HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS		QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

J. Biol. Chem., Vol. 276, Issue 30, 28562-28569, July 27, 2001

This Article Abstract Full Text (PDF) All Versions of this Article: 276/30/28562    most recent M100311200v1 Submit a Letter to Editor Alert me when this article is cited Alert me when eLetters are posted Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Stein, U. Articles by Royer, H.-D. Search for Related Content PubMed PubMed Citation Articles by Stein, U. Articles by Royer, H.-D. Social Bookmarking                      What's this?

Hyperthermia-induced Nuclear Translocation of Transcription Factor YB-1 Leads to Enhanced Expression of Multidrug Resistance-related ABC Transporters^*

Ulrike Stein^§, Karsten Jürchott, Wolfgang Walther, Stephan Bergmann, Peter M. Schlag^¶, and Hans-Dieter Royer

From the  Max-Delbrück Center for Molecular Medicine, Robert-Rössle Strasse 10, 13092 Berlin, Germany; ^¶ Charité, Humboldt-University, Campus Berlin-Buch, Robert-Rössle Clinic, Lindenberger Weg 80, 13122 Berlin, Germany; and the  Institute for Transplantation Diagnostics and Cell Therapy, Heinrich-Heine University Düsseldorf, Moorenstrasse 5, 40225 Düsseldorf, Germany

Received for publication, January 12, 2001, and in revised form, May 2, 2001

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Genotoxic stress leads to nuclear translocation of the Y-box transcription factor YB-1 and enhanced expression of the multidrug resistance gene MDR1. Because hyperthermia is used for the treatment of colon cancer in combination with chemoradiotherapy, we investigated the influence of hyperthermia on YB-1 activity and the expression of multidrug resistance-related genes. Here we report that hyperthermia causes YB-1 translocation from the cytoplasm into the nucleus of human colon carcinoma cells HCT15 and HCT116. Nuclear translocation of YB-1 was associated with increased MDR1 and MRP1 gene activity, which is reflected in strong efflux pump activity. However, a combination of hyperthermia and drug treatment effectively reduced cell survival of the HCT15 and HCT116 cells. These results demonstrate that activation of MDR1 and MRP1 gene expression and increased efflux pump activity after hyperthermia were insufficient to cause an increase in drug resistance in colon cancer cell lines. The ability of hyperthermia to abrogate drug resistance in the presence of an increase in functional MDR proteins may provide an explanation for the efficacious results seen in the clinic in colon cancer patients treated with a combination of hyperthermia and chemotherapy.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Multidrug resistance (MDR)¹ of human malignant tumors represents a major cause of cancer chemotherapy failure. The development of the MDR phenotype is often associated with increased expression of certain ATP-binding cassette transporters (ABC transporters) such as P-glycoprotein and MRP1. The multidrug resistance gene (MDR1) encodes P-glycoprotein, which causes classical MDR. In contrast, the MRP1 gene encodes the multidrug resistance-related protein (MRP1), which is associated with an atypical non-P-glycoprotein-dependent MDR phenotype (1). Although both transporter proteins function as drug-efflux pumps, they share only 15% amino acid homology, transport a non-identical spectrum of anticancer substrates, and show a different distribution in human normal and cancer tissues (2, 3).

Colorectal tumors are one of the most prevalent human malignancies and are highly drug resistant because of an MDR phenotype. Intrinsic expression of the ABC transporters P-glycoprotein and MRP1 in colon cancer has been reported (4-6). To improve the response of colon cancer to chemotherapy, other treatment modalities such as radiation and hyperthermia when used in combination with chemotherapy may be able to increase the efficacy of cancer chemotherapy (7). However, environmental stresses such as drugs, radiation, and hyperthermia harbor the potential to induce or to enhance the MDR phenotype. Induction of MDR1 gene expression by exposure to drugs (8-10) or radiation (11) were described earlier. In contrast, the influence of hyperthermia on the expression of MDR genes has not been clearly established. Two groups reported a heat-induced elevation of MDR1 gene expression, either observed following a single treatment or after repeated treatments with heat (12, 13). Induction of MRP1 expression by drugs/chemicals has also been reported (3); however, the effects of radiation and hyperthermia on MRP1 gene expression have not been explored.

Signal transduction pathways that respond to external stimuli have been extensively investigated within drug-resistant cells. Involvement of YB-1 transcription factor (14) in mediating the effects of different external stimuli has been described for a variety of chemicals and drugs such as cisplatin, mitomycin C, etoposide and also for UV light (15-17). YB-1 belongs to the family of Y-box transcription factors, which were identified by their interaction with inverted CCAAT-boxes (Y-box; 14). Translocation of YB-1 from the cytoplasm into the nucleus was observed after treatment of cell cultures with DNA-damaging agents (15) as well as by UV irradiation (17). Nuclear expression of YB-1 has been correlated with intrinsic MDR1 expression for breast carcinomas (18) and for osteosarcomas (19). For colorectal carcinomas, an enhanced coexpression of YB-1 and topoisomerase II have recently been described when compared with mucosa (20).

In the present study, we examined the subcellular distribution of YB-1 in colon carcinoma cells before and after treatment with hyperthermia. Our results show that hyperthermia causes nuclear accumulation of YB-1 and concomitant increase in MDR1 and MRP1 gene expression resulting in efflux pump activity. We further show that the increase in MDR1 and MRP1 gene expression and efflux pump activity after hyperthermia were unable to reduce the amount of drug in the cells to prehyperthermia levels. Even though treatment with hyperthermia resulted in an increase in MDR1 and MRP1 expression and function in HCT116 and HCT15 cells, paradoxically an enhancement of drug resistance was not observed. These observations are in accord with the clinical experience that demonstrates hyperthermia in combination with chemotherapy is an effective treatment for colon cancer.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Cell Lines and Hyperthermia-- The human colorectal carcinoma cell lines HCT15 and HCT116, which possess different sensitivities to MDR-related drugs (highly resistant HCT15, moderately resistant HCT116; Ref. 21), were cultured as described previously (22). Hyperthermia was performed at 40 °C and 43 °C for 15, 30, 60, and 120 min. The human breast epithelial cell line HBL-100 was cultured in RPMI 1640 supplemented with 10% fetal calf serum and L-glutamine (200 µg/ml) at 37 °C, 5% CO₂. The HBL-100 cells stably transfected with pcDNA6/YB-1 were grown with blasticidine (Invitrogen, Groningen; 5 µg/ml once a week).

