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[Cancer Research 66, 5007-5011, May 15, 2006]
© 2006 American Association for Cancer Research

Priority Reports

Novel 5' Untranslated Region Variants of BCRP mRNA Are Differentially Expressed in Drug-Selected Cancer Cells and in Normal Human Tissues: Implications for Drug Resistance, Tissue-Specific Expression, and Alternative Promoter Usage

Takeo Nakanishi1,2, Kimberly J. Bailey-Dell1, Bret A. Hassel1,3, Ken Shiozawa1,2, Daniel M. Sullivan5, Joel Turner5 and Douglas D. Ross1,2,4

1 The Program in Experimental Therapeutics, Marlene and Stewart Greenebaum Cancer Center; Departments of 2 Medicine and 3 Microbiology, University of Maryland School of Medicine; 4 Baltimore Veterans Medical Center, Baltimore, Maryland; and 5 Department of Interdisciplinary Oncology, Experimental Therapeutics Program, H. Lee Moffitt Cancer Center and Research Institute, Tampa, Florida

Requests for reprints: Takeo Nakanishi, University of Maryland Greenebaum Cancer Center, Room 9-031, Bressler Research Building, 655 West Baltimore Street, Baltimore, MD 21201. Phone: 410-328-3685; Fax: 410-328-6559; E-mail: tnakanishi{at}som.umaryland.edu.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
To investigate transcriptional activation of the breast cancer resistance protein gene (BCRP/ABCG2), we examined the 5' untranslated region of BCRP mRNA in cell lines with high BCRP transcriptional activity and in normal tissues. Human choriocarcinoma cells with high endogenous BCRP expression (JAR and BeWo) and human cancer cells (MCF-7 and Igrov1) and their BCRP-overexpressing, drug-selected, multidrug-resistant derivatives (MCF-7/AdrVp, Igrov1/MX3, and Igrov1/T8) were studied. Rapid amplification of 5'-cDNA ends-PCR (5'RACE-PCR) revealed at least three novel forms of the untranslated exon 1 (E1a, E1b, and E1c) that are spliced to a common exon 2, with differential expression of these splice variants in the drug-selected cell lines. Additionally, sequence analysis of the 5'RACE-PCR products revealed multiple transcriptional start sites for each variant, particularly in the drug-selected cells. The E1c isoform predominated in drug-selected MCF-7 cell lines and was translated more efficiently in MCF-7 cells than the E1a isoform. Varying patterns of expression of the exon 1 isoforms were observed in a variety of human tissues, suggesting that tissue-specific alternative promoters of BCRP exist. In summary, we find that BCRP overexpression in the drug-selected cells is accompanied by multiple transcriptional start sites and predominance of the more efficiently translated E1c isoform. The exon 1 variation we observe suggests that alternative promoters of the BCRP gene exist. (Cancer Res 2006; 66(10): 5007-11)


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Functional expression of the breast cancer resistance protein (BCRP/ABCG2) in human cancers and leukemia may cause multidrug resistance to a variety of cancer chemotherapeutic drugs (1–3). BCRP overexpression is frequently found in drug-selected cancer cells derived from various types of human solid tumors (1, 2, 4–6). To overcome multidrug resistance in cancer chemotherapy, it is important to understand the molecular mechanisms responsible for up-regulation of BCRP gene expression. BCRP transcription can be regulated via hypoxia (7) or estrogen (8) response elements located in the promoter region of the BCRP gene (9). However, the mechanisms underlying BCRP up-regulation in drug-resistant cancer cells remain unclear. In the present study, hence, we investigated alterations in the 5' untranslated region (UTR) of BCRP mRNA to provide clues to the mechanisms that regulate BCRP gene expression. Here, we present the differential expression of novel splice variants in the 5'UTR of BCRP mRNA in human breast cancer cell lines selected with Adriamycin and verapamil (MCF-7/AdrVp), in ovarian carcinoma cells selected with mitoxantrone (Igrov1/MX3) or topotecan (Igrov1/T8), and in normal human tissues. Interestingly, we also found evidence of multiple transcriptional start sites (TSS), predilection for the expression of a specific exon 1 splice variant in the 5'UTR of BCRP mRNA, and more efficient translation of mRNA containing that variant in the drug-selected cell lines. Exon 1 variation may also reflect the expression of BCRP in normal human tissues, suggesting that alternative promoters of the BCRP gene exist.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cell culture. Human breast cancer MCF-7 and MCF-7/AdrVp cells (1); ovarian carcinoma Igrov1, Igrov1/MX3, and Igrov1/T8 cells (4); and choriocarcinoma JAR and BeWo cells (9) were obtained and maintained as described previously.

