A SNP (rs34678167, 805 C>T, P269S) in the Human ABC Transporter ABCG2 Gene increases ABCG2-mediated Drug Resistance

A SNP (rs34678167, 805 C>T, P269S) in the Human ABC Transporter ABCG2 Gene increases ABCG2-mediated Drug Resistance | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article A SNP (rs34678167, 805 C>T, P269S) in the Human ABC Transporter ABCG2 Gene increases ABCG2-mediated Drug Resistance Shiori Sato, Megumi Tsukamoto, Kayo Ozawa, Ritsuko Imai, Hiroshi Nakagawa This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6704269/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract ABCG2 (BCRP/MXR) is an ATP-binding cassette transporter that contributes to multidrug resistance in cancer cells by extruding a wide range of substrates, including anticancer agents. Although several non-synonymous single-nucleotide polymorphisms (SNPs) in the ABCG2 gene have been characterized, the functional consequences of many remain unclear. This study investigated the impact of SNP rs34678167 (805 C>T, P269S) on ABCG2 expression and drug resistance. Using the Flp-In™ system, we established Flp-In-293 cell lines that stably express ABCG2 (P269S) variant. Quantitative real-time PCR and Western blotting revealed that the P269S variant resulted in mRNA and protein expression levels comparable to those of WT ABCG2. Drug resistance was assessed by MTT assay using mitoxantrone and SN-38. Cells expressing ABCG2 (P269S) exhibited significantly higher EC 50 values than those expressing WT ABCG2: 3.0-fold for mitoxantrone and 3.8-fold for SN-38 (p < 0.01). These results suggest that the P269S substitution enhances ABCG2-mediated drug resistance without affecting expression levels. This effect may be due to altered substrate recognition, transporter activity, or intracellular localization of the protein. Our findings underscore the importance of functionally validating genetic variants of ABCG2 and highlight the potential clinical relevance of rs34678167 in predicting drug response. This knowledge could contribute to the development of more effective therapeutic strategies tailored to the patient’s genetic background. ATP-binding cassette (ABC) transporter ABCG2 BCRP MXR Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Introduction Chemotherapy is one of the main strategies used for cancer treatment. The efficacy of chemotherapy varies between patients and cancer types, with genetic polymorphisms and mutations playing key roles. Therefore, understanding the effects of genetic polymorphisms and mutations in patients with cancer is critical for achieving successful chemotherapy outcomes. Some ABC transporters regulate drug concentration in the body and within cells by actively exporting substrates across biological membranes. ABCG2 (BCRP/MXR) was identified in a mitoxantrone-resistant human colorectal cancer cell line and an anthracycline-resistant breast cancer cell line (Allikmets et al. 1998 ; Doyle et al. 1998 ; Miyake et al. 1999 ), following the discovery of ABCB1 (Juliano and Ling 1976 ; Ueda et al. 1986 ) and ABCC1 (Cole et al. 1992 ), and also has been shown to extrude anticancer drugs (Maliepaard et al. 1999 ). ABCG2, a 72 kDa glycoprotein composed of 655 amino acids, features a single ATP-binding domain at the N-terminus and a transmembrane domain at the C-terminus (Fig. 1 ) and functions as a homodimer (Kage et al. 2002 ). ABCG2 facilitates the transport of structurally diverse endobiotics, such as dehydroepiandrosterone sulfate (Kondo et al. 2004 ), estrone-3-sulfate (Kondo et al. 2004 ; Suzuki et al. 2003 ), lumichrome (Millan-Garcia et al. 2024 ), porphyrins (Tamura et al. 2006b ; Zattoni et al. 2022 ; Zhou et al. 2005 ), and uric acid (Woodward et al. 2009 ). ABCG2 overexpression has been associated with increased resistance to several anticancer drugs, including camptothecin and its analogs (Brangi et al. 1999 ; Kage et al. 2002 ; Kawabata et al. 2001 ; Maliepaard et al. 1999 ; Tamura et al. 2006b ), epidermal growth factor receptor tyrosine kinase inhibitors (Breedveld et al. 2005 ; Burger et al. 2004 ), mitoxantrone (Kage et al. 2002 ; Mitomo et al. 2003 ; Wakabayashi et al. 2006 ), and methotrexate (Chen et al. 2003 ; Volk et al. 2002 ). In addition, recent clinical studies have indicated that ABCG2 expression levels and genetic variants can be prognostic markers for the progression and treatment response of cancers such as glioma (Raguz et al. 2024 ), hepatoma (Huang et al. 2022 ), and Non-Small Cell Lung Cancer (Wang et al. 2021 ). Our previous studies have demonstrated that amino acid substitutions can influence the intracellular stability and substrate specificity of ABCG2 (Tamura et al. 2006a ; Tamura et al. 2006b ). In addition, our previous studies have shown that 18 non-synonymous SNPs in ABCG2 affect the affinity of ABCG2 for porphyrins and methotrexate (Tamura et al. 2006b ). We have also reported that seven nonsynonymous SNPs affect the intracellular protein expression levels of ABCG2 and ABCG2-dependent anticancer drug resistance in cells (Tamura et al. 2006a ). However, the impact of approximately 100 SNPs in ABCG2 remains unclear. In this study, we present new and significant findings regarding the impact of SNP rs34678167 (805 C > T, P269S) on anticancer drug resistance in cells dependent on ABCG2. Materials and methods Construction of pcDNA5/FRT containing ABCG2 (P269S) variant cDNA To generate the expression vector pcDNA5/FRT/ABCG2 (P269S), we used site-directed mutagenesis based on the sequence information of the ABCG2 (P269S) variant obtained from the NCBI dbSNP database as previously described (Nakagawa et al. 2008 ; Tamura et al. 2006a ; Tamura et al. 2006b ; Wakabayashi et al. 2006 ). Site-directed mutagenesis was performed using PrimeSTAR® Max DNA polymerase (Takara Bio Inc., Otsu, Japan) and specific primers to prepare non-synonymous SNP variant ABCG2 (P269S) cDNA. After PCR for site-directed mutagenesis, the reaction mixture was treated with the Dpn I endonuclease to eliminate the original template plasmid pcDNA5/FRT/ABCG2 (WT) vectors. The sequences of the resulting amplicons were verified using an Applied Biosystems 3130 and 3130xl Genetic Analyzer (Applied Biosystems, Foster City, CA). Cell culture condition Flp-In-293 cells (Invitrogen, Thermo Fisher Scientific, Waltham, MA, USA) were cultured in high-glucose Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 10% heat-inactivated fetal bovine serum (FBS), 4 mM L-glutamine, 100 U/mL penicillin, 100 µg/mL streptomycin, 250 ng/mL amphotericin B, and 100 mg/mL zeocin. To select for Flp-In-293/ABCG2 (P269S) cells, 50 mg/mL hygromycin B was used instead of 100 mg/mL zeocin. The cells were maintained in a humidified incubator at 37°C with 5% CO₂, and their viability was assessed by trypan blue exclusion assay using a hemocytometer. Living cells were used in all the experiments. Zeocin was purchased from Invitrogen (Thermo Fisher Scientific, Waltham, MA), while an Antibiotic-Antimycotic Mixed Stock Solution(100×) (mixture of 10,000 U/mL penicillin, 10,000 µg/mL streptomycin, and 25,000 ng/mL amphotericin B), and high-glucose DMEM were purchased from Nacalai Tesque, Inc. (Kyoto, Japan). FBS was purchased from Equitech-Bio, Inc. (Kerrville, TX, USA). Generation of cells expressing ABCG2 (P269S) variant Flp-In-293 cells were plated in 35-mm dishes (TrueLine, Baton Rouge, LA) at a density of 1 × 10 6 cells per dish and precultured for 24 h. Cells were then co-transfected with pcDNA5/FRT/ABCG2 (P269S) and the Flp recombinase expression plasmid pOG44 vectors using Lipofectamine™-2000 according to the manufacturer's protocol. After transfection, the cells were selected with 50 mg/mL hygromycin B, and the resulting hygromycin B-resistant colonies were collected, subcultured, and used as Flp-In-293/ABCG2 (P269S) cells. Lipofectamine™-2000 and pOG44 were purchased from Invitrogen (Thermo Fisher Scientific, Waltham, MA). Hygromycin B was obtained from Nacalai Tesque, Inc. (Kyoto, Japan). Total RNA preparation and first-strand cDNA synthesis Flp-In-293/ABCG2 (WT) and Flp-In-293/ABCG2 (P269S) cells were seeded in 35-mm dishes (TrueLine, Baton Rouge, LA) at a density of 1 × 10 6 cells per dish and precultured for 3 days. Cells were collected with culture medium into 1.5 mL tubes, centrifuged (300 × g, 5 min, 4°C), and washed twice with 1 mL PBS(-). The resulting cell pellets were suspended in 600 µL of lysis/binding buffer from the High Pure RNA Isolation Kit (Roche Diagnostics, Mannheim, Germany) and stored at -80°C until total RNA preparation was performed with a High-Purity RNA Isolation Kit (Roche Diagnostics). Total RNA concentration was measured using a DU640 spectrophotometer (Beckman Coulter, Fullerton, CA, USA). Total RNA was reverse transcribed using a High-Capacity cDNA Reverse-Transcription Kit (Thermo Fisher Scientific Inc., Waltham, MA) according to the manufacturer's instructions. Quantitative evaluation of ABCG2 mRNA levels The expression levels of ABCG2 mRNA were determined using a 7500 Fast Real Time-PCR System (Applied Biosystems), where GoTaq® DNA Polymerase GoTaq® qPCR Master Mix, 2× (Promega, Tokyo, Japan) and primers for ABCG2 or GAPDH were used. The GoTaq® qPCR Master Mix, 2× was purchased from Promega (Tokyo, Japan). The primer sets for ABCG2 (HA204957-F and HA204957-R) and GAPDH (10000459 and 20000459) were purchased from Takara Bio Inc. (Otsu, Japan). MTT assay Flp-In-293/ABCG2 (WT) and Flp-In-293/ABCG2 (P269S) cells were plated in 96-well plates (Thermo Fisher Scientific) at a density of 5 × 10 3 cells/well and cultured for 24 h before exposure to different concentrations of mitoxantrone and SN-38 for 72 h. The drug concentrations ranged from 0 (control) to 1000 nM for mitoxantrone and 100 nM for SN-38. After drug exposure, cells were incubated with 500 µg/mL MTT for 3 h, lysed with 10% sodium dodecyl sulfate (SDS), and incubated overnight at 37°C in an atmosphere containing 5% CO 2 . Absorbance was measured at 570 nm using a Thermo Labsystems Multiskan Jax spectrophotometer (Thermo Fisher Scientific) with a reference wavelength of 630 nm to evaluate the amount of formazan metabolized from MTT in each well. Cell viability was calculated as a percentage of the untreated group (control) according to the absorbance at 570 nm with a reference wavelength of 630 nm. EC 50 values, representing the drug concentration required to reduce cell viability by 50%, were determined from the survival curves, and the cytotoxicity of the anti-cancer drugs (mitoxantrone and SN-38) was assessed. Mitoxantrone and SN-38 were purchased from Wako Pure Chemical Industries, Ltd. (Osaka, Japan) and were graciously provided by Yakult Honsha Co. (Tokyo, Japan), respectively. 