Immunofluorescence Analysis-- Cells were grown on slides, fixed with acetone/methanol, and preincubated with phosphate-buffered saline containing 1.5% horse serum (30 min; Vector Laboratories, Burlingame, CA). Cells were incubated with the polyclonal anti-YB-1 antibody (1:200, 30 min; Ref. 18) and with an anti-rabbit IgG-fluorescein F(ab')₂ fragment (1:200, 30 min; Roche Diagnostics, Mannheim, Germany). Specificity of the polyclonal anti-YB-1 antibody was proofed by peptide competition assay and by Western blot using whole cell lysates. For staining of nuclei 4,6-diamidino-2-phenylindole (DAPI; Roth, Karlsruhe, Germany) was added in the last incubation step. Staining was evaluated using a fluorescence microscope (Leica, Bensheim, Germany).

Confocal Laser Scanning Microscopy-- The cells were double-labeled with the polyclonal antibodies against YB-1 (1:200) and monoclonal antibodies against lamin A/C (1:50; Santa Cruz Biotechnology, Inc., Santa Cruz, CA) for 60 min and were incubated with an anti-rabbit IgG-fluorescein F(ab')₂ fragment (1:200) and an anti-mouse IgG-rhodamine F(ab')₂ fragment (1:100; Roche) for 30 min. Analysis of YB-1 subcellular distribution was performed by confocal laser scanning microscopy using the LSM 410 (Zeiss, Jena, Germany; software version 3.80) at 1000-fold magnification.

Electrophoretic Mobility Shift Assay (EMSA)-- Nuclear extracts were prepared as described (23). Briefly, 5.0 µg of each nuclear extract was incubated for 30 min at 30 °C with the ³²P-endlabeled single or double-stranded oligonucleotide. The following oligonucleotides were used: 5'-TGAGGCTGATTGGCTGGGCA-3', derived from the human MDR1 promoter sequence 86 to 67 (Y-box 82 to 73, Ref. 24); 5'-ATTTTTCTGATTGGCCAAAG-3' derived from the human HLA-DR promoter (GenBank^TM accession number M23101, nucleotides 31-50); and the nonspecific competitor oligonucleotide 5'-CCCTGTCACTTGGCCCCGCC-3' derived from the human cyclin E promoter (GenBank^TM accession number L48996, nucleotides 987-1006). The DNA-protein complexes were resolved on native 4% polyacrylamide gel electrophoresis. For autoradiography, X-Omat AR film (Kodak) was exposed overnight.

Generation of HBL-100 Transfectants with Stable Expression of a V5-tagged YB-1 cDNA-- YB-1 cDNA (25, 26) was removed from pUC19 using the EcoRI restriction endonuclease and inserted into the EcoRI site of the vector pcDNA6 (Invitrogen) thereby ensuring inframe positioning with the V5-histidine Tag of the pcDNA6 plasmid (pcDNA6/YB-1). Transfection of HBL-100 cells with pcDNA6/YB-1 was performed using LipofectAMINE (Life Technologies, Inc., Karlsruhe, Germany), and selection was done with 5 µg/ml blasticidine (Invitrogen). After 14 days, single clones were isolated. Expression of recombinant YB-1 was analyzed by immunofluorescence and Western blot analyses using monoclonal antibodies against the V5 Tag (Invitrogen).

Transient Transfection and Chloramphenicol Acetyltransferase (CAT)-ELISA-- MDR1 or MRP1 promoter-driven CAT activity was analyzed in HBL-100 cells and in HBL-100/YB-1 cells that overexpress a YB-1 cDNA under control of the cytomegalovirus promoter (pCDNA6/YB-1) by transient transfection of these cells with the following CAT reporter plasmids: for MDR1, the pCAT-MDR plasmid harboring a human MDR1 promoter sequence (207 to +158, Ref. 10; inclusively Y-box from 82 to 73, Ref. 24) in pCAT-Basic (Promega, Mannheim, Germany). MRP1 promoter-harboring CAT plasmids were kindly obtained from M. S. Center, Kansas State University, Manhattan, KS (27): pCAT-MRP/A (2008 to +103), and the deletion mutants pCAT/MRP/I (411 to +103;) and pCAT-MRP/J (91 to +103). The plasmids pCAT-Basic (promoter-less), pCAT-Control (with SV40-promoter; Promega), and transfection w/o DNA served as controls. For the transient transfections, 10 µg of plasmid DNA and 15 µg of lipofectin (Life Technologies, Inc.) were used. Cells were harvested after 96 h and lysed by 5 cycles of freeze-thaw. Cell lysates were centrifuged at 14000 rpm at 4 °C for 10 min, and 200 µl of each lysate were subjected to the CAT-ELISA in duplicate (Roche Molecular Biochemicals). Absorbance was measured at 492 nm, and CAT values were calculated from the CAT standard curve using the EasySoftG200/Easy-Fit software (SLT-Labinstruments, Crailsheim, Germany). The amount of CAT protein was normalized to the protein content of the respective lysate.

