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1 First Department of Internal Medicine
2 Department of Molecular Biology, University of Occupational and Environmental Health, School of Medicine, Yahata-nishi, Kitakyushu, Japan
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Abstract |
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 Top Abstract IntroductionResultsDiscussionExperimental proceduresReferences |
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Introduction |
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Since resistance to chemotherapy induced by P-pg is closely associated with prognosis of human malignancies (Linn et al. 1996), recent studies have helped elucidate the association of drug resistance and P-gp expression on malignant cells. P-gp is expressed on various types of tumour cells (Fojo et al. 1987), including leukaemic cells (Advani et al. 1999), CD34+ haematopoietic stem cells (Chaudhary & Roninson 1991), and epithelial cells in the liver, kidney, pancreas, gut and adrenals (Sugawara et al. 1988). On the other hand, treatment resistance is common in patients with not only haematopoietic malignancies, but also systemic autoimmune diseases, such as systemic lupus erythematosus (SLE), which sometimes leads to a poor prognosis of these diseases. However, P-gp expression on immune cells such as T cells and B cells, the functional relevance of P-gp to lymphocytes, and the regulatory mechanisms for induction of P-gp on these cells remain unclear.
We and others have reported that transcription of MDR-1 is directly regulated by human Y-box-binding protein-1 (YB-1), a MDR-1 transcription factor, and that activation of YB-1 is induced in response to genotoxic stresses (Ohga et al. 1998) such as ultraviolet light (Uchiumi et al. 1993a), anti-cancer agents (Kohno et al. 1989), serum starvation (Tanimura et al. 1992), heat shock (Miyazaki et al. 1992) and multiple drugs, including vinca alkaloids and corticosteroids (Chaudhary & Roninson 1993). However, the regulatory mechanisms of YB-1 activation and MDR-1 transcription in lymphocytes remain unclear. Furthermore, although MDR-1 is induced by various genotoxic or cytotoxic stimuli described above, it is poorly understood whether the activation stimuli such as cytokines induce MDR-1 transcription in lymphocytes. The present study was designed to investigate the transcriptional regulation of MDR-1 in lymphocytes, particularly in lymphocytes activated by interleukin (IL)-2.
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Results |
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We first examined the intracellular distribution of a transcriptional factor YB-1 in PBMCs by immunostaining using anti-YB-1 monoclonal antibody. Using confocal microscopic analysis, we observed that YB-1 was localized in the cytoplasm of PBMCs at basal conditions (Fig. 1A). Then YB-1 was translocated into the nucleus within 20 min after stimulation with IL-2. As shown in Fig. 1B, nuclear accumulation of YB-1 occurred in an IL-2-concentration-dependent manner within the range between 0.1 and 10 ng/mL. These results suggest that IL-2 activate the transcription factor YB-1.
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The nuclear localization of YB-1 is closely associated with MDR-1 gene expression in a human breast cancer cell line (Bargou et al. 1997). To test whether activated YB-1 directly affects MDR-1 gene expression in response to IL-2, we examined the expression of MDR-1 mRNA by reverse transcription-polymerase chain reaction (RT-PCR). There was a substantial increase in MDR-1 mRNA relative to ß2-microglobulin mRNA in PBMCs activated with IL-2, compared with PBMCs at basal condition (Fig. 2C). This result was consistent with that observed in the mobility shift assay and translocation of YB-1 in immunostaining.
Up-regulation of P-glycoprotein on IL-2-activated lymphocytes
Preliminary experiments showed that P-gp expression on PBMCs reached maximum levels within 3 h of incubation with IL-2 and then diminished to basal levels after 24 h of incubation (data not shown). Therefore, we evaluated the expression of P-gp after a 4-h stimulation in the following studies. We observed that expression of P-gp was augmented in a dose-dependent manner up to 10 ng/mL of IL-2 (Fig. 3). Furthermore, to investigate the expression of P-gp on lymphocytes in detail, we next performed two-colour analysis using anti-CD4, -CD8, and -CD19 antibodies and examined P-gp expression on each subset of lymphocytes. Flow cytometric analysis showed that P-gp expression was significantly augmented by IL-2 on CD4+, CD8+ and CD19+ cells (Fig. 4).
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To determine whether YB-1 is directly coupled with IL-2-induced MDR-1 gene activation, we assessed the P-gp on PBMCs transfected with YB-1 anti-sense expression plasmid (PRC/CMV AS) or control vacant vector and compared the levels of P-gp expression on PBMCs incubated with or without IL-2. In comparison with vector alone, introduction of YB-1 anti-sense significantly reduced the expression of P-glycoprotein on PBMCs. IL-2 stimulation significantly increased the expression of P-gp on control cells, but the inducibility was abolished by transfection of YB-1 anti-sense (Fig. 5).
