ABCB1 Promoter Hypomethylation, ABCB1 Expression and Acquisition of Drug Resistance

The temporal relationship between ABCB1 promoter hypomethylation, ABCB1 expression and acquisition of drug resistance

K Reed1,2, SL Hembruff2, Induced expression of the Abcb1 drug transporter often occurs in tumors in 1,2 response to chemotherapy. The role that epigenetic modifications within the JA Sprowl and ABCB1 promoter play in Abcb1 expression remains unclear. We selected 1,2,3 AM Parissenti MCF-7 cells for survival in increasing doses of chemotherapy drugs, and assessed the methylation status of 66 CpG sites within the ABCB1 promoter 1Program in Biomolecular Sciences, Laurentian University, Sudbury, ON, Canada; 2Tumour preceding, accompanying and following the onset of drug resistance. Biology Research Program, Sudbury Regional Increased ABCB1 transcript expression coincident with acquisition of Hospital, Sudbury, ON, Canada and 3Division of resistance to epirubicin or paclitaxel was temporally associated with Medical Sciences, Northern Ontario School of hypomethylation of the ABCB1 downstream promoter in the absence of Medicine, Sudbury, ON, Canada gene amplifications or changes in mRNA stability. Treatment of control MCF- Correspondence: 7 cells with demethylating and/or acetylating agents increased ABCB1 Professor AM Parissenti, Program in transcript expression. In addition to broad promoter hypomethylation, Biomolecular Sciences, Laurentian University, dramatic reductions in the methylation of specific CpG sites within the Regional Cancer Program, Sudbury Regional promoter were observed, suggesting that these sites may play a predominant Hospital, 41 Ramsey Lake Road, Sudbury, ON, Canada P3E 5J1. role in transcriptional activation through promoter hypomethylation. E-mail: [email protected] Furthermore, our data suggest that allele-specific reductions in ABCB1 promoter methylation regulate promoter usage within paclitaxel-resistant cells. This study provides strong evidence that changes in ABCB1 promoter methylation, ABCB1 promoter usage and ABCB1 transcript expression can be temporally and causally correlated with the acquisition of drug resistance in breast tumor cells. The Pharmacogenomics Journal (2010) 10, 489–504; doi:10.1038/tpj.2010.1; published online 2 February 2010

Keywords: ABCB1; promoter hypomethylation; drug resistance; breast cancer; epirubicin; paclitaxel

Introduction

Multidrug resistance (MDR) is a major reason for the failure of chemotherapy treatment in breast cancer. Tumors can have either an innate or acquired ability to evade death by multiple structurally unrelated chemotherapeutic agents.1 Increased expression and drug efflux by ATP-binding cassette (ABC) drug transporters, particularly Abcb1 (Mdr1, P-glycoprotein), have been extensively shown to mediate MDR in vitro.2,3 Although the contribution of Abcb1 to MDR Received 6 May 2009; revised 24 November 2009; accepted 21 December 2009; is well known, the mechanism by which it becomes upregulated in tumor cells published online 2 February 2010 remains unclear. ABCB1 promoter hypomethylation and drug resistance K Reed et al 490

Epigenetic control is emerging as a prominent mechanism acquisition of drug resistance. Analysis of the cell lines of gene regulation in mammalian cells. Recent studies revealed that a specific ‘threshold’ selection dose (dose-9) suggest that changes in ABCB1 expression in certain types had to be reached before any resistance to chemotherapy of cancers are correlated with epigenetic alterations. In drugs was observed (31.5 nM epirubicin, 29.1 nM doxorubi- particular, the ABCB1 promoter has been shown to be cin, 3.66 nM paclitaxel). Furthermore, acquisition of drug hypomethylated in several drug-resistant sub-lines of resistance involved multiple changes in gene expression,14 4,5 human leukemia (CCRF-CEM and HL60), human epider- including upregulation of ABCB1 expression in MCF-7EPI 6 7 moid (KB3-1) and prostate cancer (DU145) cells. Similarly, and MCF-7TAX-2 cells (but not in MCF-7DOX-2 cells even at one study examined differences in methylation between the the highest selection dose). Elevated ABCB1 transcript ABCB1 50-untranslated regions of drug-resistant and drug- expression was first detected at selection dose-9 and sensitive breast cancer cells.8 This study established a progressively increased to selection dose-1213. No other correlation between increased ABCB1 expression and hypo- drug transporters known to play a role in drug resistance methylation of the ABCB1 promoter by comparing the in vitro were expressed in MCF-7EPI and MCF-7TAX-2 cells. methylation of CpG dinucleotides within the ABCB1 down- Inhibition of Abcb1 activity using cyclosporine-A partially 8 stream promoters of MCF-7 and MCF-7/ADR cells. restored drug sensitivity in MCF-7TAX-2 and MCF-7EPI at Although the evidence presented is compelling, the conclu- selection doses-9 and/or 12, further implicating Abcb1 as a sions are suspect, since the drug-resistant cell line used in contributor to drug resistance in these cell lines.13 this study (MCF-7/ADR) has been shown to be of ovarian In this study, we examined the role of epigenetic origin and is unrelated to MCF-7 breast tumor cells.9 mechanisms in the induction of both ABCB1 gene expres-