Rapid amplification of 5'-cDNA ends-PCR. Total RNA was extracted from cells with Trizol (Invitrogen, Carlsbad, CA) and treated with DNase I. Rapid amplification of 5'-cDNA ends-PCR (RACE-PCR) was done with First Choice RLM-RACE kit (Ambion, Austin, TX). The 5' end of the BCRP transcript was amplified by nested PCR with forward primers included in the kit and BCRP gene–specific reverse primers designated as follows: outer primer, 5'- GCGGTGCTCCATTTATCAGA-3'; inner primer, 5'- TAGAAGGAGGAGTTGACATAAA-3' (RvEx4), respectively. The PCR products were visualized on 2% agarose gels (ultraPURE, Invitrogen) and were directly cloned for sequence analysis. These sequences are available at Genbank (accession nos. DQ459561-DQ459614; Supplementary Table S2).

Absolute quantification of BCRP E1a and E1c isoforms and total transcripts by quantitative reverse transcription-PCR. RNA was subjected to quantitative reverse transcription-PCR (RT-PCR; LightCycler, Roche, Indianapolis, IN), using SYBR Green I for detection of the PCR products. The sequences of the primers for total BCRP transcripts (designated FwTotal and RvTotal) were described previously (10). The forward primers used to detect the exon 1 splice variants E1a and E1c (designated FwE1a and FwE1c, respectively) are as follows: FwE1a, 5'-CAAACCCAGCTAGGTCAGACG-3'; FwE1c, 5'-GGTTAAGACCGAGCTCTATT-3'. These forward primers were paired with a common reverse primer RvEx4 (sequence described in 5' Rapid Amplification of cDNA Ends-PCR). Generally, DNase-treated RNA was subjected to the reverse transcription reaction for 25 minutes at 48°C followed by 35 PCR cycles of denaturation at 95°C for 1 seconds, annealing at 60°C for 15 seconds, and elongation at 72°C for 15 seconds. The absolute expression levels of total BCRP transcripts and each exon 1 isoform were estimated from a standard curve created by the linear calibration of the crossing points obtained from quantitative RT-PCR reactions of different known concentrations of authentic BCRP template. The percentage of a given isoform among total BCRP transcripts was determined as 100 x (expression of isoform) / (expression of total BCRP transcript).

Evaluation of translation efficiency. Three full-length BCRP cDNAs were prepared that contained either E1a (nucleotides –393 to –291), E1b (nucleotides +244 to +338), or E1c (nucleotides +292 to +529) as exon 1. MCF-7 cells were transfected for 40 hours with pcDNA3 vector (Invitrogen) constructs that contained these BCRP cDNAs downstream from the cytomegalovirus (CMV) promoter, and then cellular lysates were prepared. Protein expression of BCRP or the neomycin resistance gene NPT II contained in the pcDNA3 plasmid under control of the SV40 promoter was analyzed by Western blot, using the BXP-21 antibody to BCRP (Chemicon, Temecula, CA) or the NPT II antibody (Upstate, Charlottesville, VA), respectively.