RNA Isolation and Real Time RT-PCR-- HCT116 and HCT15 cells were harvested prior to or 0, 1, 2, 3, and 4 h posthyperthermia (43 °C for 2 h). The nuclei were isolated as described above (23). Isolation of total RNA was performed with the high pure RNA isolation kit (Roche Molecular Biochemicals) including a DNase incubation step. RNA concentrations were measured by using the RiboGreen RNA quantitation kit (Molecular Probes via MoBiTec, Göttingen, Germany) in a microplate reader and were calculated in duplicate from ribosomal RNA standard curves using the EasySoftG200/Easy-Fit software (SLT-Labinstruments). Quantitative real time RT-PCR was carried out with 50 ng of nuclear RNA (LightCycler, Roche Molecular Biochemicals). MDR1- and MRP1-specific primers were used that amplified a 167-bp product for MDR1 and a 291-bp product for MRP1 (22, 28), which were detected via intercalation of the fluorescent dye SYBR-Green (LightCycler RNA Amplification Kit, Roche Molecular Biochemicals). Copy number quantification was performed by serial dilutions of MDR1 and MRP1 transcripts (10⁵ to 10⁸ copies). In each run, total RNA from MDR1- or MRP1-overexpressing cell lines were simultaneously used. For MDR1 quantification, the cell line KBV-1 (kindly provided by M. M. Gottesman, National Cancer Institute, Bethesda, MD) was employed. For MRP1, the cell line MCF-7/VP16 (kindly obtained from E. Schneider, Wadsworth Center, Albany, NY) was used. Ratios were built in each run: MDR1 or MRP1 copy number within an unknown sample/MDR1 or MRP1 copy number of the MDR1- or MRP1-overexpressing resistant control cell line resulting in copy number/percent copy number of KBV-1 or MCF-7/VP16, respectively. To evaluate the expression of MDR1 and MRP1, we employed primers for the housekeeping genes porphobilinogen deaminase, amplifying a 121-bp RT-PCR product, which was previously used by others for normalization of MDR1 gene expression (29, 30). The quality of the RT-PCR products was controlled by melting point curve analysis.

Immunoflow Cytometry-- HCT116 and HCT15 cells were hyperthermia-treated at 40 °C or 43 °C for 1 or 2 h, respectively, and immunoflow cytometry was carried out prior to as well as 0, 1, 2, 12, 24, 48, 72, 96, and 120 h posthyperthermia. Incubation with monoclonal antibodies (for P-glycoprotein: MRK16, Syrinx-Diagnostika GmbH, Frankfurt, Germany, 1:100, and C219, Alexis Corporation, Grünberg, Germany, 2 µg per 5 × 10⁵ cells; for MRP1: MRPr1 and MRPm6, Cell Systems, St. Katharinen, Germany, 1:50), and with fluorescein-conjugated secondary antibodies (for MRK16, C219, MRPm6: a goat anti-mouse antibody, Becton Dickinson, San Jose, CA; for MRPr1, a rabbit anti-rat antibody, Sigma) have been performed as described (28). Fluorescence intensity of 1 × 10⁴ cells was measured with a FACScan flow cytometer (Becton Dickinson) and expressed as mean fluorescence/cell (difference from the mean fluorescence/cell for the antibody of interest and the respective isotype control antibody; CellQuest program, Becton Dickinson).

Adriamycin Assay-- Accumulation of the fluorescent drug adriamycin (Amersham Pharmacia Biotech, Freiburg, Germany) was used as a functional index of ABC transporter activity. HCT116 and HCT15 cells were hyperthermia-treated at 43 °C for 2 h and incubated in 50 µM adriamycin for 3 h prior to and 0, 1, 2, 12, 24, 48, 72, 96, and 120 h posthyperthermia. Measurement of fluorescence intensity of 1 × 10⁴ cells was performed as previously described (22).

Rhodamine Assay-- Hyperthermia-treated cells (43 °C, 2 h) were incubated for 15 min at 37 °C with rhodamine 123 (0.5 mg/ml; Sigma, Deisenhofen, Germany) at 0, 1, 2, 12, 24, 48, 72, 96, and 120 h posthyperthermia. After spinning, cells were split; one aliquot was kept at 4 °C (4 °C value), the other aliquot was incubated in rhodamine 123-free medium for another 1 h at 37 °C (37 °C value). Fluorescence intensity of 1 × 10⁴ cells per group was measured by using the FACScan immunoflow cytometer (Becton Dickinson, Cell Quest program). The net efflux was calculated as difference of the mean fluorescence/cell from the 4 °C values and the 37 °C values.

Cytotoxicity Assay-- For the MTT (3-(4, 5-dimethylthiazol-2yl)-2,5-diphenyltetrazolium bromide, Sigma, Deisenhofen, Germany, 5 mg/ml) colorimetric cytotoxicity assay, 8 × 10³ cells were plated into each well of 96-well microtiter plates and grown for 24 h. Hyperthermia was performed at 40 °C or 43 °C for 2 h, and cells were kept at 37 °C. After 3 days, cells were treated with adriamycin (500, 1000, 1500, 2000 ng/ml; Amersham Pharmacia Biotech, Freiburg, Germany) for another 3 days. Non-treated cells, hyperthermia-treated cells, and drug-treated cells served as controls.

Statistical Analysis-- For flow cytometry studies (immunoflow cytometry, adriamycin, and rhodamine assays) levels of statistical significance were evaluated with data from at least three experiments (each performed in duplicate) by using the non-parametric Mann-Whitney Rank Sum Test. The statistical significance of the chemosensitivity assay (MTT assay, performed in triplicate) was evaluated using the Student's t test. Statistical significance was set at the 0.05  value.

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Hyperthermia-induced Translocation of YB-1 Transcription Factor-- The localization of YB-1 was investigated in non-stressed and hyperthermia-treated (40 °C or 43 °C for 15, 30, 60, or 120 min, respectively) HCT116 and HCT15 cells. By means of immunofluorescence microscopy, YB-1 was detectable in the cytoplasm of the HCT116 (Fig. 1A) and HCT15 cells (Fig. 1E) under stress-free conditions. YB-1 was also observed in the nuclei of untreated HCT15 cells, which corresponded to the higher intrinsic drug resistance and to the higher intrinsic expression of MDR1 and MRP1 in the HCT15 cells compared with the HCT116 cells. Incubation of both cell lines at 43 °C led to an accumulation of YB-1 in the nuclei in a time-dependent manner with highest nuclear YB-1 accumulation observed immediately after the 2-h hyperthermia treatment (Fig. 1, C and G). Treatment of cells at 40 °C also resulted in a nuclear accumulation of YB-1 but at reduced levels than observed at 43 °C (data not shown). Translocation of YB-1 from the cytoplasm into the nucleus was more pronounced in HCT116 than in HCT15 cells (Fig. 1, C and G), which might indicate that hyperthermia-induced effects were cell type-specific and dependent on the intrinsic resistance status.