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) was obtained from Sigma-Aldrich Co. (St. Louis, MO). Cell lysate preparation for SDS-PAGE Flp-In-293/ABCG2 (WT) and Flp-In-293/ABCG2 (P269S) cells were plated in 35-mm dishes (TrueLine, Baton Rouge, LA) at a density of 1 × 10 6 cells per dish and precultured for 3 days. The cells were harvested with culture medium in 1.5 mL tubes, centrifuged (300 × g, 5 min, 4°C), and washed twice with 1 mL PBS(-). The resulting cell pellets were resuspended and lysed in a lysis buffer containing 50 mM Tris-HCl (pH 7.6), 5 mM EDTA (pH 8.0), 120 mM NaCl, 1% Triton X-100, 1 mM DTT, protease inhibitors, and phosphatase inhibitors. The cell lysate samples were homogenized by passing through a 27G-needle ten times. After centrifugation of the homogenate at 3,000 rpm at 4°C for 10 min, the supernatants were collected as cell lysates. The protein concentration of the supernatant was determined using bovine serum albumin as a standard by the Bradford method. Following this, 50 µg of the cell lysate was treated with PNGase F for 10 min at 37°C to remove glycomoieties from the proteins. The resulting cell lysates were mixed with SDS-PAGE sample buffer solution containing 10% (v/v) 2-mercaptoethanol (Daiichi pure chemicals Co., Ltd., Tokyo, Japan) and stored at -30°C until needed for western blotting. Evaluation of expression levels of ABCG2 Cell lysates were prepared from each cell group in triplicate and aliquots of 5 µg were mixed separately for each cell group as a cell lysate mixture. Fractionation of the mixtures was performed by SDS-PAGE on a 7.5% polyacrylamide gel, followed by transfer to nitrocellulose membranes (GE Healthcare UK Ltd., Bucks, UK). Western blotting was performed after the blocking process, in which the membranes were immersed in TBST (50 mM Tris-HCl, 150 mM NaCl, and 0.05% (v/v) Tween 20) containing skimmed milk powder at 5% (w/v) at room temperature for more than 1 h and then at 4°C overnight. After this procedure, the membranes were rinsed with TTBS and exposed to a 1:1000-diluted monoclonal anti-ABCG2 antibody (BXP-21; ALEXIS Co., Lausen, Switzerland) or anti-GAPDH antibody (anti-GAPDH-Clone 6C5 mouse monoclonal, IgG2b; American Research Products, Inc., Waltham, MA, USA) in TBST containing skimmed milk powder at 5% (w/v) with gentle shaking for 1 h at room temperature. After primary antibody treatment, the membranes were rinsed with TTBS and exposed to 1:1000-diluted HRP-conjugated anti-mouse IgG antibodies (Cell Signaling Technology, Inc., Danvers, MA, USA) in 5% (w/v) skim milk powder-containing TBST with gentle shaking for 1 h at room temperature. Blots were visualized using Western Lighting Chemiluminescent Reagent Plus (PerkinElmer Life and Analytical Sciences, Boston, MA, USA) and detected using WSE-6100 LuminoGraph I (Atto Corp., Tokyo, Japan). The ImageJ software was used to quantify the signal intensities associated with ABCG2 or GAPDH (Wayne Rasband, Bethesda, MD, USA). Statistical analysis JSTAT (version 20.0), developed by Masato Sato, was used for statistical analyses using one-way ANOVA and Tukey's honestly significant difference (HSD) test. In all analyses, statistical significance was set at p T, P269S) on ABCG2 protein expression and ABCG2-dependent drug resistance, we established Flp-In-293 cells expressing ABCG2 (P269S) (Figs. 1 and 2 ). Total RNA was extracted from Flp-In-293/Mock, Flp-In-293/ABCG2 (WT), and Flp-In-293/ABCG2 (P269S) cells, and their quality was evaluated by comparing the A260/A280 ratios and qPCR threshold cycles across samples. As the quality of the total RNA produced was consistent, the levels of ABCG2 mRNA were normalized to those of GAPDH, and the successful integration of ABCG2 (P269S) cDNA at the FRT site within the genomic DNA was confirmed. The results of qPCR showed that ABCG2 mRNA levels in ABCG2 cDNA-transfected cells [Flp-In-293/ABCG2 (WT) and Flp-In-293/ABCG2 (P269S) cells] were more than 100 times higher than those in Flp-In-293/Mock cells (Fig. 3 ). ABCG2 mRNA levels in Flp-In-293/ABCG2 (P269S) cells were comparable to those in Flp-In-293/ABCG2 (WT) cells, indicating that the Flp-In™ system functioned as expected. Western blotting was performed to determine ABCG2 protein expression levels in Flp-In-293/ABCG2 (WT) and Flp-In-293/ABCG2 (P269S) cells. PNGase F was used to remove glycosylation and accurately assess the protein expression levels. As shown in Fig. 4 , ABCG2 protein levels corresponded with ABCG2 mRNA expression, confirming significantly higher ABCG2 expression in Flp-In-293/ABCG2 (WT) and Flp-In-293/ABCG2 (P269S) cells than in Flp-In-293/Mock cells. In contrast, ABCG2 protein levels in Flp-In-293/ABCG2 (P269S) cells were similar to those in Flp-In-293/ABCG2 (WT) cells. Evaluation of drug resistance in cells expressing ABCG2 (P269S) To investigate the impact of rs34678167 (805 C > T, P269S) on ABCG2-mediated drug resistance, we performed an MTT assay to assess cellular responses to mitoxantrone and SN-38 as detailed in the Materials and Methods section. The cells were treated with varying concentrations of these anticancer drugs, and their EC 50 values were determined. Consistent with the results of previous studies (Tamura et al. 2006a ; Tamura et al. 2006b ; Wakabayashi et al. 2006 ), Flp-In-293/ABCG2 (WT) cells exhibited increased resistance to mitoxantrone and SN-38 compared with Flp-In-293/Mock cells (Fig. 5 and Table 1 ). The EC 50 values for mitoxantrone and SN-38 were 4.6- and 5.5-fold higher, respectively, in Flp-In-293/ABCG2 (WT) cells than in Flp-In-293/Mock cells (Table 1 ). Similarly, Flp-In-293/ABCG2 (P269S) cells showed significant resistance to mitoxantrone and SN-38 compared with Flp-In-293/Mock cells (Fig. 5 and Table 1 ). EC 50 values indicated that Flp-In-293/ABCG2 (P269S) cells were 3.0- and 3.8-fold more resistant to mitoxantrone and SN-38, respectively, than Flp-In-293/ABCG2 (WT) cells (Table 1 ). These findings suggest that rs34678167 (805 C > T, P269S) enhances ABCG2-mediated drug resistance, although it does not significantly alter ABCG2 mRNA and protein expression levels. Table 1 Anticancer drug registance profiles (EC 50 ) of the cells. Compounds EC 50 (nM) Mock cells ABCG2 (WT) cells ABCG2 (P269S) cells Mitoxantrone 4.1 ± 1.4 18.8 ± 3.8 * 55.8 ± 10.0 *, ** SN-38 3.1 ± 1.5 16.9 ± 3.8 * 64.3 ± 6.6 *, ** SN-38, 7-ethyl-10-hydroxy-camptothecin. EC 50 values from each of the five experiments are shown. Data are expressed as mean ± S.D. (n = 5). One-way ANOVA and Tukey's HSD test were employed for statistical analysis. [*; p < 0.05 compared to the Mock group, **; p < 0.05 compared with the wild-type (WT)]. Discussion Generation of human ABCG2 (P269S)-expressing cells using the Flp-In™ system The emergence of drug resistance in cancer cells and its inter-individual variability have been attributed, in part, to the overexpression of ABC transporters and the presence of specific single-nucleotide polymorphisms (SNPs) in their genes (Nakagawa et al. 2008 ; Tamura et al. 2006a ; Tamura et al. 2006b ; Wakabayashi et al. 2006 ). Our previous studies demonstrated that several non-synonymous SNPs in ABCG2 , such as rs2231137 (V12M), rs2231142 (Q141K), rs1061018 (F208S), rs3116448 (S248P), rs1354553769 (S441N), and rs192169063 (F489L), can influence drug resistance in cells expressing ABCG2 (Tamura et al. 2006a ). In this study, we used the Flp-In™ system to generate cells expressing the rs34678167 (P269S) variant of ABCG2. This system enables site-specific integration of a single copy of cDNA at the FRT site in the telomeric region of chromosome 12 in Flp-In-293 cells (Tamura et al. 2006a ; Wirth and Hauser 2004 ), providing a controlled platform for quantitatively assessing the functional consequences of individual SNPs on drug resistance. Although we did not directly confirm the genomic integration site and copy number of the cDNA in Flp-In-293/ABCG2 (P269S) cells, the