RT-PCR of normal human tissues. Snap-frozen samples of normal human tissue were obtained from the University of Maryland Greenebaum Cancer Center Tissue Bank shared service in accordance with our Institutional Review Board. These samples were from 11 organs, each from different individuals. Total RNA was reverse-transcribed with PowerScript (BD Clonetech, Palo Alto, CA) using an oligo-dT16 primer followed by 36 PCR cycles in the presence of the forward primers for E1a or E1c and the reverse primer RvEx4. The forward primer for E1b (FwE1b) was 5'-GGTTCCGGGCGCGCAGGAGG-3'. The primers used for total BCRP mRNA expression were as described in Absolute Quantification of BCRP E1a and E1c Isoforms and Total Transcripts by Quantitative Reverse Transcription-PCR. PCR products were visualized on 2% agarose gels.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Transcriptional up-regulation of BCRP in drug-selected cell lines. BCRP protein is overexpressed in the drug-selected MCF-7/AdrVp, Igrov1/MX3, and Igrov1/T8 cells compared with their parental cell lines (Fig. 1A ). High endogenous BCRP expression is found in human BeWo and JAR choriocarcinoma cells, as reported previously (9). In the drug-selected cells, the high BCRP protein expression is caused by transcriptional up-regulation and not by gene amplification stabilization of BCRP mRNA or protein (MCF-7/AdrVp), or demethylation of the known CpG island in the BCRP promoter (ref. 9; see Supplementary Data). Furthermore, 5'RACE-PCR studies did not reveal evidence of rearrangement with another constitutively active gene in the 5'UTR region of the drug-selected cells, as has been observed for the MDR1 gene (11). However, the 5'RACE-PCR studies did reveal evidence of variant forms of the untranslated exon 1. Because leader exon variation is considered a manifestation of alternative promoter usage (12), we examined the 5'UTR of BCRP mRNA in detail, using 5'RACE-PCR methodology.


Figure 1
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  		Figure 1. A, Western blot analysis of BCRP in various parental and drug-selected cell lines. Each lane contains 25 µg of total cell lysate. B, 5'RACE-PCR products from various parental and drug-selected cell lines were visualized by electrophoresis on 2% agarose gels. 5'RACE-PCR was done once for each cell line. Representative of electrophoretic displays of two individual nested PCR studies. The 5'RACE-PCR products have the following theoretical sizes (including 38 bp of 5'RACE adapter): E1a, 497 bp; E1b, 489 bp; 5' truncated E1c isoform with TSS of +451, 473 bp. The larger 5'-RACE PCR products detected in drug-resistant cell lines contained various sizes of the larger E1c isoforms, ranging from 580 to 680 bp. C, sequence analysis of cloned 5'RACE-PCR products from MCF-7 and MCF-7/AdrVp cells. Unique 5' leader exons isolated from MCF-7 cells and MCF-7/AdrVp cells are diagrammed along with the number of clones isolated with that sequence (far right). The number in brackets at the left of each bar shows the TSS for each species that was designated relative to the TSS (+1) described in our previous study (9). MED-1 and iMED refer to multiple start site element downstream and inverted MED-1, respectively.

 
5'UTR variants and multiple TSS. 5'RACE-PCR was done for the drug-sensitive MCF-7 and Igrov1 cells, the placental-derived JAR and BeWo cells, and the drug-selected MCF-7/AdrVp, Igrov1/MX3, and Igrov/T8 cells. PCR product(s) of ~450 to 500 bp predominated in all lines (Fig. 1B), except for Igrov1 cells, where no significant PCR product was amplified. Interestingly, additional larger PCR products (up to 650 bp) were observed only in the drug-selected cell lines, suggesting that the 5'UTR of BCRP mRNA differs in these cells compared with parental cells (Fig. 1A).

Sequence analysis of clones of these PCR products revealed at least three 5'UTR variants of exon 1 (designated as E1a, E1b, and E1c) that were alternatively spliced to a common exon 2, compliant with the GT-AG rule (Fig. 1C). Based on our previously described TSS (+1; ref. 9), splice sites for E1a, E1b, and E1c were calculated at nucleotide –291, +338, and +529, respectively (Fig. 1C). The +529 site was the original exon 1-exon 2 junction reported previously (9). In MCF-7 cells, each exon 1 variant generally has a distinct major TSS: E1a starts at nucleotide –393, E1b starts at +244, and three of four clones of E1c began at +344 (Fig. 1C). In contrast, the TSS for the exon 1 variants in MCF-7/AdrVp cells are more heterogeneous than those in the parental cells such that no single TSS could be assigned for a given exonal variant (Fig. 1C). Furthermore, longer forms of E1c (extending as far as nucleotide +244 upstream) were observed in MCF-7/AdrVp cells (accession nos. DQ459561-DQ459590; Supplementary Table S2).