View larger version (60K): [in this window] [in a new window]   Fig. 1.   Indirect immunofluorescence (A-H) and confocal laser scanning microscopy (I-L) of YB-1 localization in HCT116 and HCT15 cells prior to and posthyperthermia. Hyperthermia was performed with the HCT116 (A-D, I, K) and HCT15 (E-H, J, L) cells at 43 °C for 2 h (C, D, G, H, K, L). Cells were stained using a peptide-specific polyclonal antibody against YB-1 (A, C, E, G; green, I-L) and a monoclonal antibody against lamin A/C (red, I-L). Staining of the nuclei was done using 4,6-diamidino-2-phenylindole (DAPI) (B, D, F, H).

Using confocal laser scanning microscopy, YB-1 was mainly detectable in the cytoplasm of non-stressed HCT15 and HCT116 cells and additionally to a low extent within the nuclei of HCT15 cells, which confirmed the observations made with immunofluorescence (Fig. 1, I and J). Highest rates of hyperthermia-induced YB-1 translocation were observed immediately posthyperthermia in both cell lines (Fig. 1, K and L). These results were verified by Western blot using cytoplasmic and nuclear fractions of non-treated and hyperthermia-treated cells. Furthermore, a block of de novo protein synthesis using cycloheximide prior to hyperthermia did not influence the heat-induced nuclear accumulation of YB-1 (data not shown). Based on these observations, we concluded that YB-1 will be activated under hyperthermic conditions resulting in a translocation of YB-1 into the nucleus.

Hyperthermia-induced Binding of YB-1 to MDR1 Promoter Sequences-- To examine whether nuclear translocation of YB-1 resulted in elevated binding activity to the human MDR1 promoter, nuclear extracts of non-treated and hyperthermia-treated HCT116 and HCT15 cells were analyzed by EMSA. Because it is known that YB-1 binds preferentially to single-stranded oligonucleotides (14), a radiolabeled single-stranded oligonucleotide containing the Y-box region from the MDR1 promoter sequence 86 to 67 was employed (Fig. 2, lanes 1-10). Retarded DNA-protein complexes were observed in the parental and in the hyperthermia-treated cells of both lines (lanes 1-4). To ensure that YB-1 interacts with the MDR1 promoter, supershift assays were performed. The preincubation of the nuclear extracts with increasing amounts of anti-YB-1 antibodies abolished protein-DNA binding, demonstrating that YB-1 is an integral part of this complex (data not shown).

View larger version (43K): [in this window] [in a new window]   Fig. 2.   EMSA with nuclear extracts of HCT116 and HCT15 cells prior to and posthyperthermia (43 °C for 2 h). Equal amounts (5 µg) of nuclear extracts were analyzed using radiolabeled single-stranded (lanes 1-10) or double-stranded (lanes 11-15) oligonucleotides, both originating from the Y-box of the human MDR1 promoter. For competition experiments, unlabeled oligonucleotides originating from the Y-box of the MDR1 promoter (lanes 5, 6), from the Y-box of the human HLA-DR promoter (lanes 7, 8), or from the human cyclin E promoter (nonspecific oligonucleotide; lanes 9, 10) were used in 50-fold and 100-fold excess, respectively.

In general, the signals for the YB-1-specific major DNA-protein complex were found to be higher in the non-treated and in the hyperthermia-treated HCT15 cells (lanes 3, 4) compared with the HCT116 cells (lanes 1, 2). The higher levels of YB-1 specific major DNA-protein complex in the HCT15 cells correspond to the higher intrinsic expression of MDR1 in the HCT15 cells. In both cell lines, hyperthermia resulted in an increase of the YB-1-specific DNA-protein complex (lanes 2, 4) demonstrating that translocation of YB-1 into the nucleus led to an increased binding to the MDR1 gene promoter.

Sequence specificity of binding to the MDR1 promoter Y-box was demonstrated by competition experiments. Competitor oligonucleotides containing the Y-box region of the MDR1 promoter prevented formation of a retarded YB-1-DNA complex (lanes 5 and 6). Identical results were obtained with a Y-box from the HLA-DR promoter (lanes 7 and 8). However, a control oligonucleotide from the cyclin E promoter did not affect YB-1 complexes (lanes 9 and 10).

Because the transcription factor NF-Y has similar binding sites as YB-1, the role of NF-Y was investigated under hyperthermic conditions. A radiolabeled double-stranded oligonucleotide was employed, which also originated from the Y-box region of the MDR1 promoter sequence (same sequence as the single-stranded oligo; Fig. 2, lanes 11-15). The figure shows that NF-Y activity was not affected by hyperthermia because no differences were found between either non-treated cell lines (lanes 12 and 14) or between the non-treated and hyperthermia-treated cells (lanes 12-15). Based on these observations, it is unlikely that NF-Y is involved in the hyperthermia-caused modulation of MDR1 expression in the analyzed cell lines.

MDR1 and MRP1 Promoter Activity in YB-1-transfected Cells-- Because translocation, nuclear accumulation, and binding of YB-1 to MDR1 promoter sequences were observed to be hyperthermia inducible, the effect of YB-1 on MDR1 and MRP1 promoter-driven CAT expression was examined in a breast epithelial cell line, which was engineered to overexpress YB-1 (HBL-100/YB-1). To investigate the function of YB-1 in MDR1 and MRP1 gene control, parental and YB-1-overexpressing HBL-100 cells were transfected with the MDR1 promoter-harboring CAT construct pCAT-MDR and with CAT constructs containing MRP1 promoter variants, pCAT-MRP/A, pCAT-MRP/I, and pCAT-MRP/J.

Transfection with pCAT-MDR led to a 30-fold increase of CAT protein expression in the parental and in the YB-1-overexpressing HBL-100 cells, when compared with the pCAT-Control-transfected parental or YB-1-overexpressing HBL-100 cells, respectively (Fig. 3). However, the MDR1 promoter-driven CAT protein expression was almost 3-fold higher in the YB-1-overexpressing cells when compared with the parental HBL-100 cells. Thus, MDR1 promoter-driven CAT expression is directly dependent on the amount of available YB-1 within the cells.