Another study identified changes in ABCB1 promoter sion and drug resistance in MCF-7EPI and MCF-7TAX-2 cells. methylation in doxorubicin-resistant MCF-7 cells, compared We report that gene amplification and mRNA stabilization with parental MCF-7 cells.10 However, the conclusions of do not contribute to increased ABCB1 expression at or above this study were based on assessment of only a few the dose required to achieve drug resistance. In contrast, methylation sites.10 Consequently, a rigorous, comprehen- bisulfite sequencing data indicated that broad hypomethy- sive investigation of the role of ABCB1 promoter methyla- lation of the ABCB1 downstream promoter temporally tion in the induction of drug resistance in breast cancer cell correlated with acquisition of drug resistance in MCF-7EPI lines has yet to be conducted. and MCF-7TAX-2 cells. Through profiling DNA methylation Another major issue that remains unaddressed for any changes at 66 CpG sites during the acquisition of drug tumor cell type is the temporal relationship between resistance, we were able to determine a common pattern of changes in promoter DNA methylation and both induction drug-induced epigenetic changes as well as key sites that of gene expression and acquisition of drug resistance. Many have dramatically reduced methylation associated with studies have simply compared drug-sensitive tumor cells ABCB1 transcription. Moreover, our data indicate that with tumor cells selected for resistance to high concentra- allele-specific changes in the methylation of the ABCB1 tions of chemotherapy agents.8,11,12 These and other studies downstream promoter can regulate promoter usage within have not examined whether changes in the methylation the ABCB1 gene in drug-resistant MCF-7 cells. Taken status of CpG dinucleotides (in any gene) are temporally or together, this study provides compelling evidence that causally correlated to acquisition of drug resistance. In acquisition of drug resistance in breast tumor cells can be addition, most epigenetic studies have assessed changes in temporally and causally related to reductions in the ABCB1 promoter methylation in response to selection for methylation of the ABCB1 downstream promoter. resistance to a single chemotherapy drug (primarily doxorubicin). Thus it is not clear at present whether selection for resistance to a variety of classes of ABCB1-inducing chemotherapy agents results in changes in ABCB1 promoter Materials and methods methylation. A novel system to study changes in ABCB1 promoter Cell culture and drug treatments methylation during acquisition of drug resistance in breast MCF-7DOX-2, MCF-7EPI and MCF-7TAX-2 cells were created by cancer cells has been developed within our laboratory. This selecting MCF-7 cells for survival in increasing concentra- system consists of three panels of isogenic MCF-7 cell lines tions (doses) of doxorubicin, epirubicin or paclitaxel, selected for survival in increasing concentrations (doses) of respectively, until maximum resistance was obtained as epirubicin, doxorubicin or paclitaxel until maximum resis- previously described.13 Aliquots of cells were stored at each tance is achieved (MCF-7EPI, MCF-7DOX-2 and MCF-7TAX-2 of the 12 selection doses. Similarly, 12 MCF-7CC sub-lines cells, respectively).13 Cells were also ‘selected’ in the absence were produced by ‘selection’ in the absence of drug. of drug to serve as co-cultured control cells (MCF-7CC cells). MCF-7CC cells were maintained in Dulbecco’s modified Samples of cells were stored at each drug dose during the Eagles’ medium supplemented with 10% FBS at 37 1C and selection process, which enabled us to study the precise 5% CO2, while MCF-7DOX-2, MCF-7EPI and MCF-7TAX-2 cells events preceding, accompanying and following acquisition were maintained in an identical medium supplemented of drug resistance, including whether epigenetic changes with the chemotherapeutic drugs (at the various doses to (such as promoter hypomethylation) are correlated with maintain selective pressure).13