We also found a longer E1c isoform (+316) and multiple TSS (+316, +344, and +451) in Igrov1/T8 cells. In JAR and BeWo cells, we found a predominance of E1c isoforms, but 5' truncated forms (TSS +344 or higher) were found more frequently. Two clones from BeWo cells had no exon 1 at all. Five of six clones isolated from JAR cells were 5' truncated forms of E1c with TSS of +449 or greater (accession nos. DQ459591-DQ459614; Supplementary Table S2).

Proportion of the E1 isoforms among BCRP transcripts. To evaluate the absolute frequency of expression of BCRP exon 1 variants in parental and drug-selected MCF-7 cells, total BCRP mRNA transcripts and BCRP mRNA transcripts containing the E1a or E1c isoforms were determined by quantitative RT-PCR, using the primer pairs diagrammed in Fig. 2A . The forward primers FwE1a and FwE1c were oligodeoxynucletides corresponding to the 3'-most regions of E1a and E1c, which enabled us to detect all exonal forms regardless of TSS. Because any forward primer within the E1b isoform would amplify both E1b and the longest E1c isoforms (+244/+529), the E1b variant was not studied in this context; the proportion of E1b was determined by subtraction (see Fig. 2B legend).


Figure 2
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  		Figure 2. A, diagram of primer system used to quantify variant forms BCRP exon 1. Each primer used in this study is displayed with its nucleotide residues calculated based on the TSS (+1). The reverse primer (RvEx4) for the exon 1 variants corresponds to sequence in exon 4. The forward and reverse primers for total BCRP mRNA transcripts (FwTotal and RvTotal, respectively) correspond to sequence in exons 11 and 13, respectively. Each white line shown in E1c variant represents TSS found. B, the percentage of BCRP mRNA transcripts containing the E1a, E1b, or E1c exon 1 variants among total BCRP mRNA transcripts in parental (open columns) and drug-selected MCF-7 cells (closed columns). The absolute number of total BCRP mRNA transcripts and mRNA transcripts containing the E1a and E1c exon 1 variant forms was determined by quantitative RT-PCR using the primers described in (A). The percentage of E1b transcripts was determined by subtracting (% E1a + % E1c) from 100. Columns, mean of 5 to 10 individual PCR assays; bars, SE. *, P < 0.05; ***, P < 0.001 by t test.

 
The E1c isoform was increased in the drug-resistant cells and comprised 47% of the total BCRP mRNA transcripts in MCF-7/AdrVp but only 23% of transcripts in parental MCF-7 cells (Fig. 2B). In contrast, the E1a isoform was found to be only 6% of total BCRP transcripts in MCF-7/AdrVp compared with 38% in MCF-7 cells. In Igrov/T8 and Igrov/MX3 cells, the E1c isoform comprised 71% and 66% of total BCRP mRNA transcripts, respectively.

Evaluation of translational efficiency of the exon 1 isoforms. To explore whether these exon 1 variants relate to the efficiency of expression of BCRP, we constructed full-length BCRP cDNAs that contained E1a (–393/–291), E1b (+244/+338), or E1c (+292/+529) as exon 1. These cDNAs were cloned downstream of the constitutive CMV promoter in the pcDNA3 vector and were transfected into MCF-7 cells. As expected, no statistically significant difference was observed in the level of transcribed mRNA from the three different isoforms under these conditions (Fig. 3A ). Furthermore, the mRNA generated from each BCRP exon 1 isoform construct was translatable (Fig. 3B). However, the median value of BCRP protein expression from mRNA containing the E1c isoform was higher than mRNA containing E1a or E1b as exon 1 (Fig. 3C). In the case of the E1c versus the E1a isoform, the difference was statistically significant (P < 0.05). These data show for the first time that BCRP protein expression can be regulated at the posttranscriptional level as the result of variations in the 5'UTR of the mRNA.