View larger version (17K): [in this window] [in a new window]   Fig. 3.   CAT reporter gene ELISA of MDR1 or MRP1 promoter-driven CAT expression in parental () and stably pcDNA6/YB-1-transfected () HBL-100 cells. HBL-100/YB-1 cells overexpress a YB-1 cDNA under the control of the CMV promoter (pcDNA6/YB-1). Transient transfection was performed using the plasmid pCAT-MDR harboring a MDR1 promoter sequence (207 to +158, inclusively Y-box from 82 to 73) within the plasmid pCAT-Basic. For MRP1, pCAT-MRP/A (2008 to +103) and the deletion mutants pCAT-MRP/I (411 to +103) and pCAT-MRP/J (91 to +103) were employed. pCAT-Basic (promoter-less), pCAT-Control (with SV40-promoter), and transfection w/o DNA served as controls. The amount of CAT protein was normalized to the protein content of the respective lysate.

Transfection with the MRP1 promoter-harboring constructs also resulted in an elevated CAT expression in YB-1-overexpressing HBL-100 cells compared with the parental cells (about 6-fold), which was rather similar for all three sublines transfected with the MRP1 promoter CAT constructs pCAT-MRP/A, -/I, and -/J. However, the amount of CAT protein in HBL-100/YB-1 cells was much lower in the MRP promoter transfectants (about one-tenth) compared with those of the pCAT-MDR transfectants.

The data obtained with the CAT reporter constructs indicate that both MDR1- and MRP1-driven CAT expression is dependent on the availability of YB-1, albeit to a somewhat different extent. Because the Y-box was identified within the MDR1 promoter but not within the MRP1 promoter, the MRP1 promoter may harbor additional sequences that bind YB-1. To prove this assumption, we used three overlapping single-stranded oligonucleotides representing the MRP1 promoter fragment J as competitors in EMSA with a Y-box from the MDR1 promoter. The common feature of these oligonucleotides is a GC content higher than 80%. Each of these MRP1 promoter oligonucleotides prevented formation of YB-1-containing retarded complexes, indicating that YB-1 specifically binds to the MRP1 promoter (data not shown). Thus, YB-1 is involved in regulating expression of both MDR1 and MRP1 genes.

Expression of ABC Transporters MDR1/P-glycoprotein and MRP1-- Hyperthermia-induced effects on the expression of MDR1 mRNA/P-glycoprotein and MRP1 mRNA/MRP1 were evaluated by real time RT-PCR and immunoflow cytometry. At the RNA level, hyperthermia-induced (43 °C for 2 h) increases in expression of the MDR1 and MRP1 genes have been observed in both colon carcinoma cell lines (Fig. 4). MDR1 mRNA expression increased by 4.5-fold (Fig. 4A), and MRP1 mRNA expression increased by 2-fold (Fig. 4B). Transcript levels of both genes were strongly increased immediately or 1 h posthyperthermia.

View larger version (35K): [in this window] [in a new window]   Fig. 4.   Influence of hyperthermia (43 ° for 24 h) on the expression of MDR1 mRNA/P-glycoprotein (A, C, E) and MRP1 mRNA/MRPI (B, D, F) in HCT116 () and HCT15 () cells. A and B, nuclear RNA was isolated prior to and 0, 1, 2, 3, and 4 h posthyperthermia, and quantitative real time RT-PCR was performed. Copy numbers of MDR1 or MRP1 were given as percentage of the MDR1-overexpressing KBV-1 cell line or of the MRP1-overexpressing MCF-7/VP16 cell line. CF, immunoflow cytometry was performed prior to and 0, 1, 2, 12, 24, 48, 72, 96, and 120 h posthyperthermia. For detection of P-glycoprotein, the monoclonal antibodies MRK16 (C) and C219 (E) were employed, and for MRP1, the monoclonal antibodies MRPr1 (D) and MRPm6 (F) were used.

Significant induction of P-glycoprotein was observed for HCT116 and HCT15 cells after treatment with hyperthermia at 43 °C for 2 h (Fig. 4, C and E). In HCT116 cells, P-glycoprotein expression was induced up to 5.5-fold when measured with MRK16 (p = 0.00216) and up to 9.5-fold when detected with C219 (p = 0.000583). In HCT15 cells, expression of P-glycoprotein was enhanced about 1.5-fold when determined with MRK16, or 3-fold when measured with C219 (p = 0.0286).

MRP1 expression was also found to be inducible by hyperthermia with the highest induction rates after treatment at 43 °C for 2 h (Fig. 4, D and F). In HCT116 cells, MRP1 expression was 6.5-fold (p = 0.00216) or 4.5-fold (p = 0.00216) increased when determined with MRPr1 or MRPm6, respectively. A 2.5-fold (MRPr1, p = 0.0286) or 1.7-fold (MRPm6, p = 0.0571) elevation of MRP1 was detected in HCT15 cells.

When hyperthermia was carried out at 40 °C or for shorter times at 43 °C, either little or no enhancement of P-glycoprotein and MRP1 expression was observed (data not shown). Thus, the hyperthermia-induced increase in ABC transporter expression occurred in a temperature- and time-dependent manner.

Functional Activity of ABC Transporters-- To evaluate whether the hyperthermia-induced expression levels of P-glycoprotein and MRP1 correspond with an increase in efflux pump function, adriamycin (Fig. 5A) and rhodamine (Fig. 5, B and C) were performed. Unexpectedly, accumulation of adriamycin was elevated within both cell lines at 120 h posthyperthermia (Fig. 5A): 1.4-fold in HCT116 (p = 0.0571) and 1.7-fold in HCT15 (p = 0.0286) when compared with the respective parental cell line, although an enhanced hyperthermia-induced expression of both ABC transporters was shown at these time points.