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Generation of mammalian cell clones pended in 100 ml of phosphate-buffered saline containing Individual clonal isolates of MCF-7CC and MCF-7TAX-2 cells 1% bovine serum albumin (PBA). A 5-ml volume of MRK16 (selected to dose-12) were prepared by seeding cells at low antibody was incubated with the cell suspension for 1 h. The density in semi-solid media (1.8% methylcellulose (Sargent cells were washed twice in PBA and resuspended in 60 mlof Welch Laboratories, Buffalo Grove, IL) in 1 Â IMDM media PBA. A 2-ml volume of fluorescein isothiocyanate-conjugated (Princess Margaret Hospital, Toronto, ON) and 30% fetal goat anti-mouse secondary antibody (ID Labs) was added bovine serum) so as to obtain colonies originating from a and the cells were incubated in the dark at 4 1C for 30 min. single cell. Colony formation was permitted for approxi- Cells were then washed twice in PBA, resuspended in 100 ml mately 10 days, after which single colonies were selected of 1% paraformaldehyde and incubated at room tempera- and seeded in 96 well plates containing Dulbecco’s modified ture for 10 min. A 400-ml volume of PBA was added. Ten Eagles’ medium. Cells were allowed to adhere to 96-well thousand events were examined with the flow cytometer plates for 4 days, after which MCF-7TAX-2 cells were (Cytomics FC 500; Beckman Coulter, Mississauga, ON, transferred to identical medium containing 99 nM paclitaxel Canada) in three independent experiments. Background

(dose-12 concentration). MCF-7CC cells were maintained fluorescence was determined for MCF-7CC cells using an and propagated in drug-free media. The cells were propa- isotypic control antibody as the primary antibody. gated sequentially in 96-well plates, 24-well plates, six-well plates and T-25 cm2 flasks (Sarstedt, Montreal, QC, Canada), Quantitative PCR with genomic DNA with replating when cells reached 60–80% confluence. Seven Differences in the copy number of the ABCB1 gene were and nine clonal isolates of MCF-7CC and MCF-7TAX-2 cells, assessed in quantitative PCR experiments using 0.5 ng of respectively, were propagated for further experimentation. genomic DNA extracted from MCF-7CC, MCF-7EPI and MCF- 16 7TAX-2 cells at selection dose-12, as previously described. A Immunohistochemistry standard curve for ABCB1 was generated using 2, 1, 0.5, 0.25 The expression levels of the Abcb1 protein in MCF-7CC, and 0.125 ng of genomic DNA. The mean quantity of ABCB1 MCF-7EPI and MCF-7TAX-2 cells were first examined using gene was normalized to the S28 gene. Fold changes were immunofluorescence confocal microscopy following stan- then calculated using normalized MCF-7EPI and MCF-7TAX-2 dard approaches. MCF-7CC, MCF-7EPI and MCF-7TAX-2 cells values related to the MCF-7CC cell line. were selected to dose-12 (852 nM epirubicin or 99.0 nM paclitaxel in the latter two cell lines, respectively) and Fluorescence in situ hybridization grown on sterile coverslips in six-well tissue culture plates. The number of copies of ABCB1 relative to a chromosome-7

