with introduced BCRP was a lung cancer cell line in our study and a breast cancer cell line in a previous study, the different genetic background of the two cell lines are also likely to cause different cross-resistant patterns by the BCRP. This cellular factor might be either another half-transporter that functions by dimerizing with BCRP or endogeneous small molecules to be cotransported with the substrates (25)
. (c) Cellular metabolism or modification of the drug is also a possible factor for the transport mechanism of the drug. MRP1 (25
, 26)
and MRP2 (27)
are known to recognize the glutathione-conjugated compounds and glucuronidated compounds as substrates, respectively. Such metabolic pathways might contribute to the resistance mediated by BCRP in some cell lines.
Our results also did not show resistance to topotecan of BCRP-transfected PC-13 cell lines. This is consistent with a previous finding that the forced expression of BCRP in breast cancer cell lines resulted in little or no resistance to topotecan (28) and further supported the notion that BCRP alone is not sufficient for resistance to topotecan.
In conclusion, we demonstrated involvement of the ABC half-transporter BCRP in the indolocarbazle topo I poisons, NB-506 and J-107088. Our results strongly suggested that BCRP is an efflux pump of NB-506 and J-1070788. Although the expression of drug efflux pumps causes resistance of tumor cells to chemotherapeutic drugs, they also play a physiological role in the host defense mechanism against toxic xenobiotics by preventing the entry of the drugs into important organs (29 , 30) . Thus, its expression determines the toxicity to the drugs as well as tumor sensitivity to the drug. The BCRP transcript was detected in various organs, although its expression was low, except in the placenta (7) . On the other hand, expression of this protein in a panel of human tumor was very low or undetectable (11) , although additional studies are required to conclude the expression status in human cancer. Considering these points, it is possible that BCRP contributes to reduction of the toxicity of indolocarbazole anticancer agents such as J-107088, which have a wide therapeutic window (31) .
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 To whom requests for reprints should be addressed, at Banyu Tsukuba Research Institute, 3 Okubo, Tsukuba-shi, Ibaraki, 300-2611, Japan. 
2 The abbreviations used are: ABC, ATP-binding cassette; P-gp, P-glycoprotein; MRP, multidrug resistance-associated protein; BCRP, breast cancer-resistant protein; MXR, mitoxantrone resistance; ABCP, placenta-specific ABC transporter; MDR, multidrug resistance; topo, topoisomerase; DNP, 2,4-dinitrophenol; EST, expressed sequence tag; GAPDH, glyceraldehyde-3-phosphate dehydrogenase.
Received 12/ 4/00. Accepted 2/ 7/01.
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, LY/NR1 cells; 
, LY/NR2 cells. B, effect of energy depletion on the accumulation and efflux of NB-506 in drug-resistant LY cells. For study of the effect of energy depletion on the accumulation, 50 µM NB-506 was incorporated into cells for 60 min in the absence (
) of DNP as described in "Materials and Methods." For determination of drug efflux from the cells, they were incubated with 50 µM [14C]NB-506 for 60 min in energy-depleted conditions, washed, and then incubated in drug-free medium for 15 min (
). Bars, SE from three independent experiments.
) were preloaded with 10 and 75 µM [14C]J-107088, respectively, for 120 min. The J-107088 remaining in the cells after incubation is indicated by the percentage of the count at 0 time. Bars, SE from three independent experiments.