comparable levels of ABCG2 mRNA expression between ABCG2 (P269S)- and (WT)-expressing cells (Fig. 3 ) suggested successful and equivalent integration. Although it remains possible that exogenous expression of ABCG2 (P269S) might influence the expression of other ABC transporters, this model remains valid for evaluating the specific impact of rs34678167 (805 C > T, P269S) on ABCG2-mediated drug resistance. Drug resistance profiles of the ABCG2 (P269S)-expressing cells ABCG2 has been shown to mediate the efflux of a broad range of substrates, including anticancer drugs such as camptothecin and its analogs (Brangi et al. 1999 ; Kage et al. 2002 ; Maliepaard et al. 1999 ; Tamura et al. 2006b ), epidermal growth factor receptor tyrosine kinase inhibitors (Breedveld et al. 2005 ; Burger et al. 2004 ), mitoxantrone (Kage et al. 2002 ; Mitomo et al. 2003 ; Wakabayashi et al. 2006 ), and methotrexate (Chen et al. 2003 ; Volk et al. 2002 ), as well as endogenous compounds like dehydroepiandrosterone sulfate (Kondo et al. 2004 ), estrone-3-sulfate (Kondo et al. 2004 ; Suzuki et al. 2003 ), lumichrome (Millan-Garcia et al. 2024 ), porphyrins (Tamura et al. 2006b ; Zattoni et al. 2022 ; Zhou et al. 2005 ), and uric acid (Woodward et al. 2009 ). As shown in Fig. 5 and Table 1 , our results confirmed that Flp-In-293/ABCG2 (WT) cells exhibited higher resistance to mitoxantrone and SN-38 than Flp-In-293/Mock cells, consistent with the findings of previous studies (Brangi et al. 1999 ; Maliepaard et al. 1999 ; Mitomo et al. 2003 ; Tamura et al. 2006a ; Tamura et al. 2006b ; Wakabayashi et al. 2006 ). Likewise, Flp-In-293/ABCG2 (P269S) cells also showed increased resistance to these drugs, validating the functionality of transfected ABCG2 (P269S) (Fig. 5 and Table 1 ). Several previous studies have assessed the transport activity of ABCG2 (P269S) using prepared membrane vesicles. While some studies have reported similar transport activities between ABCG2 (P269S) and ABCG2 (WT) prepared from HEK293 cells for substrates such as estrone-3-sulfate and methotrexate (Kondo et al. 2004 ) (Higashino et al. 2017 ), others have observed reduced transport activity of ABCG2 (P269S) prepared from Sf9 cells (Lee et al. 2007 ). These inconsistent findings suggest that the substrate specificity and transport activity of ABCG2 (P269S) may vary depending on the experimental condition and the cellular protein expression system. In this study, we used the Flp-In™ system to achieve a uniform cellular background, enabling a more accurate comparison between cells expressing ABCG2 (WT) and those expressing ABCG2 (P269S) compared to conventional transfection methods with the pcDNA3.1 vector. Our results showed that despite similar mRNA and protein expression levels, Flp-In-293/ABCG2 (P269S) cells exhibited a distinct drug resistance profile compared to Flp-In-293/ABCG2 (WT) cells. This implies that the amino acid substitution derived from rs34678167 (805 C > T, P269S) may affect the functional properties of ABCG2, such as substrate recognition, intracellular localization or ATP binding affinity, thereby altering intracellular drug accumulation as suggested by other substitutions [ABCG2 (V12M, Q141K, F208S, S248P, F431L, S441N, or F489L)] (Nakagawa et al. 2008 ; Tamura et al. 2006b ). Further studies are required to elucidate the molecular basis of these functional differences. Investigations into the subcellular localization, ATPase activity, and transport kinetics of ABCG2 (P269S), as well as the direct measurement of intracellular drug accumulation, will be crucial for uncovering the precise mechanisms underlying altered drug resistance. Conclusion In this study, we employed a quantitative and site-specific expression system to evaluate the impact of SNP rs34678167 (805 C > T, P269S) on the human ABCG2 gene on drug resistance profiles of cells. Using the Flp-In™ system, we were able to generate isogenic cell lines expressing ABCG2 (WT), ABCG2 (P269S), and others (Nakagawa et al. 2008 ; Tamura et al. 2006a ; Tamura et al. 2006b ; Wakabayashi et al. 2006 ), thereby eliminating variability due to differences in integration sites or copy number. Our findings demonstrated that rs34678167 (805 C > T, P269S) conferred enhanced resistance to the ABCG2 substrates mitoxantrone and SN-38, despite exhibiting mRNA and protein expression levels comparable to those of ABCG2 (WT). These results suggest that the amino acid substitution at position 269 may influence the functional properties of ABCG2, such as substrate interaction or ATP hydrolysis efficiency, rather than affect its expression. This study contributes to a deeper understanding of how non-synonymous SNPs in ABCG2 can modulate drug resistance phenotypes, and underscores the importance of considering such genetic variations in the context of personalized cancer chemotherapy. Ultimately, these insights may support the development of effective therapeutic strategies and diagnostic tools tailored to individual genetic profiles. Abbreviations ABC, ATP-binding cassette; BCRP, breast cancer resistance protein; BSA, bovine serum albumin; D-MEM, high-glucose Dulbecco’s modified Eagle’s medium; DTT, dithiothreitol; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; FCS, fetal calf serum; FRT, Flp recombination target; HRP, horseradish peroxidase; MTT, 3-(4,5-dimethyl-2-thiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide; MXR, mitoxantrone resistance-associated protein; PBS, phosphate-buffered saline without both Ca 2+ and Mg 2+ ; RT-PCR, reverse transcriptase-polymerase chain reaction; SNP, single nucleotide polymorphism; TBS, Tris-buffered saline; TTBS, TBS with 0.05% (v/v) Tween 20; WT, wild type. Declarations Author Contributions : conceptualization, H.N.; validation, S.S., M.T., and K.O.; formal analysis, S.S., M.T., and K.O.; investigation, S.S., M.T., and K.O.; resources, H.N.; data curation, H.N.; writing—original draft preparation, S.S.; writing—review and editing, H.N.; visualization, S.S. and R.I.; supervision, H.N.; project administration, H.N.; funding acquisition, H.N. All authors have read and agreed to the published version of the manuscript. Funding : This research was funded by Aichi Cancer Research Foundation and Chubu University Grant D (DII28IIM02). Megumi Tsukamoto is a Research Associates at the Chubu University, Japan. Acknowledgments : The authors thank Yakult Honsha Co., Ltd. (Tokyo, Japan) for providing SN-38. Conflicts of Interest : The authors declare no conflict of interest. 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(2008) Ubiquitin-mediated proteasomal degradation of non-synonymous SNP variants of human ABC transporter ABCG2 Biochem J 411:623-631 Raguz M, Tarle M, Muller D, Tomasovic-Loncaric C, Chudy H, Marinovic T, Chudy D (2024) ABCG2 Expression as a Potential Survival Predictor in Human Gliomas Int J Mol Sci 25 doi:10.3390/ijms25063116 Suzuki M, Suzuki H, Sugimoto Y, Sugiyama Y (2003) ABCG2 transports sulfated conjugates of steroids and xenobiotics J Biol Chem 278:22644-22649 doi:10.1074/jbc.M212399200 Tamura A, Wakabayashi K, Onishi Y, Nakagawa H, Tsuji M, Matsuda Y, Ishikawa T (2006a) Genetic polymorphisms of human ABC transporter ABCG2: development of the standard method for functional validation of SNPs by using the Flp recombinase system J Exp Ther Oncol 6:1-11 Tamura A, Watanabe M, Saito H, Nakagawa H, Kamachi T, Okura I, Ishikawa T (2006b) Functional validation of the genetic polymorphisms of human ATP-binding cassette (ABC) transporter ABCG2: identification of alleles that are defective in porphyrin transport Mol Pharmacol 70:287-296 doi:10.1124/mol.106.023556 Ueda K, Cornwell MM, Gottesman MM, Pastan I, Roninson IB, Ling V, Riordan JR (1986) The mdr1 gene, responsible for multidrug-resistance, codes for P-glycoprotein Biochem Biophys Res Commun 141:956-962 Volk EL, Farley KM, Wu Y, Li F, Robey RW, Schneider E (2002) Overexpression of wild-type breast cancer resistance protein mediates methotrexate resistance Cancer Res 62:5035-5040 Wakabayashi K, Nakagawa H, Adachi T, Kii I, Kobatake E, Kudo A, Ishikawa T (2006) Identification of cysteine residues critically involved in homodimer formation and protein expression of human ATP-binding cassette transporter ABCG2: a new approach using the flp recombinase system J Exp Ther Oncol 5:205-222 Wang L et al. (2021) A pharmacogenetics study of platinum-based chemotherapy in lung cancer: ABCG2 polymorphism and its genetic interaction with SLC31A1 are associated with response and survival J Cancer 12:1270-1283 doi:10.7150/jca.51621 Wirth D, Hauser H (2004) Flp-mediated integration of expression cassettes into FRT-tagged chromosomal loci in mammalian cells Methods Mol Biol 267:467-476 doi:10.1385/1-59259-774-2:467 Woodward OM, Kottgen A, Coresh J, Boerwinkle E, Guggino WB, Kottgen M (2009) Identification of a urate transporter, ABCG2, with a common functional polymorphism causing gout Proc Natl Acad Sci U S A 106:10338-10342 doi:10.1073/pnas.0901249106 Zattoni IF et al. (2022) A new porphyrin as selective substrate-based inhibitor of breast cancer resistance protein (BCRP/ABCG2) Chem Biol Interact 351:109718 doi:10.1016/j.cbi.2021.109718 Zhou S, Zong Y, Ney PA, Nair G, Stewart CF, Sorrentino BP (2005) Increased expression of the Abcg2 transporter during erythroid maturation plays a role in decreasing cellular protoporphyrin IX levels Blood 105:2571-2576 doi:10.1182/blood-2004-04-1566 Additional Declarations No competing interests reported. Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-6704269","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":468276005,"identity":"0628d7ff-97be-4cf9-a5dd-03b02bba2c66","order_by":0,"name":"Shiori Sato","email":"","orcid":"","institution":"Chubu University","correspondingAuthor":false,"prefix":"","firstName":"Shiori","middleName":"","lastName":"Sato","suffix":""},{"id":468276006,"identity":"4bba8974-b34e-4567-9e65-8e689e3299fa","order_by":1,"name":"Megumi Tsukamoto","email":"","orcid":"","institution":"Chubu University","correspondingAuthor":false,"prefix":"","firstName":"Megumi","middleName":"","lastName":"Tsukamoto","suffix":""},{"id":468276007,"identity":"2980fd26-4643-4795-bb39-c5e4d30eafb5","order_by":2,"name":"Kayo Ozawa","email":"","orcid":"","institution":"Chubu University","correspondingAuthor":false,"prefix":"","firstName":"Kayo","middleName":"","lastName":"Ozawa","suffix":""},{"id":468276008,"identity":"96fdba55-1410-4ea9-9e4e-772d59adb7c5","order_by":3,"name":"Ritsuko Imai","email":"","orcid":"","institution":"Chubu University","correspondingAuthor":false,"prefix":"","firstName":"Ritsuko","middleName":"","lastName":"Imai","suffix":""},{"id":468276009,"identity":"61b9691a-3991-4af9-ad7b-7a9b25295c1d","order_by":4,"name":"Hiroshi Nakagawa","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA+klEQVRIiWNgGAWjYBACAwYGZjCDn/kAiDoAEgEBNsJaJNsSSNVicAxVC25gLt382ODHL7vEzcfYH35gqLmTuF0igfHDDwa+PFxaLOccM07s7UtO3HaMx1iC4dizxJ0zEpglexjYinE67EaC8QHeHubcbfd72BgYGw4nbriRwCAN9EtiA04t6Z8P/u2pz93cxv4MpoX5N34tOcbJPD8O525gYzCDaWEjYEtOsbFsw/H6GSC/JBw7bLyz52GbZY8BPr+kb5Z886famL8NGGIfag7LbmdPPnzjR8UxnCEGBoxtUEYChNsAjybc4A+mUA0BLaNgFIyCUTCCAADFA17lPvB1mwAAAABJRU5ErkJggg==","orcid":"","institution":"Chubu University","correspondingAuthor":true,"prefix":"","firstName":"Hiroshi","middleName":"","lastName":"Nakagawa","suffix":""}],"badges":[],"createdAt":"2025-05-20 06:23:16","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6704269/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6704269/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":84366692,"identity":"2c6992c3-38e7-45e8-86a5-3d5781b027a4","added_by":"auto","created_at":"2025-06-11 06:10:27","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":4421384,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSchematic illustration of human ABCG2 and the location of SNP (rs34678167, 805 C\u0026gt;T, P269S).\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eArrow, location of the SNP (rs34678167, 805 C\u0026gt;T, P269S); ABC, ATP binding cassette (nucleotide binding domain).\u003c/p\u003e","description":"","filename":"Fig1.png","url":"https://assets-eu.researchsquare.com/files/rs-6704269/v1/3148d36417e1960748d0d983.png"},{"id":84366715,"identity":"28371efd-35f5-44f4-a680-6a411bd3886f","added_by":"auto","created_at":"2025-06-11 06:10:28","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":27792981,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSchematic illustration of the pcDNA5/FRT/ABCG2 vector (A) and electropherogram of DNA Sequencing (B).\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A) Partial cDNA sequences at positions 800 to 810 in \u003cem\u003eABCG2 (WT)\u003c/em\u003e and \u003cem\u003e(P269S)\u003c/em\u003e cDNA are indicated. The base at position 805 is indicated by bold underline. ABCG2, \u003cem\u003eABCG2\u003c/em\u003e cDNA; BGH, bovine growth hormone; CMV, cytomegalovirus; pA, polyadenylation sequence; pUC ori, pUC vector origin of replication; SV40, simian virus 40. (B) Electropherograms confirming nucleotide substitution (C\u0026gt;T) at position 805 in \u003cem\u003eABCG2 (WT)\u003c/em\u003ecDNA. The nucleotide at position 805 is indicated by the arrows.\u003c/p\u003e","description":"","filename":"Fig2.png","url":"https://assets-eu.researchsquare.com/files/rs-6704269/v1/b53af220944e30e098575434.png"},{"id":84366700,"identity":"35bde9bb-a767-4a5d-957f-ec9b2a300d03","added_by":"auto","created_at":"2025-06-11 06:10:28","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":523101,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003emRNA levels of ABCG2 in cells established using the Flp-In™ system.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe data are shown as a ratio to the GAPDH mRNA level in the cells and were normalized by the ABCG2/GAPDH ratio. The mean ± S.D. (n = 5) is shown. Statistical analysis was performed using one-way analysis of variance and Tukey's HSD test (*; p \u0026lt; 0.01 compared with the mock group).\u003c/p\u003e","description":"","filename":"Fig3.png","url":"https://assets-eu.researchsquare.com/files/rs-6704269/v1/e3a84e6bad8e4c07883c1652.png"},{"id":84366695,"identity":"169faf84-55f7-409b-8193-5e8ae9525fae","added_by":"auto","created_at":"2025-06-11 06:10:27","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":8347063,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eThe expression levels of ABCG2 in cells established using the Flp-In™ system.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe expression levels of ABCG2 and GAPDH were determined by western blotting as de-scribed in the Materials and Methods section. The experiments were independently performed on more than two occasions. Data are expressed as mean ± S.D. (n = 3). Statistical analyses were performed using one-way ANOVA and Tukey's HSD test (*; p \u0026lt; 0.05 compared with the Mock group).\u003c/p\u003e","description":"","filename":"Fig4.png","url":"https://assets-eu.researchsquare.com/files/rs-6704269/v1/0ec4ed77c1769b54aebac15d.png"},{"id":84366694,"identity":"232759dd-ce30-4b7e-891a-4fb2f3897630","added_by":"auto","created_at":"2025-06-11 06:10:27","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":24716798,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eAnticancer drug resistance profiles of cells established using the Flp-In™ system.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A) Representatives images of five experiments are shown. Data are expressed as mean ± S.D. (n = 4). (B) EC\u003csub\u003e50\u003c/sub\u003e values from each of five experiments are shown. The data are expressed as mean values ± S.D. (n = 5). One-way ANOVA and Tukey's HSD test were employed for statistical analysis. Different letters indicate significant differences (p \u0026lt; 0.05) between the groups.\u003c/p\u003e","description":"","filename":"Fig5.png","url":"https://assets-eu.researchsquare.com/files/rs-6704269/v1/bc5c3ebece97d254e4034585.png"}],"financialInterests":"No competing interests reported.","formattedTitle":"\u003cp\u003e\u003cstrong\u003eA SNP (rs34678167, 805 C\u0026gt;T, P269S) in the Human ABC Transporter \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eABCG2\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e Gene increases ABCG2-mediated Drug Resistance\u003c/strong\u003e\u003c/p\u003e","fulltext":[{"header":"Introduction","content":"\u003cp\u003eChemotherapy is one of the main strategies used for cancer treatment. The efficacy of chemotherapy varies between patients and cancer types, with genetic polymorphisms and mutations playing key roles. Therefore, understanding the effects of genetic polymorphisms and mutations in patients with cancer is critical for achieving successful chemotherapy outcomes.\u003c/p\u003e \u003cp\u003eSome ABC transporters regulate drug concentration in the body and within cells by actively exporting substrates across biological membranes. ABCG2 (BCRP/MXR) was identified in a mitoxantrone-resistant human colorectal cancer cell line and an anthracycline-resistant breast cancer cell line (Allikmets et al. \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1998\u003c/span\u003e; Doyle et al. \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e1998\u003c/span\u003e; Miyake et al. \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e1999\u003c/span\u003e), following the discovery of ABCB1 (Juliano and Ling \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e1976\u003c/span\u003e; Ueda et al. \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e1986\u003c/span\u003e) and ABCC1 (Cole et al. \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e1992\u003c/span\u003e), and also has been shown to extrude anticancer drugs (Maliepaard et al. \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e1999\u003c/span\u003e). ABCG2, a 72 kDa glycoprotein composed of 655 amino acids, features a single ATP-binding domain at the N-terminus and a transmembrane domain at the C-terminus (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e) and functions as a homodimer (Kage et al. \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2002\u003c/span\u003e). ABCG2 facilitates the transport of structurally diverse endobiotics, such as dehydroepiandrosterone sulfate (Kondo et al. \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2004\u003c/span\u003e), estrone-3-sulfate (Kondo et al. \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2004\u003c/span\u003e; Suzuki et al. \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2003\u003c/span\u003e), lumichrome (Millan-Garcia et al. \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2024\u003c/span\u003e), porphyrins (Tamura et al. \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2006b\u003c/span\u003e; Zattoni et al. \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Zhou et al. \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2005\u003c/span\u003e), and uric acid (Woodward et al. \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2009\u003c/span\u003e). ABCG2 overexpression has been associated with increased resistance to several anticancer drugs, including camptothecin and its analogs (Brangi et al. \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e1999\u003c/span\u003e; Kage et al. \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2002\u003c/span\u003e; Kawabata et al. \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2001\u003c/span\u003e; Maliepaard et al. \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e1999\u003c/span\u003e; Tamura et al. \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2006b\u003c/span\u003e), epidermal growth factor receptor tyrosine kinase inhibitors (Breedveld et al. \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2005\u003c/span\u003e; Burger et al. \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2004\u003c/span\u003e), mitoxantrone (Kage et al. \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2002\u003c/span\u003e; Mitomo et al. \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2003\u003c/span\u003e; Wakabayashi et al. \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2006\u003c/span\u003e), and methotrexate (Chen et al. \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2003\u003c/span\u003e; Volk et al. \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2002\u003c/span\u003e). In addition, recent clinical studies have indicated that ABCG2 expression levels and genetic variants can be prognostic markers for the progression and treatment response of cancers such as glioma (Raguz et al. \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2024\u003c/span\u003e), hepatoma (Huang et al. \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2022\u003c/span\u003e), and Non-Small Cell Lung Cancer (Wang et al. \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2021\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eOur previous studies have demonstrated that amino acid substitutions can influence the intracellular stability and substrate specificity of ABCG2 (Tamura et al. \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2006a\u003c/span\u003e; Tamura et al. \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2006b\u003c/span\u003e). In addition, our previous studies have shown that 18 non-synonymous SNPs in \u003cem\u003eABCG2\u003c/em\u003e affect the affinity of ABCG2 for porphyrins and methotrexate (Tamura et al. \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2006b\u003c/span\u003e). We have also reported that seven nonsynonymous SNPs affect the intracellular protein expression levels of ABCG2 and ABCG2-dependent anticancer drug resistance in cells (Tamura et al. \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2006a\u003c/span\u003e). However, the impact of approximately 100 SNPs in \u003cem\u003eABCG2\u003c/em\u003e remains unclear. In this study, we present new and significant findings regarding the impact of SNP rs34678167 (805 C\u0026thinsp;\u0026gt;\u0026thinsp;T, P269S) on anticancer drug resistance in cells dependent on ABCG2.\u003c/p\u003e"},{"header":"Materials and methods","content":"\u003cp\u003eConstruction of pcDNA5/FRT containing ABCG2 (P269S) variant cDNA\u003c/p\u003e \u003cp\u003eTo generate the expression vector pcDNA5/FRT/ABCG2 (P269S), we used site-directed mutagenesis based on the sequence information of the ABCG2 (P269S) variant obtained from the NCBI dbSNP database as previously described (Nakagawa et al. \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2008\u003c/span\u003e; Tamura et al. \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2006a\u003c/span\u003e; Tamura et al. \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2006b\u003c/span\u003e; Wakabayashi et al. \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2006\u003c/span\u003e). Site-directed mutagenesis was performed using PrimeSTAR\u0026reg; Max DNA polymerase (Takara Bio Inc., Otsu, Japan) and specific primers to prepare non-synonymous SNP variant ABCG2 (P269S) cDNA. After PCR for site-directed mutagenesis, the reaction mixture was treated with the Dpn I endonuclease to eliminate the original template plasmid pcDNA5/FRT/ABCG2 (WT) vectors. The sequences of the resulting amplicons were verified using an Applied Biosystems 3130 and 3130xl Genetic Analyzer (Applied Biosystems, Foster City, CA).\u003c/p\u003e \u003cp\u003eCell culture condition\u003c/p\u003e \u003cp\u003eFlp-In-293 cells (Invitrogen, Thermo Fisher Scientific, Waltham, MA, USA) were cultured in high-glucose Dulbecco\u0026rsquo;s Modified Eagle Medium (DMEM) supplemented with 10% heat-inactivated fetal bovine serum (FBS), 4 mM L-glutamine, 100 U/mL penicillin, 100 \u0026micro;g/mL streptomycin, 250 ng/mL amphotericin B, and 100 mg/mL zeocin. To select for Flp-In-293/ABCG2 (P269S) cells, 50 mg/mL hygromycin B was used instead of 100 mg/mL zeocin. The cells were maintained in a humidified incubator at 37\u0026deg;C with 5% CO₂, and their viability was assessed by trypan blue exclusion assay using a hemocytometer. Living cells were used in all the experiments.\u003c/p\u003e \u003cp\u003eZeocin was purchased from Invitrogen (Thermo Fisher Scientific, Waltham, MA), while an Antibiotic-Antimycotic Mixed Stock Solution(100\u0026times;) (mixture of 10,000 U/mL penicillin, 10,000 \u0026micro;g/mL streptomycin, and 25,000 ng/mL amphotericin B), and high-glucose DMEM were purchased from Nacalai Tesque, Inc. (Kyoto, Japan). FBS was purchased from Equitech-Bio, Inc. (Kerrville, TX, USA).\u003c/p\u003e \u003cp\u003eGeneration of cells expressing ABCG2 (P269S) variant\u003c/p\u003e \u003cp\u003eFlp-In-293 cells were plated in 35-mm dishes (TrueLine, Baton Rouge, LA) at a density of 1 \u0026times; 10\u003csup\u003e6\u003c/sup\u003e cells per dish and precultured for 24 h. Cells were then co-transfected with pcDNA5/FRT/ABCG2 (P269S) and the Flp recombinase expression plasmid pOG44 vectors using Lipofectamine\u0026trade;-2000 according to the manufacturer's protocol. After transfection, the cells were selected with 50 mg/mL hygromycin B, and the resulting hygromycin B-resistant colonies were collected, subcultured, and used as Flp-In-293/ABCG2 (P269S) cells.\u003c/p\u003e \u003cp\u003eLipofectamine\u0026trade;-2000 and pOG44 were purchased from Invitrogen (Thermo Fisher Scientific, Waltham, MA). Hygromycin B was obtained from Nacalai Tesque, Inc. (Kyoto, Japan).\u003c/p\u003e \u003cp\u003eTotal RNA preparation and first-strand cDNA synthesis\u003c/p\u003e \u003cp\u003eFlp-In-293/ABCG2 (WT) and Flp-In-293/ABCG2 (P269S) cells were seeded in 35-mm dishes (TrueLine, Baton Rouge, LA) at a density of 1 \u0026times; 10\u003csup\u003e6\u003c/sup\u003e cells per dish and precultured for 3 days. Cells were collected with culture medium into 1.5 mL tubes, centrifuged (300 \u0026times; g, 5 min, 4\u0026deg;C), and washed twice with 1 mL PBS(-). The resulting cell pellets were suspended in 600 \u0026micro;L of lysis/binding buffer from the High Pure RNA Isolation Kit (Roche Diagnostics, Mannheim, Germany) and stored at -80\u0026deg;C until total RNA preparation was performed with a High-Purity RNA Isolation Kit (Roche Diagnostics). Total RNA concentration was measured using a DU640 spectrophotometer (Beckman Coulter, Fullerton, CA, USA). Total RNA was reverse transcribed using a High-Capacity cDNA Reverse-Transcription Kit (Thermo Fisher Scientific Inc., Waltham, MA) according to the manufacturer's instructions.\u003c/p\u003e \u003cp\u003eQuantitative evaluation of ABCG2 mRNA levels\u003c/p\u003e \u003cp\u003eThe expression levels of ABCG2 mRNA were determined using a 7500 Fast Real Time-PCR System (Applied Biosystems), where GoTaq\u0026reg; DNA Polymerase GoTaq\u0026reg; qPCR Master Mix, 2\u0026times; (Promega, Tokyo, Japan) and primers for ABCG2 or GAPDH were used.\u003c/p\u003e \u003cp\u003eThe GoTaq\u0026reg; qPCR Master Mix, 2\u0026times; was purchased from Promega (Tokyo, Japan). The primer sets for ABCG2 (HA204957-F and HA204957-R) and GAPDH (10000459 and 20000459) were purchased from Takara Bio Inc. (Otsu, Japan).\u003c/p\u003e \u003cp\u003eMTT assay\u003c/p\u003e \u003cp\u003eFlp-In-293/ABCG2 (WT) and Flp-In-293/ABCG2 (P269S) cells were plated in 96-well plates (Thermo Fisher Scientific) at a density of 5 \u0026times; 10\u003csup\u003e3\u003c/sup\u003e cells/well and cultured for 24 h before exposure to different concentrations of mitoxantrone and SN-38 for 72 h. The drug concentrations ranged from 0 (control) to 1000 nM for mitoxantrone and 100 nM for SN-38. After drug exposure, cells were incubated with 500 \u0026micro;g/mL MTT for 3 h, lysed with 10% sodium dodecyl sulfate (SDS), and incubated overnight at 37\u0026deg;C in an atmosphere containing 5% CO\u003csub\u003e2\u003c/sub\u003e. Absorbance was measured at 570 nm using a Thermo Labsystems Multiskan Jax spectrophotometer (Thermo Fisher Scientific) with a reference wavelength of 630 nm to evaluate the amount of formazan metabolized from MTT in each well. Cell viability was calculated as a percentage of the untreated group (control) according to the absorbance at 570 nm with a reference wavelength of 630 nm. EC\u003csub\u003e50\u003c/sub\u003e values, representing the drug concentration required to reduce cell viability by 50%, were determined from the survival curves, and the cytotoxicity of the anti-cancer drugs (mitoxantrone and SN-38) was assessed.