Cells were fixed in 4% paraformaldehyde, permeabilized centromere probe was investigated in MCF-7CC, MCF-7EPI with 0.2% Triton X-100 for 5 min and incubated in blocking and MCF-7TAX-2 cells selected to dose-12, as previously buffer (10% horse serum, 1% bovine serum albumin, 0.02% described.16 The gene copy number was determined using a NaN3 in phosphate-buffered saline) for 1 h at room tem- Metasystem-equipped microscope with Metafer4/Metacyte perature. A mouse monoclonal Abcb1 antibody (UIC2; Santa software v3.3.101. A minimum of 105 cells were analyzed Cruz Biotechnology, Santa Cruz, CA, USA) was then added for each cell line. at a 1:10 dilution in standard Tris-buffered saline with 1% bovine serum albumin, followed by addition of a fluorescein mRNA stability isothiocyanate-conjugated goat anti-mouse secondary anti- The stability of ABCB1 mRNA was determined in MCF-7CC, body (ID Labs, London, ON, Canada) at a 1:500 dilution in MCF-7EPI and MCF-7TAX-2 cells selected to dose-9 in four Tris-buffered saline, 1% bovine serum albumin. The cover- independent experiments. Cells grown to 40% confluence slips were mounted on glass microscope slides for observa- were treated with actinomycin-D at the concentration that tion by confocal microscopy (LSM 510 Meta; Carl Zeiss, inhibited incorporation of [5,63H]-UTP at or above 95% À1 À1 Toronto, ON, Canada) with a 63 Â objective lens. The laser (10 mgml for MCF-7CC and MCF-7TAX-2 cells, 100 mgml line used for excitation was Argon 488 nm and the band- for MCF-7EPI cells). RNA was extracted at 0, 1, 2, 4, 6 and pass filter setting used for detection was LP 505. All slides in 24-h post treatment using an RNeasy kit (Qiagen, Mississau- each experiment were examined on the same day, with ga, ON, Canada) as per manufacturer’s protocols. Transcript triplicate experiments performed. levels were then determined by quantitative reverse transcription PCR (Q-PCR) as previously described.13 Flow cytometry Abcb1 protein expression was further examined by flow Bisulfite sequencing PCR cytometry in MCF-7CC, MCF-7DOX-2, MCF-7EPI and MCF- The methylation status of CpG sites within the ABCB1 7TAX-2 cells (selected to dose-12) using the Abcb1-specific downstream promoter was determined by bisulfite sequen- primary antibody MRK16 (Kamiya Biomedical Company, cing PCR (BSP), as previously described,16 with genomic 15 Seattle, WA, USA) as previously described. For each cell DNA extracted from MCF-7CC cells at selection doses 1, 8 line, 2.5 Â 105 cells were placed in a 10-cm tissue culture and 12. ABCB1 promoter methylation was also examined in plate and the cells permitted to grow for 48 h. The cells were MCF-7DOX2 cells at selection doses 8 and 12, and in both released from their plates by trypsin/EDTA treatment, MCF-7EPI and MCF-7TAX-2 cells at selection doses 8, 9 and 12. washed twice with phosphate-buffered saline and resus- The methylation status of the upstream promoter was not

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investigated due to the paucity of CpG islands in this ABCB1 expression and promoter usage region.16 Sequencing of positive clones was performed by To investigate the relative abundance of ABCB1 transcripts Mobix DNA sequencing service (McMaster University, derived from the upstream and downstream promoters, Hamilton, ON, Canada). Changes in methylation at in- Q-PCR experiments were performed using cDNA prepared dividual CpG sites were calculated by subtracting the from RNA isolated from MCF-7CC, MCF-7EPI and MCF-7TAX-2 average percentage methylation for the lower dose from cells (selected to dose-12) and primers specific for transcripts the higher dose at each site. derived from the upstream promoter or from both promoters as previously described.16 Duplicate experiments with Effects of DNA methylase or histone deacetylase inhibitors on triplicate samples were performed. ABCB1 expression in MCF-7CC cells Analysis of ABCB1 expression and ABCB1 upstream MCF-7CC cells (‘selected’ to dose-12) were seeded at less than promoter usage within 12 clonal isolates of MCF-7CC and 50% confluence and allowed to adhere overnight at 37 1C MCF-7TAX-2 cells (selected to dose-12) was conducted as prior to treatment with 5-aza-20-deoxycytidine (Aza) previously described.16 Mean quantities were determined for (Methylation Ltd, Port Orange, FL, USA; 1 mM) for 96 h triplicate wells and normalized to S28 expression. Fold (alone or in combination with the histone deacetylase changes within 12 clones of MCF-7CC and MCF-7TAX-2 cells inhibitor trichostatin-A (TSA; Sigma Laboratories, Oakville, were calculated relative to the normalized mean quantity of ON, Canada; 50 ng mlÀ1) for 48 h). Media and/or Aza/TSA their respective populations. replacement occurred every 24 h. DNA demethylation by Aza treatment was confirmed by bisulfite sequencing PCR Statistical analysis (using 10 bacterial clones) while acetylation by TSA treat- A Gaussian distribution was tested using the D’Agostino and ment was confirmed by measuring histone acetylation using Pearson omnibus normality test to determine the use of a cell-based ELISA kit, as per manufacturer’s protocol parametric or non-parametric statistical tests. Significance (CycLex Co Ltd, Nagano, Japan). RNA was extracted from was then tested with Mann–Whitney non-parametric two- the cells at 96 h, reverse-transcribed into cDNA and Q-PCR tailed t-tests (a ¼ 0.05). The significance of percentage experiments performed using cDNA from untreated, methylation between MCF-7CC and MCF-7TAX-2 biological