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To investigate the association between expression of P-gp and exclusion of drugs through P-gp, intracellular and extracellular concentration of dexamethasone was determined as described in Experimental procedures. IL-2 simulation resulted in the significant decrease of intracellular dexamethasone on PBMCs during observed periods (3–30 min) as shown in Fig. 6. To confirm the functional involvement of P-gp in the decrease of intracellular dexamethasone, we added cyclosporin A, a competitive inhibitor of P-gp, to IL-2 stimulated PBMCs. Excretion of dexamethasone in PBMCs inhibited by cyclosporin A in a concentration-dependent manner, up to 100 ng/mL of cyclosporin A (Fig. 7).
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Discussion |
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Furthermore, when dexamethasone was added to lymphocytes in vitro, excretion of dexamethasone was up-regulated and intracellular dexamethasone was reduced in IL-2-activated lymphocytes, corresponding to higher expression of P-gp. However, the addition of cyclosporin A to the culture, a competitive inhibitor of P-gp (List et al. 2002; Fedeli et al. 1989; Zacherl et al. 1994), inhibited excretion of dexamethasone in a dose-dependent manner, implying that P-gp induced on IL-2-activated lymphocytes play a functional role in the drug-excretion in lymphocytes, which leads to resistance to multiple drugs such as corticosteroids.
The role of MDR-1 and P-gp in tumour cells and haematopoietic malignancies is well known and the regulation of MDR-1 gene by IL-2 varies among cancer cells (Burton et al. 1994; Johannessen et al. 2000; Stein et al. 1996). However, P-gp expression, its functional relevance and the regulatory mechanisms of P-gp in normal or activated lymphocytes still remain unclear. SLE, a representative systemic autoimmune disease, is characterized by activation of T cells and B cells and the presence of activated helper T cells and the Th1/Th2 imbalance are involved in the pathogenesis of SLE (Huang et al. 1988; Akahoshi et al. 1999). We and others also reported that the numbers of lymphocytes producing cytokines such as IL-2 and serum levels of these cytokines are increased in patients with active SLE (Tanaka et al. 1988; Dau et al. 1991; Horwitz et al. 1994). The increased IL-2 levels in SLE patients usually fall below the threshold, whereas they remain at high levels in patients who respond poorly to treatments and continue to have high disease activity. However, the mechanisms of drug resistance in SLE are largely unclear.
We here propose that the sequential events in IL-2 activated lymphocytes, consisted of YB-1 activation by IL-2, MDR-1 transcription, P-gp expression and excretion of intracellular dexamethasone, could be relevant to poor responsiveness to several immunosuppressants and corticosteroids in SLE patients with a high disease activity. Indeed, we observed that P-gp expression on lymphocytes markedly increased in active SLE patients (data not shown). We further propose that cyclosporin A could be used not only as an inhibitor of NF-AT-dependent IL-2 transcription but also as the competitive inhibitor of P-gp in activated lymphocytes of SLE patients, since dexamethasone concentration in IL-2-activated lymphocytes was recovered by low dose of cyclosporin A. Taken together, the regulation of P-gp on lymphocytes could provide a novel therapeutic strategy in patients with multidrug resistance, including patients in active stages of systemic autoimmune diseases as well as progressive states of leukaemia/lymphoma.
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Experimental procedures |
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We isolated peripheral blood mononuclear cells (PBMCs) from healthy donors by density gradient centrifugation using Lymphocyte Separation Medium 50494 (Pharmacia Biotech, Uppsala, Sweden) as previously described (Tanaka et al. 1997; 1999). We confirmed that purified PBMCs containing more than 90% of lymphocytes (CD4, CD8 or CD20 positive cells) and less than 10% of CD14 positive monocytes by immunostaining.
The study was approved by the human subject research committee of the University of Occupational and Environmental Health, School of Medicine, and informed consent was obtained from all donors who enrolled in the study.