Figure 3
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  		Figure 3. Translational efficiency of the first exon of BCRP mRNA transcripts was determined in MCF-7 cells transiently transfected with BCRP cDNAs containing E1a, E1b, or E1c as exon 1, housed in the pcDNA3 vector. A, total BCRP mRNA expression was determined by quantitative RT-PCR in each transfectant. BCRP mRNA expression was normalized to that of ß-actin. Columns, mean value of six or seven separate quantitative RT-PCR reactions carried out for each BCRP transfectant type; bars, SE. BCRP mRNA expression is given relative to that of MCF-7 cells transfected with empty pcDNA3 vector. B, representative Western blot analysis of BCRP and NPT II expression in MCF-7 cells transfected with BCRP cDNAs containing different forms of exon 1. Thirty micrograms of total cell lysate was loaded into each lane. C, quantification of BCRP protein translated from mRNA with differing leader exons. The intensity of BCRP or NPTII on the Western blots (as shown in B) was quantified by densitometer (SI, Molecular Dynamics, Sunnyvale, CA), using ImageQuant analytic software (Molecular Dynamics), and the expression of BCRP protein was normalized to that of NPT II on the same blot. The individual values of normalized BCRP expression obtained from seven individual Western blots for each transfected exon 1 variant form are shown along with the median value. *, P < 0.05 by Wilcoxon rank sum test (two sided).

 
Tissue distribution of BCRP 5'UTR isoforms. To elucidate the physiologic significance of the exon 1 splice variants of BCRP mRNA, the expression of these variants and of total BCRP mRNA was measured in 11 different normal human tissues by conventional RT-PCR using the primer pairs shown in Fig. 2A. BCRP mRNA transcripts were detectable in 10 tissues; no PCR product was detected in the pancreas. Tissue-specific variations in the expression of the different exon 1 variants of BCRP were seen (Fig. 4A ). In lung, small intestine, colon, prostate, and testis, all three isoforms were detected. The E1c isoform predominated in kidney, liver, and prostate. Because the E1b primers detect E1b and long species of E1c, size heterogeneity of the PCR products was sometimes observed with the use of these primers. For example, additional PCR products of ~640 bp were found for the E1b primer pair in lung, small intestine, and MCF-7/AdrVp cells, implying the existence of large species of the E1c isoform (as is known to be the case for MCF-7/AdrVp cells). In kidney, liver, ovary, and muscle, no PCR products were amplified with the E1a primer pair. In MCF-7 and MCF-7/AdrVp cells, all exon 1 species were expressed, with greater expression of E1c in MCF-7/AdrVp cells, reflecting our 5'RACE-PCR data.


Figure 4
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  		Figure 4. A, tissue-specific distribution of different 5'UTR isoforms of BCRP mRNA. PCR was done using the primer sets described in Fig. 2A. PCR products for total BCRP (186 bp), ß-actin (238 bp), and the E1a (377 bp), E1b (448 bp), and E1c (379 bp) isoforms of the BCRP 5'UTR from 10 different human adult tissues and MCF-7 cell lines were visualized in 2% agarose gels. The FwE1b/RvEx4 primer pair will also produce a 639-bp product if long forms of E1c are present. Thirty-six cycles of PCR were used. Positive controls were done using authentic cDNA for the desired PCR assay as template. As a negative control, PCR was done in absence of templates. PCR was repeated at least twice for each specimen from a given tissue. Representative PCR bands of data obtained for each tissue type. The number in parentheses after each tissue refers to the number of samples studied for that tissue type, each from a different individual. B, plot of the exon 1 variants of the BCRP gene based on cDNA clones or expressed sequence tags reported in the Genbank database containing the exon 1-exon 2 splice junction (see Supplementary Table S1 for details regarding source files).

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The present study suggests transcriptional and/or splicing mechanism(s) are altered and may contribute to up-regulation of BCRP gene expression in cancer cells with acquired drug resistance. To the best of our knowledge, this is the first report showing the differential expression of novel 5'UTR variants of functional BCRP transcripts with differing translation efficiency in human cancer cells and normal tissues.