Aza-treated and Aza/TSA-treated MCF-7CC cells and primers clones and their respective populations was tested relative to specific for ABCB1 or S28, as previously described.17 A their populations by one-way analysis of variance (a ¼ 0.05) minimum of three independent experiments containing and Tukey’s post hoc analysis. Pearson correlation coeffi- triplicate samples were performed. Genomic DNA was used cients (r) were calculated for ABCB1 gene expression and for bisulfite sequencing as described above. ABCB1 promoter methylation.

Pyrosequencing of DNA from mammalian cell clones Results To determine whether strong variations in the methylation of the ABCB1 downstream promoter existed within drug- Increased Abcb1 protein levels upon acquisition of drug resistance resistant cells, pyrosequencing was used to analyze the in MCF-7EPI and MCF-7TAX-2 cells methylation status of 66 CpG sites within the ABCB1 Examination of Abcb1 protein expression by flow cytometry promoter of various clones isolated from MCF-7CC and and immunofluorescence microscopy confirmed our

MCF-7TAX-2 cells. CpG sites 32–49 of the ABCB1 promoter previous findings that MCF-7CC and MCF-7DOX-2 cells were selected for analysis by pyrosequencing. This region in selected to dose-12 did not express appreciable amounts of bisulfite-treated DNA from all clones (as well as CpG- Abcb1 protein (Figures 1a and b). In contrast, MCF-7EPI and methylated Jurkat genomic DNA and Aza-treated Jurkat MCF-7TAX-2 cells at selection dose-12 expressed the Abcb1 genomic DNA controls from New England Biolabs, Picker- protein predominantly at the cell membrane (Figure 1b). ing, ON, Canada) was amplified by PCR using the following Abcb1 protein levels were significantly higher in MCF-7EPI 0 primers: ADS834 forward 5 -TTATGGGGATTAAGTGGGGT cells than in MCF-7TAX-2 cells (Figure 1a), and expression was TA-30 and ADS834 reverse biotinylated 50-GGTAAAGTTTAG detected in all cells for both cell lines rather than in select AATGYGTTGTTAG-30, where Y could be a C or a T members of the population (Figure 1b). depending on the methylation status at that CpG site. The

PCR reaction mixture contained 3 mM MgCl2, 200 mM dNTP, ABCB1 gene amplification and RNA stability are not factors 9pM forward and reverse primers, 0.75 U of HotStar Taq contributing to increased Abcb1 protein levels in MCF-7EPI and Polymerase (Qiagen), 1 Â PCR buffer and 1 ml of bisulfite- MCF-7TAX-2 cells treated DNA per 30 ml reaction volume. The PCR cycles were Differences in the copy number of the ABCB1 gene were as follows: 95 1C for 15 min, 45 cycles of 95 1C for 30 s, 48 1C undetectable in quantitative PCR experiments using for 30 s, 72 1C for 30 s, followed by a final extension at 72 1C genomic DNA extracted from MCF-7CC,MCF-7EPI and for 5 min. The pyrosequencing design and reactions were MCF-7TAX-2 cells at selection dose-12 (Figure 2a). Data from conducted by EpigenDx (Worcester, MA, USA) using the fluorescence in situ hybridization (FISH) studies conducted 0 following pyrosequencing primers: ADS834 FS1:5 -AGTGGT with MCF-7CC, MCF-7EPI and MCF-7TAX-2 cells at selection ATTGGATTATG-30, ADS834 FS2:50-GGGATTTGTTTTTTGA-30 dose-12 confirmed this lack of ABCB1 amplification. and ADS834 FS3:50-GGGTGGGAGGAAGTA-30. For all three cell lines, the majority of cells had four