\u003c/p\u003e \u003cp\u003eMitoxantrone and SN-38 were purchased from Wako Pure Chemical Industries, Ltd. (Osaka, Japan) and were graciously provided by Yakult Honsha Co. (Tokyo, Japan), respectively. 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) was obtained from Sigma-Aldrich Co. (St. Louis, MO).\u003c/p\u003e \u003cp\u003eCell lysate preparation for SDS-PAGE\u003c/p\u003e \u003cp\u003eFlp-In-293/ABCG2 (WT) and Flp-In-293/ABCG2 (P269S) cells were plated in 35-mm dishes (TrueLine, Baton Rouge, LA) at a density of 1 \u0026times; 10\u003csup\u003e6\u003c/sup\u003e cells per dish and precultured for 3 days. The cells were harvested with culture medium in 1.5 mL tubes, centrifuged (300 \u0026times; g, 5 min, 4\u0026deg;C), and washed twice with 1 mL PBS(-). The resulting cell pellets were resuspended and lysed in a lysis buffer containing 50 mM Tris-HCl (pH 7.6), 5 mM EDTA (pH 8.0), 120 mM NaCl, 1% Triton X-100, 1 mM DTT, protease inhibitors, and phosphatase inhibitors. The cell lysate samples were homogenized by passing through a 27G-needle ten times. After centrifugation of the homogenate at 3,000 rpm at 4\u0026deg;C for 10 min, the supernatants were collected as cell lysates. The protein concentration of the supernatant was determined using bovine serum albumin as a standard by the Bradford method. Following this, 50 \u0026micro;g of the cell lysate was treated with PNGase F for 10 min at 37\u0026deg;C to remove glycomoieties from the proteins. The resulting cell lysates were mixed with SDS-PAGE sample buffer solution containing 10% (v/v) 2-mercaptoethanol (Daiichi pure chemicals Co., Ltd., Tokyo, Japan) and stored at -30\u0026deg;C until needed for western blotting.\u003c/p\u003e \u003cp\u003eEvaluation of expression levels of ABCG2\u003c/p\u003e \u003cp\u003eCell lysates were prepared from each cell group in triplicate and aliquots of 5 \u0026micro;g were mixed separately for each cell group as a cell lysate mixture. Fractionation of the mixtures was performed by SDS-PAGE on a 7.5% polyacrylamide gel, followed by transfer to nitrocellulose membranes (GE Healthcare UK Ltd., Bucks, UK). Western blotting was performed after the blocking process, in which the membranes were immersed in TBST (50 mM Tris-HCl, 150 mM NaCl, and 0.05% (v/v) Tween 20) containing skimmed milk powder at 5% (w/v) at room temperature for more than 1 h and then at 4\u0026deg;C overnight. After this procedure, the membranes were rinsed with TTBS and exposed to a 1:1000-diluted monoclonal anti-ABCG2 antibody (BXP-21; ALEXIS Co., Lausen, Switzerland) or anti-GAPDH antibody (anti-GAPDH-Clone 6C5 mouse monoclonal, IgG2b; American Research Products, Inc., Waltham, MA, USA) in TBST containing skimmed milk powder at 5% (w/v) with gentle shaking for 1 h at room temperature. After primary antibody treatment, the membranes were rinsed with TTBS and exposed to 1:1000-diluted HRP-conjugated anti-mouse IgG antibodies (Cell Signaling Technology, Inc., Danvers, MA, USA) in 5% (w/v) skim milk powder-containing TBST with gentle shaking for 1 h at room temperature. Blots were visualized using Western Lighting Chemiluminescent Reagent Plus (PerkinElmer Life and Analytical Sciences, Boston, MA, USA) and detected using WSE-6100 LuminoGraph I (Atto Corp., Tokyo, Japan). The ImageJ software was used to quantify the signal intensities associated with ABCG2 or GAPDH (Wayne Rasband, Bethesda, MD, USA).\u003c/p\u003e \u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analysis\u003c/h2\u003e \u003cp\u003eJSTAT (version 20.0), developed by Masato Sato, was used for statistical analyses using one-way ANOVA and Tukey's honestly significant difference (HSD) test. In all analyses, statistical significance was set at p\u0026thinsp;\u0026lt;\u0026thinsp;0.05.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eExpression levels of ABCG2 mRNA and protein in cells expressing ABCG2 (P269S)\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003cp\u003eTo quantitatively evaluate the effect of SNP rs34678167 (805 C\u0026thinsp;\u0026gt;\u0026thinsp;T, P269S) on ABCG2 protein expression and ABCG2-dependent drug resistance, we established Flp-In-293 cells expressing ABCG2 (P269S) (Figs.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e and \u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). Total RNA was extracted from Flp-In-293/Mock, Flp-In-293/ABCG2 (WT), and Flp-In-293/ABCG2 (P269S) cells, and their quality was evaluated by comparing the A260/A280 ratios and qPCR threshold cycles across samples. As the quality of the total RNA produced was consistent, the levels of ABCG2 mRNA were normalized to those of GAPDH, and the successful integration of ABCG2 (P269S) cDNA at the FRT site within the genomic DNA was confirmed.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe results of qPCR showed that ABCG2 mRNA levels in ABCG2 cDNA-transfected cells [Flp-In-293/ABCG2 (WT) and Flp-In-293/ABCG2 (P269S) cells] were more than 100 times higher than those in Flp-In-293/Mock cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). ABCG2 mRNA levels in Flp-In-293/ABCG2 (P269S) cells were comparable to those in Flp-In-293/ABCG2 (WT) cells, indicating that the Flp-In\u0026trade; system functioned as expected.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eWestern blotting was performed to determine ABCG2 protein expression levels in Flp-In-293/ABCG2 (WT) and Flp-In-293/ABCG2 (P269S) cells. PNGase F was used to remove glycosylation and accurately assess the protein expression levels. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e, ABCG2 protein levels corresponded with ABCG2 mRNA expression, confirming significantly higher ABCG2 expression in Flp-In-293/ABCG2 (WT) and Flp-In-293/ABCG2 (P269S) cells than in Flp-In-293/Mock cells. In contrast, ABCG2 protein levels in Flp-In-293/ABCG2 (P269S) cells were similar to those in Flp-In-293/ABCG2 (WT) cells.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eEvaluation of drug resistance in cells expressing ABCG2 (P269S)\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003cp\u003eTo investigate the impact of rs34678167 (805 C\u0026thinsp;\u0026gt;\u0026thinsp;T, P269S) on ABCG2-mediated drug resistance, we performed an MTT assay to assess cellular responses to mitoxantrone and SN-38 as detailed in the Materials and Methods section. The cells were treated with varying concentrations of these anticancer drugs, and their EC\u003csub\u003e50\u003c/sub\u003e values were determined.\u003c/p\u003e \u003cp\u003eConsistent with the results of previous studies (Tamura et al. \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2006a\u003c/span\u003e; Tamura et al. \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2006b\u003c/span\u003e; Wakabayashi et al. \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2006\u003c/span\u003e), Flp-In-293/ABCG2 (WT) cells exhibited increased resistance to mitoxantrone and SN-38 compared with Flp-In-293/Mock cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e and Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). The EC\u003csub\u003e50\u003c/sub\u003e values for mitoxantrone and SN-38 were 4.6- and 5.5-fold higher, respectively, in Flp-In-293/ABCG2 (WT) cells than in Flp-In-293/Mock cells (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). Similarly, Flp-In-293/ABCG2 (P269S) cells showed significant resistance to mitoxantrone and SN-38 compared with Flp-In-293/Mock cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e and Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). EC\u003csub\u003e50\u003c/sub\u003e values indicated that Flp-In-293/ABCG2 (P269S) cells were 3.0- and 3.8-fold more resistant to mitoxantrone and SN-38, respectively, than Flp-In-293/ABCG2 (WT) cells (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). These findings suggest that rs34678167 (805 C\u0026thinsp;\u0026gt;\u0026thinsp;T, P269S) enhances ABCG2-mediated drug resistance, although it does not significantly alter ABCG2 mRNA and protein expression levels.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eAnticancer drug registance profiles (EC\u003csub\u003e50\u003c/sub\u003e) of the cells.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"13\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c8\" colnum=\"8\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c9\" colnum=\"9\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c10\" colnum=\"10\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c11\" colnum=\"11\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c12\" colnum=\"12\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c13\" colnum=\"13\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eCompounds\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"12\" nameend=\"c13\" namest=\"c2\"\u003e \u003cp\u003eEC\u003csub\u003e50\u003c/sub\u003e (nM)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003ctr\u003e \u003cth align=\"left\" colspan=\"3\" nameend=\"c4\" namest=\"c2\"\u003e \u003cp\u003eMock\u003c/p\u003e \u003cp\u003ecells\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colspan=\"3\" nameend=\"c8\" namest=\"c6\"\u003e \u003cp\u003eABCG2 (WT)\u003c/p\u003e \u003cp\u003ecells\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c9\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colspan=\"3\" nameend=\"c12\" namest=\"c10\"\u003e \u003cp\u003eABCG2 (P269S) cells\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c13\"\u003e\u0026nbsp;\u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMitoxantrone\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e4.