Our 5'RACE-PCR did not find any species with a TSS of +1. Our previous work (9) assigned this TSS based on a BCRP mRNA sequence deposited in the Genbank database (AF151530). The reference sequence for BCRP/ABCG2 mRNA (NM_004827) starts at +56; however, we did not recover this species either. A BLAST search of mRNA clones and/or expressed sequence tags in Genbank against BCRP exon 2 and the 5'-upstream region of BCRP reveals the presence of the E1a, E1b and E1c exon 1 variants we report here, with a preponderance of E1c forms (Fig. 4B; Supplementary Table S1). Additionally, multiple TSS, particularly for the E1c variant, are likely, as reflected by the great heterogeneity in the length of the E1c variants displayed in Fig. 4B. Only three clones corresponding to E1c start before +200 (+3 and +56); the majority of E1c transcripts start between +240 and +450 (Fig. 4B; Supplementary Table S1). This is in good agreement with our 5'RACE-PCR data, suggesting that the truncated forms (TSS +244 or higher) are common amongst BCRP E1c transcripts. Only one clone in Genbank corresponds to the E1a variant (Fig. 4B).

In addition to the variants shown in Fig. 4B, four BCRP mRNA clones were found in Genbank that lacked exon 1 entirely, as we observed in two mRNA clones from BeWo cells (designated as E1–; Supplementary Table S1). Surprisingly, our Genbank search also revealed four clones of BCRP mRNA with an exon 1 variant that was located 73 kb upstream from the current +1 TSS (designated as E1-Upstream, or E1u; Supplementary Table S1). We did not observe this variant in any of the 5'RACE-PCR clones isolated in this study. Future studies investigating the tissue specificity and potential promoter elements upstream to the TSS for E1– (e.g., within intron 1) and for E1U exon 1 variants are warranted.

Transactivation and overexpression of the human MDR1 gene in drug-selected cells has been reported to be the result of gene rearrangements within the 5'UTR region (11). However, our 5'RACE studies do not support such rearrangements in drug-selected cells that overexpress BCRP. Nevertheless, our data do not rule out the possibility that rearrangements or translocations may occur that result in a constitutively transactivated gene promoter being placed immediately upstream from the BCRP TSS.

Heterogeneity of the TSS for exon 1 observed in drug-resistant MCF-7/AdrVp cells reflects previous reports that multiple TSS of hMDR1 were often used in cells selected with actinomycin D (13), where response elements for RNA polymerase II trans-activating proteins designated MED-1 or iMED were attributed to the multiple TSS and up-regulation of gene expression (14, 15). Indeed, these elements are located in the BCRP promoter region (Fig. 1C), in proximity to the BCRP exon 1 variants we describe. Further study of the role these response elements play in BCRP gene expression is reasonable.

Generally, splice variants of untranslated leader exons are associated with alternative promoter usage, which can result in a diversity of gene expression, including tissue- or cell type-specific gene expression, and the efficiency with which the mRNA with differing leader exons is translated (12, 16), as illustrated by expression of the MDM2 proto-oncogene (17). Therefore, our finding of variation in the expression of BCRP leader exons among different tissues is reasonable and suggests that alternative and perhaps tissue-specific promoters may control the expression of BCRP. Future study is required to identify the precise location of alternative promoters involved in BCRP expression, which may shed light upon the mechanism(s) for BCRP up-regulation in different tissues and in multidrug-resistant cancer cells.


   Acknowledgments
 
Grant support: Department of Veterans Affairs Merit Review (D.D. Ross).

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


   Footnotes
 
Note: Supplementary data for this article are available at Cancer Research Online (http://cancerres.aacrjournals.org/).

Sequence data obtained from the 5' RACE-PCR studies are deposited in the Genbank database, under accession nos. DQ459561 to DQ459614.

Parts of this work was presented to the 94th and 95th Annual Meetings of the American Association for Cancer Research, July 2003, Washington, District of Columbia and March 2004, Orlando, Florida.

K.J. Bailey-Dell is currently at the Department of Molecular and Cellular Biology, Roswell Park Cancer Institute, Buffalo, NY 14263.