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Decreased methylation of the ABCB1 downstream promoter temporally correlated with increased ABCB1 expression and the acquisition of drug resistance

Bisulfite sequencing data indicated that relative to MCF-7CC and MCF-7DOX-2 cells, the ABCB1 downstream promoters in MCF-7EPI and MCF-7TAX-2 cells were significantly hypomethylated upon acquisition of drug resistance (dose-9) and at the highest selection dose (dose-12). This conclusion was based on an assessment of the total and average percentage ABCB1 promoter methylation across the cell lines at selection doses 8, 9 and 12 (Figure 3a). Similar conclusions were drawn by examining the average methylation of specific CpG sites in the ABCB1 promoter of the various cell lines at selection doses 8, 9 and 12 (Figures 3d–f). The methylation data for each bacterial clone used to determine the minimum mean percent ABCB1 promoter methylation

for MCF-7CC cells, MCF-7EPI cells at selection doses 8, 9 and 12, and MCF-7TAX-2 cells at selection doses 8, 9 and 12, are shown in Figure 4. Pyrosequencing analysis confirmed

significant hypomethylation of MCF-7TAX-2 cells relative to MCF-7CC cells at selection dose-12 (Table 1). No significant differences in ABCB1 downstream promoter methylation were observed across the cell lines prior to acquisition of drug resistance (selection dose 8) (Figures 3a, c and d). The average percentage methylation of the ABCB1 downstream

promoter in MCF-7CC cells at selection doses 1, 8 and 12 (representative of low, moderate and high selection doses) did not change significantly during selection (P ¼ 0.29) and were pooled (Figures 3a and b). The average percentage

methylation for the 59 clones from MCF-7CC cells was 70.1%

Figure 1 Expression of the Abcb1 drug transporter in MCF-7CC, across the selection doses (Figure 3a). MCF-7TAX-2 and MCF-7EPI cells. (a) Abcb1 protein expression was Interestingly, when two populations (a hypermethylated measured in MCF-7CC (blue), MCF-7DOX-2 (pink), MCF-7EPI (green) population (X50% of CpG sites methylated) and a hypo- and MCF-7TAX-2 cells (light blue) cells (dose-12) by flow cytometry methylated population (o50% of CpG sites methylated)) using an anti-Abcb1 primary antibody (MRK16) and a goat anti-mouse were defined, differences in ABCB1 downstream promoter fluorescein isothiocyanate-conjugated secondary antibody. Background methylation between MCF-7 cells and MCF-7 or fluorescence using an isotypic control antibody is shown in red. CC EPI MCF-7 cells were enhanced for the hypomethylated (b) Expression of Abcb1 protein in MCF-7 , MCF-7 and MCF-7 TAX-2 CC EPI TAX- population (Figure 3c and data not shown). Both hyper- 2 cells (dose-12) was also measured by imunofluorescence confocal microscopy using an anti-Abcb1 primary antibody (UIC2) and a methylated and hypomethylated populations occurred fluorescein isothiocyanate-conjugated goat anti-mouse secondary in similar proportions in drug-resistant MCF-7EPI and MCF- antibody. 7TAX-2 cells when the classifier of 50% methylation was used (Figure 4). Two prominent populations were not

seen in MCF-7CC cells, at any selection dose, as only 8 of the 59 clones had ABCB1 downstream promoters that were green spots representing the presence of four chromosome-7 methylated below 50% (Figure 4a). The average percentage centromeres (Figure 2b). More variability was observed methylation of the eight hypomethylated MCF-7CC bacterial for the ABCB1/ABCB4 gene probe within MCF-7CC, clones was 43.1%. The lowest level of ABCB1 promoter MCF-7EPI and MCF-7TAX-2 cells, as signal splitting was methylation in MCF-7CC cells was 39.4%. In contrast, the more common with this probe. Most cells had between 3 mean percentage methylation of the ABCB1 downstream and 5 hybridized red spots, indicating no significant promoter in MCF-7EPI cells at selection doses 9 and 12 was increase in ABCB1 gene copy number (Figure 2c). Represen- 15.5 and 21.4%, respectively. The minimum level of tative FISH images for MCF-7CC, MCF-7EPI and MCF-7TAX-2 methylation was 0 and 1.5%, respectively, and the median cells are shown in Figure 2 (panels d–f, respectively). percentage methylation was 1.5 and 18.2%, respectively. In addition, experiments with actinomycin-D to block gene Similarly, average percentage methylation of the ABCB1 transcription revealed no differences among MCF-7CC, downstream promoter in MCF-7TAX-2 cells at selection MCF-7EPI and MCF-7TAX-2 cells (selected to dose level-9) in doses 9 and 12 was 34.8 and 3.0%, respectively, while terms of the stability (half-life) of ABCB1 transcripts minimum average percentage methylation was 33.3 and 0%, (Figures 2g and h). respectively.