View larger version (20K): [in this window] [in a new window]   Fig. 5.   Influence of hyperthermia (43 °C for 2 h) on the functional activity of the ABC transporters in HCT15 (A, B) and HCT116 (A, C) cells. A, adriamycin assay; demonstrating the accumulation of adriamycin (50 µM) after 3 h in previously hyperthermia-treated cells as measured prior to, and 0, 1, 2, 12, 24, 48, 72, 96, and 120 h posthyperthermia. B and C, rhodamine 123 assay; demonstrating the accumulation of rhodamine 123 (0.5 mg/ml) after 15 min at 37 °C when cells were kept at 4 °C afterwards (4 °C value) versus the rhodamine accumulation when these cells extruded the drug for another h at 37 °C (37 °C value) as well as the net efflux (difference of 4 °C and 37 °C values). Rhodamine assay was performed prior to and 0, 1, 2, 12, 24, 48, 72, 96, and 120 h posthyperthermia.

To further analyze whether drug accumulation and efflux is increased under hyperthermic conditions, the rhodamine assay (15-min incubation in rhodamine, 1-h incubation in rhodamine-free medium) was performed. Although the overall accumulation of rhodamine was increased in the hyperthermia-treated cells, the net efflux was also increased in both cell lines. In HCT116 cells, a 4.5-fold elevation (p = 0.0794) and in HCT15 cells, a 2.3-fold increase (p = 0.0286) in the net efflux was determined, reflecting the functional activity of the hyperthermia-induced P-glycoprotein. However, hyperthermia-induced P-glycoprotein expression was apparently not sufficient to prevent drug accumulation in HCT15 and HCT116 colon carcinoma cells.

Chemosensitivity of Hyperthermia-treated Cells-- Next, chemosensitivity toward the MDR-associated drug adriamycin was analyzed in untreated and hyperthermia-treated HCT15 and HCT116 cells 3 days posthyperthermia (43 °C for 2 h; Fig. 6, A and B). Hyperthermia by itself did not significantly affect cell survival of HCT15 cells and led to 20% reduced cell survival of HCT116 cells. However, a dose-dependent decrease in cell survival was observed in the presence of adriamycin; for HCT15 within the concentration range of 1000-2000 ng/ml and for HCT116 within 500 and 2000 ng/ml. After treatment with 2000 ng/ml adriamycin, survival was 68% for HCT15 and 52% for HCT116 cells when compared with untreated cells. These values reflect the intrinsic resistance of the highly multidrug-resistant HCT15 cells and of the moderately multidrug-resistant HCT116 cells. Cell survival decreased significantly to 16% in HCT 15 cells when treated with the combination of hyperthermia and adriamycin when compared with adriamycin alone (p = 0.0007). The same observation was made for HCT116 cells by comparison of hyperthermia- and drug-treated cells versus exclusively drug-treated cells, showing a decrease in cell survival by 35% (p = 0.0067). Thus, the ID₅₀ (inhibitory dose, with respect to hyperthermia) or the IC₅₀ (with respect to drug treatment) was not reached with the sole treatment of either hyperthermia (43 °C for 2 h) or adriamycin alone (concentration range up to 2000 ng/ml), whereas the combination of both reached the ID/IC₅₀ within the same drug concentration range for both cell lines. Therefore, pretreatment with hyperthermia did significantly increase the cytotoxic effects of adriamycin in both colon carcinoma cell lines, confirming the data obtained with the functional assays. By contrast, mild hyperthermia (40 °C for 2 h) did not lead to any significant modulation of cell survival when used alone (compared with untreated cells) or in combination with adriamycin (compared with adriamycin-treated cells; data not shown).

View larger version (28K): [in this window] [in a new window]   Fig. 6.   Influence of hyperthermia on chemosensitivity toward the MDR-associated drug adriamycin of the HCT15 (A) and HCT116 (B) human colon carcinoma cells. Hyperthermia-treated (43 °C for 2 h) cells were kept at 37 °C. After 72 h, cells were treated with adriamycin at different concentrations (500, 1000, 1500, 2000 ng/ml) for another 72 h. The MTT assay was carried out, and cell survival of hyperthermia- and drug-treated cells () was calculated with respect to non-treated cells (100% ), hyperthermia-treated cells , and drug-treated cells (without hyperthermia ).

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Hyperthermia-induced translocation of the transcription factor YB-1 from the cytoplasm into the nucleus was observed in HCT15 and HCT116 cells and was found to be time- and temperaturedependent with highest translocation rates observed at 43 °C compared with 40 °C. The mechanism of YB-1 translocation as a reaction to external stress stimuli has also been reported after treatment of cell cultures with DNA-damaging agents (15) or UV irradiation (17). Thus, translocation caused by environmental stress factors is a general activation mechanism of YB-1. The activities of a variety of other transcription factors such as NF-B and STAT1 are also regulated by intracellular redistribution. Moreover, the nuclear translocation of DNA-binding proteins as a response to hyperthermia has also been reported for the heat shock transcription factors HSF1 and HSF3 (31).

It was previously shown that YB-1 is involved in the regulation of P-glycoprotein expression in human breast carcinomas and osteosarcomas (18, 19). The promoter of the MDR1 gene contains a Y-box, which is responsible for basal MDR1 expression (24). In this study, the direct hyperthermia-induced binding of YB-1 to the Y-box of the MDR1 gene promoter was demonstrated in HCT15 and HCT116 cells. Previous reports discussed the DNA double strand-binding protein NF-Y as a potential regulator of MDR1 expression acting via the CCAAT-box (32). NF-Y was also shown to be involved in mediating effects of several stimuli such as sodium butyrate or UV irradiation (33, 34). In this study, however, hyperthermia did not alter the binding of NF-Y to the Y-box of the MDR1 promoter, suggesting that NF-Y is not involved in hyperthermia-regulated transcriptional control of the MDR1 gene.