Received 12/21/05. Revised 3/27/06. Accepted 4/ 3/06.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Doyle LA, Yang W, Abruzzo LV, et al. A multidrug resistance transporter from human MCF-7 breast cancer cells. Proc Natl Acad Sci U S A 1998;95:15665–70.[Abstract/Free Full Text]
  2. Miyake K, Mickley L, Litman T, et al. Molecular cloning of cDNAs which are highly overexpressed in mitoxantrone-resistant cells: demonstration of homology to ABC transport genes. Cancer Res 1999;59:8–13.[Abstract/Free Full Text]
  3. Doyle LA, Ross DD. Multidrug resistance mediated by the breast cancer resistance protein BCRP (ABCG2). Oncogene 2003;22:7340–58.[CrossRef][Medline]
  4. Maliepaard M, van Gastelen MA, de Jong LA, et al. Overexpression of the BCRP/MXR/ABCP gene in a topotecan-selected ovarian tumor cell line. Cancer Res 1999;59:4559–63.[Abstract/Free Full Text]
  5. Robey RW, Medina-Perez WY, Nishiyama K, et al. Overexpression of the ATP-binding cassette half-transporter, ABCG2 (MXR/BCRP/ABCP1), in flavopiridol-resistant human breast cancer cells. Clin Cancer Res 2001;7:145–52.[Abstract/Free Full Text]
  6. Volk EL, Farley KM, Wu Y, et al. Overexpression of wild-type breast cancer resistance protein mediates methotrexate resistance. Cancer Res 2002;62:5035–40.[Abstract/Free Full Text]
  7. Krishnamurthy P, Ross DD, Nakanishi T, et al. The stem cell Marker Bcrp/ABCG2 enhances hypoxic cell survival through interactions with Heme. J Biol Chem 2004;279:24218–25.[Abstract/Free Full Text]
  8. Ee PLR, Kamalakaran S, Tonetti D, et al. Identification of a novel estrogen response element in the breast cancer resistance protein (ABCG2) gene. Cancer Res 2004;64:1247–51.[Abstract/Free Full Text]
  9. Bailey-Dell KJ, Hassel B, Doyle LA, et al. Promoter characterization and genomic organization of the human breast cancer resistance protein (ATP-binding cassette transporter G2) gene. Biochim Biophys Acta 2001;1520:234–41.[Medline]
  10. Nakanishi T, Karp JE, Tan M, et al. Quantitative analysis of breast cancer resistance protein and cellular resistance to flavopiridol in acute leukemia patients. Clin Cancer Res 2003;9:3320–8.[Abstract/Free Full Text]
  11. Mickley LA, Spengler BA, Knutsen TA, et al. Gene rearrangement: a novel mechanism for MDR-1 gene activation. J Clin Invest 1997;99:1947–57.[Medline]
  12. Ayoubi T, Van De Ven W. Regulation of gene expression by alternative promoters. FASEB J 1996;10:453–60.[Abstract]
  13. Ince TA, Scotto KW. Differential utilization of multiple transcription start points accompanies the overexpression of the P-glycoprotein-encoding gene in Chinese hamster lung cells. Gene 1995;156:287–90.[Medline]
  14. Ince TA, Scotto KW. A conserved downstream element defines a new class of RNA polymerase II promoters. J Biol Chem 1995;270:30249–52.[Abstract/Free Full Text]
  15. Labialle S, Gayet L, Marthinet E, et al. Transcriptional regulation of the human MDR1 gene at the level of the inverted MED-1 promoter region. Ann N Y Acad Sci 2002;973:468–71.[CrossRef][Medline]
  16. Kozak M. Structural features in eukaryotic mRNAs that modulate the initiation of translation. J Biol Chem 1991;266:19867–70.[Free Full Text]
  17. Barak Y, Gottlieb E, Juven-Gershon T, et al. Regulation of mdm2 expression by p53: alternative promoters produce transcripts with nonidentical translation potential. Genes Dev 1994;8:1739–49.[Abstract/Free Full Text]



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