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u0026plusmn;\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e1.4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e18.8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e\u0026plusmn;\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e3.8\u003csup\u003e*\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c10\"\u003e \u003cp\u003e55.8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003e\u0026plusmn;\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c12\"\u003e \u003cp\u003e10.0\u003csup\u003e*, **\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c13\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSN-38\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e3.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u0026plusmn;\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e1.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e16.9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e\u0026plusmn;\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e3.8\u003csup\u003e*\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c10\"\u003e \u003cp\u003e64.3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003e\u0026plusmn;\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c12\"\u003e \u003cp\u003e6.6\u003csup\u003e*, **\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c13\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003ctfoot\u003e \u003ctr\u003e\u003ctd colspan=\"13\"\u003eSN-38, 7-ethyl-10-hydroxy-camptothecin. EC\u003csub\u003e50\u003c/sub\u003e values from each of the five experiments are shown. Data are expressed as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;S.D. (n\u0026thinsp;=\u0026thinsp;5). One-way ANOVA and Tukey's HSD test were employed for statistical analysis. [*; p\u0026thinsp;\u0026lt;\u0026thinsp;0.05 compared to the Mock group, **; p\u0026thinsp;\u0026lt;\u0026thinsp;0.05 compared with the wild-type (WT)].\u003c/td\u003e\u003c/tr\u003e \u003c/tfoot\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eGeneration of human ABCG2 (P269S)-expressing cells using the Flp-In\u0026trade; system\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003cp\u003eThe emergence of drug resistance in cancer cells and its inter-individual variability have been attributed, in part, to the overexpression of ABC transporters and the presence of specific single-nucleotide polymorphisms (SNPs) in their genes (Nakagawa et al. \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2008\u003c/span\u003e; Tamura et al. \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2006a\u003c/span\u003e; Tamura et al. \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2006b\u003c/span\u003e; Wakabayashi et al. \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2006\u003c/span\u003e). Our previous studies demonstrated that several non-synonymous SNPs in \u003cem\u003eABCG2\u003c/em\u003e, such as rs2231137 (V12M), rs2231142 (Q141K), rs1061018 (F208S), rs3116448 (S248P), rs1354553769 (S441N), and rs192169063 (F489L), can influence drug resistance in cells expressing ABCG2 (Tamura et al. \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2006a\u003c/span\u003e). In this study, we used the Flp-In\u0026trade; system to generate cells expressing the rs34678167 (P269S) variant of ABCG2. This system enables site-specific integration of a single copy of cDNA at the FRT site in the telomeric region of chromosome 12 in Flp-In-293 cells (Tamura et al. \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2006a\u003c/span\u003e; Wirth and Hauser \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2004\u003c/span\u003e), providing a controlled platform for quantitatively assessing the functional consequences of individual SNPs on drug resistance. Although we did not directly confirm the genomic integration site and copy number of the cDNA in Flp-In-293/ABCG2 (P269S) cells, the comparable levels of ABCG2 mRNA expression between ABCG2 (P269S)- and (WT)-expressing cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e) suggested successful and equivalent integration. Although it remains possible that exogenous expression of ABCG2 (P269S) might influence the expression of other ABC transporters, this model remains valid for evaluating the specific impact of rs34678167 (805 C\u0026thinsp;\u0026gt;\u0026thinsp;T, P269S) on ABCG2-mediated drug resistance.\u003cdiv class=\"BlockQuote\"\u003e\u003cp\u003eDrug resistance profiles of the ABCG2 (P269S)-expressing cells\u003c/p\u003e\u003c/div\u003e\u003c/p\u003e \u003cp\u003eABCG2 has been shown to mediate the efflux of a broad range of substrates, including anticancer drugs such as camptothecin and its analogs (Brangi et al. \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e1999\u003c/span\u003e; Kage et al. \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2002\u003c/span\u003e; Maliepaard et al. \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e1999\u003c/span\u003e; Tamura et al. \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2006b\u003c/span\u003e), epidermal growth factor receptor tyrosine kinase inhibitors (Breedveld et al. \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2005\u003c/span\u003e; Burger et al. \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2004\u003c/span\u003e), mitoxantrone (Kage et al. \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2002\u003c/span\u003e; Mitomo et al. \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2003\u003c/span\u003e; Wakabayashi et al. \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2006\u003c/span\u003e), and methotrexate (Chen et al. \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2003\u003c/span\u003e; Volk et al. \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2002\u003c/span\u003e), as well as endogenous compounds like dehydroepiandrosterone sulfate (Kondo et al. \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2004\u003c/span\u003e), estrone-3-sulfate (Kondo et al. \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2004\u003c/span\u003e; Suzuki et al. \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2003\u003c/span\u003e), lumichrome (Millan-Garcia et al. \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2024\u003c/span\u003e), porphyrins (Tamura et al. \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2006b\u003c/span\u003e; Zattoni et al. \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Zhou et al. \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2005\u003c/span\u003e), and uric acid (Woodward et al. \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2009\u003c/span\u003e). As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e and Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e, our results confirmed that Flp-In-293/ABCG2 (WT) cells exhibited higher resistance to mitoxantrone and SN-38 than Flp-In-293/Mock cells, consistent with the findings of previous studies (Brangi et al. \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e1999\u003c/span\u003e; Maliepaard et al. \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e1999\u003c/span\u003e; Mitomo et al. \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2003\u003c/span\u003e; Tamura et al. \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2006a\u003c/span\u003e; Tamura et al. \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2006b\u003c/span\u003e; Wakabayashi et al. \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2006\u003c/span\u003e). Likewise, Flp-In-293/ABCG2 (P269S) cells also showed increased resistance to these drugs, validating the functionality of transfected ABCG2 (P269S) (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e and Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eSeveral previous studies have assessed the transport activity of ABCG2 (P269S) using prepared membrane vesicles. While some studies have reported similar transport activities between ABCG2 (P269S) and ABCG2 (WT) prepared from HEK293 cells for substrates such as estrone-3-sulfate and methotrexate (Kondo et al. \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2004\u003c/span\u003e) (Higashino et al. \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2017\u003c/span\u003e), others have observed reduced transport activity of ABCG2 (P269S) prepared from Sf9 cells (Lee et al. \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2007\u003c/span\u003e). These inconsistent findings suggest that the substrate specificity and transport activity of ABCG2 (P269S) may vary depending on the experimental condition and the cellular protein expression system.