The crucial role of YB-1 in MDR1 regulation was demonstrated by using stably YB-1-transfected HBL-100 cells. In HBL-100/YB-1 cells MDR1 promoter-driven CAT gene expression was YB-1-dependent. However, YB-1 overexpression resulted in enhanced MRP1 promoter-driven CAT expression levels as well, although no Y-box motif was identified in the human MRP1 promoter (27). This suggested that YB-1 interacts with unknown MRP1 promoter elements. EMSA revealed that YB-1 binds to oligonucleotides derived from the MRP1 promoter fragment J. These oligonucleotides are all very GC-rich (about 80%). The interaction of recombinant YB-1 with similar GC-rich oligonucleotides was described recently (35). Therefore we conclude that YB-1 may interact with the MRP1 promoter directly and that the hyperthermia-induced translocation of YB-1 into the nucleus may result in an YB-1-dependent activation of the MRP1 promoter.

Our data show that after hyperthermia, P-glycoprotein and MRP1 protein levels were strongly up-regulated. It has been previously reported that heat shock activates MDR1 gene expression (12, 13). Our data provide a mechanism that shows how YB-1 links hyperthermia to gene activation. Furthermore, this is the first report to show that YB-1 is involved in regulating transcription of the MRP1 gene. Although several cis elements have been identified in the promoter of the MDR1 gene, which respond to environmental stress (36-38), no such elements were detected in the MRP1 promoter (27). Thus, the observed YB-1 interaction with the MRP1 promoter provides a potential mechanism how environmental stress leads to MRP1 gene activation.

Increased transcription of MDR1 and MRP1 in response to hyperthermia was associated with elevated levels of the corresponding proteins and strongly increased efflux pump activity. Despite this result, we observed accumulation of anticancer drugs in hyperthermia-treated colon cancer cells. In this case, however, increased pump activity did not lead to an enhanced MDR phenotype which has been described for chemoresistance mechanisms (39).

Furthermore, mechanisms other than the induction of the ABC transporters might possibly be involved in the development/regulation of the drug resistance; e.g. the hyperthermia-induced phosphorylation of P-glycoprotein (40) or heat-dependent factors regulating the membrane topology of a P-glycoprotein sequence (41). In addition, other protection mechanisms against environmental stress may be present in the colon carcinoma cells (6, 42). Moreover, general effects of thermal stress such as the induction of epithelial permeability and induction of apoptosis (43) may also affect survival.

In summary, we demonstrate here that hyperthermia induces YB-1 translocation from the cytoplasm into the nucleus leading to an enhanced YB-1 binding to the MDR1 promoter sequence, followed by an increase in the expression levels of MDR1/P-glycoprotein and MRP1/MRP1 expression. However, the hyperthermia-induced increases in these drug resistance genes and functional proteins did not result in drug resistance following treatment with adriamycin and hyperthermia. In contrast, combination of hyperthermia and drug treatment resulted in a significant reduction in cell survival of both colon carcinoma cell lines. Further studies using patient samples are required to evaluate the effect of hyperthermia and chemotherapy on MDR genes and proteins in a clinically relevant situation to assess the risk of inducing MDR proteins, which may potentially complicate subsequent chemotherapy (44). Moreover, analyses of patient samples would provide information necessary for determining the appropriate regimen of chemotherapy with respect to potential hyperthermia-induced resistance gene expression.

	ACKNOWLEDGEMENTS

We thank L. Malcherek and L. Bauer for excellent technical assistance. Critical reading of the manuscript by R. H. Shoemaker, National Cancer Institute, Frederick, MD and by C. M. Laurencot, National Cancer Institute, Bethesda, MD is gratefully acknowledged.

	FOOTNOTES

^* This work was supported by Grant SFB 273 from the Deutsche Forschungsgemeinschaft (to U. S.) and by a grant from the Berliner Krebshilfe (to H.-D. R.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

^§ To whom correspondence should be addressed: Max-Delbrück Center for Molecular Medicine, Robert-Rössle Strasse 10, 13092 Berlin, Germany. Tel.: 49-30-9406-3432; Fax: 49-30-9406-2780; E-mail: ustein@mdc-berlin.de.

Published, JBC Papers in Press, May 21, 2001, DOI 10.1074/jbc.M100311200

	ABBREVIATIONS

The abbreviations used are: MDR, multidrug resistance; ABC transporters, ATP-binding cassette transporters; CAT, chloramphenicol acetyltransferase; EMSA, electrophoretic mobility shift assay; MDR1, multidrug resistance gene; MRP1, gene that encodes the multidrug resistance-related protein MRP1; MTT, 3-(4,5-dimethylthiazol-2yl)-2,5-diphenyltetrazolium bromide; ELISA, enzyme-linked immunosorbent assay; RT-PCR, reverse transcription-polymerase chain reaction.

	REFERENCES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

CiteULike

Complore

Connotea

Del.icio.us

Digg

Reddit

Technorati

This article has been cited by other articles:

				S. Das, R. Chattopadhyay, K. K. Bhakat, I. Boldogh, K. Kohno, R. Prasad, S. H. Wilson, and T. K. Hazra Stimulation of NEIL2-mediated Oxidized Base Excision Repair via YB-1 Interaction during Oxidative Stress J. Biol. Chem., September 28, 2007; 282(39): 28474 - 28484. [Abstract] [Full Text] [PDF]

				K. Mantwill, N. Kohler-Vargas, A. Bernshausen, A. Bieler, H. Lage, A. Kaszubiak, P. Surowiak, T. Dravits, U. Treiber, R. Hartung, et al. Inhibition of the Multidrug-Resistant Phenotype by Targeting YB-1 with a Conditionally Oncolytic Adenovirus: Implications for Combinatorial Treatment Regimen with Chemotherapeutic Agents. Cancer Res., July 15, 2006; 66(14): 7195 - 7202. [Abstract] [Full Text] [PDF]

				T. Fujita, K.-i. Ito, H. Izumi, M. Kimura, M. Sano, H. Nakagomi, K. Maeno, Y. Hama, K. Shingu, S.-i. Tsuchiya, et al. Increased Nuclear Localization of Transcription Factor Y-Box Binding Protein 1 Accompanied by Up-Regulation of P-glycoprotein in Breast Cancer Pretreated with Paclitaxel Clin. Cancer Res., December 15, 2005; 11(24): 8837 - 8844. [Abstract] [Full Text] [PDF]