Immunostaining and confocal microscopy analysis
PBMCs were plated in a 12-well culture dish (2 x 105 cells/well) and incubated for 20 min at 37 °C in the presence or absence of 10 ng/mL of recombinant human IL-2 (Becton Dickinson Labware, Mountain View, CA, USA) in RPMI 1640 (Nissui, Tokyo, Japan) containing 5% FCS (Bio-Pro, Karlsruhe, Germany). The cells were then treated with 4% formaldehyde (Sigma Aldrich Japan, Tokyo) in FACS medium for 15 min and then with 0.1% saponin (Sigma Aldrich Japan) in FACS medium. The cells thus obtained were incubated with a specific antibody (Ab) against YB-1 (a binding protein to the Y box and CCAAT box, which is critical for the cis-regulatory element that regulates drug-induced MDR-1 gene expression (Ohga et al. 1998)) for 30 min at 4 °C. Subsequently, the cells were incubated with FITC-conjugated anti-rabbit IgG Ab at saturating concentrations in FACS medium. We performed confocal analysis of YB-1 using a LSM 410 invert Laser Scan Microscope (Carl Zeiss Microscope Systems, Germany).
Gel shift assay
Nuclear extracts from PBMCs were prepared as previously described (Ohga et al. 1998) and then incubated with or without 10 ng/mL of recombinant human IL-2. In the next step, 4 µg of nuclear protein were preincubated for 20 min at room temperature in 15 µL of buffer (10 mM Tris-HCl, pH 7.5, 1 mM ethylenediaminetetraacetic acid [EDTA], 1 mM 2-mercaptoethanol, 4% glycerol, and 40 mM NaCl) containing 0.5 µg of poly(dI-dC) (Pharmacia Biotech, Uppsala, Sweden) and a P32-end-labelled double-stranded oligonucleotide containing the YB-1 consensus binding site (5'-GGGCAGTTTTAGCCAGCTCCTCCCTA-3', 5'-GGGGTAGGGAGGAGCTGGCTAAAACT-3') as previously described (Dignam et al. 1983). The reaction mixtures were electrophoresed on 4% polyacrylamide gels in 0.25 x TAE buffer. For the supershift experiments, nuclear proteins were incubated with a specific antibody (Ab) against YB-1 (a binding protein to the Y box and CCAAT box, which is critical for the cis-regulatory element that regulates drug-induced MDR-1 gene expression (Ohga et al. 1998) before adding the P32-end-labelled double-stranded oligonucleotide containing the YB-1 consensus binding site (YB-1 oligo). For the cold competition assay, 25-fold molecular excess of double stranded YB-1 oligo or irrelevant double stranded oligonucleotide were preincubated with nuclear extract before the addition of hot probes.
Reverse transcription-polymerase chain reaction
After 4 h of incubation with or without 10 ng/mL of IL-2, total cellular RNA from PBMCs was isolated by a single step isolation procedure with ISOGEN (Wako, Osaka, Japan) and stored purified total RNA at –80 °C. Five hundred ng of total RNA were reverse transcribed at 42 °C for 30 min. Amplification with specific primers for MDR-1 and ß2 microglobulin was performed in an iCycler (Bio-Rad, Richmond, CA, USA) for 30 cycles of 45 s at 94 °C for denaturing, 45 s at 55 °C for annealing and 90 s at 72 °C for extension. The primer sequences were as follows: human ß2-microglobulin forward 5'-ACCCCCACTGAAAAAGATGA-3', reverse 5'-ATCTTCAAACCTCCATGATG-3'; human MDR-1 forward 5'-CCCATCATTGCAATAGCAGG-3', reverse 5'-GTTCAAACTTCTGCTCCTGA-3'. Amplified products were electrophoresed with Marker 4 (Nippon Gene, Tokyo, Japan) on 3% agarose gels.
Flow cytometric analysis
Staining and flow cytometric analysis of PBMCs were conducted by standard procedures as previously described using a FACScan (Becton Dickinson) (Tanaka et al. 1997; 1999,). Briefly, PBMCs (2 x 105 cells/well) were initially incubated with polyclonal 
-globulin (10 µg/mL, Yoshitomi Pharmaceutical Co.) for the blocking of Fc-receptors and then incubated with MRK-16, a specific monoclonal antibody (mAb) against P-gp (Hamada & Tsuruo 1986), followed by FITC-conjugated anti-mouse IgG Ab (Fujisawa, Osaka, Japan) in FACS medium consisting of phosphate-buffered saline (PBS), 0.5% HSA, and 0.2% NaN3 (Sigma Aldrich Japan). For the two-colour analysis, we incubated PBMCs with phycoerythrin (PE)-conjugated CD4 mAb, CD8 mAb or CD19 mAb (Fujisawa, Osaka, Japan) after blocking of free anti-mouse IgG-binding sites with irrelevant antibodies. Monoclonal antibodies-two-colour-stained cells were detected by electronic gating based on their CD4, CD8 or CD19 expression using a FACScan. Amplification of mAb-binding was provided by a three-decade logarithmic amplifier. Quantification of the cell surface antigens on one cell was performed using QIFIKIT beads (Dako, Kyoto, Japan) as reported previously (Tanaka et al. 1996).