\u003c/p\u003e \u003cp\u003eIn this study, we used the Flp-In\u0026trade; system to achieve a uniform cellular background, enabling a more accurate comparison between cells expressing ABCG2 (WT) and those expressing ABCG2 (P269S) compared to conventional transfection methods with the pcDNA3.1 vector. Our results showed that despite similar mRNA and protein expression levels, Flp-In-293/ABCG2 (P269S) cells exhibited a distinct drug resistance profile compared to Flp-In-293/ABCG2 (WT) cells. This implies that the amino acid substitution derived from rs34678167 (805 C\u0026thinsp;\u0026gt;\u0026thinsp;T, P269S) may affect the functional properties of ABCG2, such as substrate recognition, intracellular localization or ATP binding affinity, thereby altering intracellular drug accumulation as suggested by other substitutions [ABCG2 (V12M, Q141K, F208S, S248P, F431L, S441N, or F489L)] (Nakagawa et al. \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2008\u003c/span\u003e; Tamura et al. \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2006b\u003c/span\u003e). Further studies are required to elucidate the molecular basis of these functional differences. Investigations into the subcellular localization, ATPase activity, and transport kinetics of ABCG2 (P269S), as well as the direct measurement of intracellular drug accumulation, will be crucial for uncovering the precise mechanisms underlying altered drug resistance.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eIn this study, we employed a quantitative and site-specific expression system to evaluate the impact of SNP rs34678167 (805 C\u0026thinsp;\u0026gt;\u0026thinsp;T, P269S) on the human \u003cem\u003eABCG2\u003c/em\u003e gene on drug resistance profiles of cells. Using the Flp-In\u0026trade; system, we were able to generate isogenic cell lines expressing ABCG2 (WT), ABCG2 (P269S), and others (Nakagawa et al. \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2008\u003c/span\u003e; Tamura et al. \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2006a\u003c/span\u003e; Tamura et al. \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2006b\u003c/span\u003e; Wakabayashi et al. \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2006\u003c/span\u003e), thereby eliminating variability due to differences in integration sites or copy number. Our findings demonstrated that rs34678167 (805 C\u0026thinsp;\u0026gt;\u0026thinsp;T, P269S) conferred enhanced resistance to the ABCG2 substrates mitoxantrone and SN-38, despite exhibiting mRNA and protein expression levels comparable to those of ABCG2 (WT). These results suggest that the amino acid substitution at position 269 may influence the functional properties of ABCG2, such as substrate interaction or ATP hydrolysis efficiency, rather than affect its expression. This study contributes to a deeper understanding of how non-synonymous SNPs in \u003cem\u003eABCG2\u003c/em\u003e can modulate drug resistance phenotypes, and underscores the importance of considering such genetic variations in the context of personalized cancer chemotherapy. Ultimately, these insights may support the development of effective therapeutic strategies and diagnostic tools tailored to individual genetic profiles.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cp\u003eABC, ATP-binding cassette; BCRP, breast cancer resistance protein; BSA, bovine serum albumin; D-MEM, high-glucose Dulbecco’s modified Eagle’s medium; DTT, dithiothreitol; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; FCS, fetal calf serum; FRT, Flp recombination target; HRP, horseradish peroxidase; MTT, 3-(4,5-dimethyl-2-thiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide; MXR, mitoxantrone resistance-associated protein; PBS, phosphate-buffered saline without both Ca\u003csup\u003e2+\u003c/sup\u003e and Mg\u003csup\u003e2+\u003c/sup\u003e; RT-PCR, reverse transcriptase-polymerase chain reaction; SNP, single nucleotide polymorphism; TBS, Tris-buffered saline; TTBS, TBS with 0.05% (v/v) Tween 20; WT, wild type.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAuthor Contributions\u003c/strong\u003e: conceptualization, H.N.; validation, S.S., M.T., and K.O.; formal analysis, S.S., M.T., and K.O.; investigation, S.S., M.T., and K.O.; resources, H.N.; data curation, H.N.; writing—original draft preparation, S.S.; writing—review and editing, H.N.; visualization, S.S. and R.I.; supervision, H.N.; project administration, H.N.; funding acquisition, H.N. All authors have read and agreed to the published version of the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e: This research was funded by Aichi Cancer Research Foundation and Chubu University Grant D (DII28IIM02). Megumi Tsukamoto is a Research Associates at the Chubu University, Japan.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgments\u003c/strong\u003e: The authors thank Yakult Honsha Co., Ltd. (Tokyo, Japan) for providing SN-38.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflicts of Interest\u003c/strong\u003e: The authors declare no conflict of interest.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eAllikmets R, Schriml LM, Hutchinson A, Romano-Spica V, Dean M (1998) A human placenta-specific ATP-binding cassette gene (ABCP) on chromosome 4q22 that is involved in multidrug resistance Cancer Res 58:5337-5339\u003c/li\u003e\n\u003cli\u003eBrangi M et al. 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(2021) A pharmacogenetics study of platinum-based chemotherapy in lung cancer: ABCG2 polymorphism and its genetic interaction with SLC31A1 are associated with response and survival J Cancer 12:1270-1283 doi:10.7150/jca.51621\u003c/li\u003e\n\u003cli\u003eWirth D, Hauser H (2004) Flp-mediated integration of expression cassettes into FRT-tagged chromosomal loci in mammalian cells Methods Mol Biol 267:467-476 doi:10.1385/1-59259-774-2:467\u003c/li\u003e\n\u003cli\u003eWoodward OM, Kottgen A, Coresh J, Boerwinkle E, Guggino WB, Kottgen M (2009) Identification of a urate transporter, ABCG2, with a common functional polymorphism causing gout Proc Natl Acad Sci U S A 106:10338-10342 doi:10.1073/pnas.0901249106\u003c/li\u003e\n\u003cli\u003eZattoni IF et al. (2022) A new porphyrin as selective substrate-based inhibitor of breast cancer resistance protein (BCRP/ABCG2) Chem Biol Interact 351:109718 doi:10.1016/j.cbi.2021.109718\u003c/li\u003e\n\u003cli\u003eZhou S, Zong Y, Ney PA, Nair G, Stewart CF, Sorrentino BP (2005) Increased expression of the Abcg2 transporter during erythroid maturation plays a role in decreasing cellular protoporphyrin IX levels Blood 105:2571-2576 doi:10.1182/blood-2004-04-1566\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":false,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"ATP-binding cassette (ABC) transporter, ABCG2, BCRP, MXR","lastPublishedDoi":"10.21203/rs.3.rs-6704269/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6704269/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eABCG2 (BCRP/MXR) is an ATP-binding cassette transporter that contributes to multidrug resistance in cancer cells by extruding a wide range of substrates, including anticancer agents. Although several non-synonymous single-nucleotide polymorphisms (SNPs) in the \u003cem\u003eABCG2\u003c/em\u003e gene have been characterized, the functional consequences of many remain unclear. This study investigated the impact of SNP rs34678167 (805 C\u0026gt;T, P269S) on ABCG2 expression and drug resistance. Using the Flp-In™ system, we established Flp-In-293 cell lines that stably express ABCG2 (P269S) variant. Quantitative real-time PCR and Western blotting revealed that the P269S variant resulted in mRNA and protein expression levels comparable to those of WT ABCG2. Drug resistance was assessed by MTT assay using mitoxantrone and SN-38. Cells expressing ABCG2 (P269S) exhibited significantly higher EC\u003csub\u003e50\u003c/sub\u003e values than those expressing WT ABCG2: 3.0-fold for mitoxantrone and 3.8-fold for SN-38 (p \u0026lt; 0.01). These results suggest that the P269S substitution enhances ABCG2-mediated drug resistance without affecting expression levels. This effect may be due to altered substrate recognition, transporter activity, or intracellular localization of the protein. Our findings underscore the importance of functionally validating genetic variants of ABCG2 and highlight the potential clinical relevance of rs34678167 in predicting drug response. This knowledge could contribute to the development of more effective therapeutic strategies tailored to the patient’s genetic background.\u003c/p\u003e","manuscriptTitle":"A SNP (rs34678167, 805 C\u0026gt;T, P269S) in the Human ABC Transporter ABCG2 Gene increases ABCG2-mediated Drug Resistance","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-06-11 06:10:22","doi":"10.21203/rs.3.rs-6704269/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"28b6dfdd-a227-465b-8278-547d1f6ac0eb","owner":[],"postedDate":"June 11th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2026-01-02T15:54:14+00:00","versionOfRecord":[],"versionCreatedAt":"2025-06-11 06:10:22","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-6704269","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6704269","identity":"rs-6704269","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}