				C. R.C. van Roeyen, F. Eitner, S. Martinkus, S. R. Thieltges, T. Ostendorf, D. Bokemeyer, B. Luscher, J. M. Luscher-Firzlaff, J. Floege, and P. R. Mertens Y-Box Protein 1 Mediates PDGF-B Effects in Mesangioproliferative Glomerular Disease J. Am. Soc. Nephrol., October 1, 2005; 16(10): 2985 - 2996. [Abstract] [Full Text] [PDF]

				Z. H. Lu, J. T. Books, and T. J. Ley YB-1 Is Important for Late-Stage Embryonic Development, Optimal Cellular Stress Responses, and the Prevention of Premature Senescence Mol. Cell. Biol., June 1, 2005; 25(11): 4625 - 4637. [Abstract] [Full Text] [PDF]

				K. Matsumoto, K. J. Tanaka, and M. Tsujimoto An Acidic Protein, YBAP1, Mediates the Release of YB-1 from mRNA and Relieves the Translational Repression Activity of YB-1 Mol. Cell. Biol., March 1, 2005; 25(5): 1779 - 1792. [Abstract] [Full Text] [PDF]

				S. H. Thorne, G. Brooks, Y.-L. Lee, T. Au, L. F. Eng, and T. Reid Effects of Febrile Temperature on Adenoviral Infection and Replication: Implications for Viral Therapy of Cancer J. Virol., January 1, 2005; 79(1): 581 - 591. [Abstract] [Full Text] [PDF]

				M. Kuwano, Y. Oda, H. Izumi, S.-J. Yang, T. Uchiumi, Y. Iwamoto, M. Toi, T. Fujii, H. Yamana, H. Kinoshita, et al. The role of nuclear Y-box binding protein 1 as a global marker in drug resistance Mol. Cancer Ther., November 1, 2004; 3(11): 1485 - 1492. [Abstract] [Full Text] [PDF]

				P. S. Holm, H. Lage, S. Bergmann, K. Jurchott, G. Glockzin, A. Bernshausen, K. Mantwill, A. Ladhoff, A. Wichert, J. S. Mymryk, et al. Multidrug-resistant Cancer Cells Facilitate E1-independent Adenoviral Replication: Impact for Cancer Gene Therapy Cancer Res., January 1, 2004; 64(1): 322 - 328. [Abstract] [Full Text] [PDF]

				K. Jurchott, S. Bergmann, U. Stein, W. Walther, M. Janz, I. Manni, G. Piaggio, E. Fietze, M. Dietel, and H.-D. Royer YB-1 as a Cell Cycle-regulated Transcription Factor Facilitating Cyclin A and Cyclin B1 Gene Expression J. Biol. Chem., July 18, 2003; 278(30): 27988 - 27996. [Abstract] [Full Text] [PDF]

				U. Raffetseder, B. Frye, T. Rauen, K. Jurchott, H.-D. Royer, P. L. Jansen, and P. R. Mertens Splicing Factor SRp30c Interaction with Y-box Protein-1 Confers Nuclear YB-1 Shuttling and Alternative Splice Site Selection J. Biol. Chem., May 9, 2003; 278(20): 18241 - 18248. [Abstract] [Full Text] [PDF]

				M. P. Nekrasov, M. P. Ivshina, K. G. Chernov, E. A. Kovrigina, V. M. Evdokimova, A. A. M. Thomas, J. W. B. Hershey, and L. P. Ovchinnikov The mRNA-binding Protein YB-1 (p50) Prevents Association of the Eukaryotic Initiation Factor eIF4G with mRNA and Inhibits Protein Synthesis at the Initiation Stage J. Biol. Chem., April 11, 2003; 278(16): 13936 - 13943. [Abstract] [Full Text] [PDF]

				K. Higashi, Y. Inagaki, N. Suzuki, S. Mitsui, A. Mauviel, H. Kaneko, and I. Nakatsuka Y-box-binding Protein YB-1 Mediates Transcriptional Repression of Human alpha 2(I) Collagen Gene Expression by Interferon-gamma J. Biol. Chem., February 7, 2003; 278(7): 5156 - 5162. [Abstract] [Full Text] [PDF]

				U. Stein, K. Jurchott, M. Schlafke, and P. Hohenberger Expression of Multidrug Resistance Genes MVP, MDR1, and MRP1 Determined Sequentially Before, During, and After Hyperthermic Isolated Limb Perfusion of Soft Tissue Sarcoma and Melanoma Patients J. Clin. Oncol., August 1, 2002; 20(15): 3282 - 3292. [Abstract] [Full Text] [PDF]

				L. A. Sonna, J. Fujita, S. L. Gaffin, and C. M. Lilly Molecular Biology of Thermoregulation: Invited Review: Effects of heat and cold stress on mammalian gene expression J Appl Physiol, April 1, 2002; 92(4): 1725 - 1742. [Abstract] [Full Text] [PDF]

				M. Safak, B. Sadowska, R. Barrucco, and K. Khalili Functional Interaction between JC Virus Late Regulatory Agnoprotein and Cellular Y-Box Binding Transcription Factor, YB-1 J. Virol., March 19, 2002; 76(8): 3828 - 3838. [Abstract] [Full Text] [PDF]

				P. S. Holm, S. Bergmann, K. Jurchott, H. Lage, K. Brand, A. Ladhoff, K. Mantwill, D. T. Curiel, M. Dobbelstein, M. Dietel, et al. YB-1 Relocates to the Nucleus in Adenovirus-infected Cells and Facilitates Viral Replication by Inducing E2 Gene Expression through the E2 Late Promoter J. Biol. Chem., March 15, 2002; 277(12): 10427 - 10434. [Abstract] [Full Text] [PDF]

This Article Abstract Full Text (PDF) All Versions of this Article: 276/30/28562    most recent M100311200v1 Submit a Letter to Editor Alert me when this article is cited Alert me when eLetters are posted Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Stein, U. Articles by Royer, H.-D. Search for Related Content PubMed PubMed Citation Articles by Stein, U. Articles by Royer, H.-D. Social Bookmarking                      What's this?