Transfection of anti-sense oligonucleotides of YB-1 in PBMCs
YB-1 anti-sense expression plasmid (PRC/CMV AS) was constructed as previously described (Ohga et al. 1996). We transfected 2 µg of PRC/CMV AS or control vacant vector into 2 x 106 PBMCs in a six-well culture dish using a cationic liposome-mediated transfection method, with cationic lipid reagents (DMRIE-C, Life Technologies, Rockville, MD, USA) according to the instructions provided by the manufacturer (Itoh et al. 1993; Rodriguez-Viciana et al. 1997; Tamada et al. 1997). Forty-eight h after transfection, the cells were used for the following experiments.
Dexamethasone accumulation
[14C]n-Butanol(Toho Biochemical, Tokyo, Japan; 1.61 mCi/mmol) diluted with unlabelled butanol (Sigma Aldrich Japan) at a concentration of 0.5 MBq/mL [3H]-dexamethasone (PerkinElmer Life Sciences, Boston, MA. USA; 40.0 Ci/mmol) was dissolved in Dimethyl sulphoxide (DMSO; Nacalai tesque, Tokyo, Japan) before diluting with PBS (final concentration of DMSO was 0.1%). PBMCs incubated with or without 10 ng/mL of IL-2 for 4 h at 37 °C were resuspended in PBS with 7 mM of dextrose for ATP supply, which is dispensable in this assay (Richard & John 1993), at a cell density of 5 x 106 cells/mL. In the next step, PBMCs incubated with 5.0 x 10–5 M of [14C]n-Butanol and 3.0 x 10–8M of [3H]-dexamethasone for 0-30 min time range at 37 °C. For competitive studies with cyclosporin A, PBMCs were incubated with 0-100 ng/mL of cyclosporin A (Novartis Pharmaceutical, Japan Co., Tokyo, Japan) for 15 min before incubated with [14C]n-Butanol and [3H]-dexamethasone. Cyclosporin A was dissolved in DMSO before diluting with PBS (final concetration of DMSO was 0.03%). After incubation with IL-2 and cyclosporin A, 100 µL of aliquots were layered on 80 µL of the mixture of lauryl bromide and silicone oil (mixture ratio 2 : 1, Nacalai tesque, Tokyo, Japan) in an Eppendorf tube (Assist, Tokyo, Japan). After centrifugation at 10 000 r.p.m. for 2 min, the aliquots were rapidly frozen in liquid nitrogen, the frozen tube was cut between medium-mixture borders. We thereby obtain an upper layer as medium fraction and a lower layer as cell fraction. The obtained cell fractions were melted with soluene-350 and 10 mL of HIONIC-FLUOR (Packard, Meriden, USA) was added. The medium fractions were mixed with 10 mL of mixtures of toluene (Wako, Osaka, Japan), methanol (Wako, Osaka, Japan), ethylene glycol monoethyl ether (Nacalai tesque, Tokyo, Japan) and PERMAFLUOR (Packard, Meriden, USA; mixture ratio 200 : 50 : 50 : 12). Radioactivity of each fraction was counted with scintillation counter. C/M ratio, which is an index of intracellular dexamethasone concentration and extracellular concentration ratio, was computed using the following formula: C/M ratio = [(3H in cell fraction/14C in cell fraction)/(3H in medium fraction/14C in medium fraction)].
Statistical analysis
Student's t-test was used to compare data between two groups. One-way ANOVA and Bonferroni correction were used to compare data between three or more groups. Values are expressed as mean ± SD P < 0.05 was considered statistically significant.
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Acknowledgements |
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Footnotes |
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* Correspondence: E-mail: tanaka{at}med.uoeh-u.ac.jp
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References |
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Received: 16 August 2004
Accepted: 14 September 2004
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) or without (
) 10 ng/mL of IL-2 for 4 h (D). Each value represents the number of molecules expressed per cell, calculated using standard QIFIKIT beads. Data represent mean ± SD of five independent experiments. Statistical analysis was performed using the paired t-test. *P < 0.05, **P < 0.01.
) 10 ng/mL of IL-2 for 4 h. Each points represent mean ± SD of five independent experiments. Statistical analysis was performed using the non-paired t-test. *P < 0.05, **P < 0.01.