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Reductions in methylation were observed broadly across resistance (dose-9) and beyond (dose-12), both in compar-

CpG sites within the ABCB1 downstream promoter of ison to MCF-7CC cells ‘selected’ to equivalent doses MCF-7EPI and MCF-7TAX-2 cells at acquisition of drug (Figures 3e and f) and in comparison to MCF-7EPI and

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80 150 Dose 8 MCF-7 MCF-7 * CC CC MCF-7 MCF-7 60 DOX-2 EPI * MCF-7 MCF-7TAX-2 * * EPI MCF-7TAX-2 100 40

20 50 Average Methylation (%) Methylation Average

0 (%) Methylation Average

cc d8 d8 d9 d8 d9 d12 d12 d12 Aza -2 X-2 EPI EPI -2 cc O X-2 EPI X 0 MCF-7 D O TAX-2 TAX D TA 02040 60 80 MCF-7 MCF-7 MCF-7 MCF-7 MCF-7MCF-7 MCF-7 MCF-7 MCF-7 CpG Site Cell line & Dose

100 150 Dose 9 MCF-7CC MCF-7 75 EPI MCF-7TAX-2 100 50

25 50 Average Methylation (%) Methylation Average

0 (%) Methylation Average

C d8 d8 d9 d8 d9 C d12 d12 d12 0 Aza -2 -2 X-2 EPI EPI C O X-2 EPI X-2 C D O 7 TAX TAX 0608020 40 MCF-7 D TA MCF-7 MCF-7 CpG Site MCF-7MCF-7 MCF-7MCF- MCF-7 MCF-7 MCF-7 Cell line & Dose

90 150 Dose 10 80 MCF-7CC MCF-7CC MCF-7 70 EPI MCF-7EPI MCF-7 MCF-7 60 * TAX-2 TAX-2 100 50 * MCF-7CC Aza 40 * 30 * 20 50

Average Methylation (%) Methylation Average 10 *

0 (%) Methylation Average . . . .

Total Total Total Total 0

HypomethHypermeth.HypomethHypermeth.HypomethHypermeth.HypomethHypermeth. 06020 40 80 CpG Site Dose 12 Populations Figure 3 Quantification of DNA methylation levels within the ABCB1 downstream promoter in various cell lines and in response to Aza treatment as measured by BSP. (a) The average level of ABCB1 downstream promoter methylation was assessed for specific bacterial clones housing amplified ABCB1 downstream promoter DNA from MCF-7CC,MCF-7DOX-2,MCF-7EPI and MCF-7TAX-2 cells (nX16) selected to dose level (d)8,9or12andMCF-7CC cells treated with Aza (MCF-7CC Aza, n ¼ 10). Genomic DNA was isolated from the above cells, bisulfite-treated and the ABCB1 downstream promoter region was amplified by PCR. The amplified DNAs were cloned into bacterial expression vectors, which were used to transform bacterial cells. After isolation of 20 clones of bacterial transformants, the ABCB1 downstream promoter region within the plasmid of each bacterial clone was then sequenced to determine the methylation status of each CpG site. The percentage promoter methylation was determined by calculating the percentage of CpG sites that are methylated of 66 possible sites for all bacterial clones (n), and then calculating the average methylation from all clones assessed for a particular cell line and dose. (b)The average methylation values for each of several bacterial clones housing amplified ABCB1 downstream promoter DNA from the above cell lines, with or without Aza treatment. The mean methylation value for all of the clones (horizontal line) is also depicted. (c) The mean percent methylation was also calculated after clones were classified as hypermethylated (X50% of sites methylated) or hypomethylated (p50% of sites methylated). The symbol *denotes changes in the level of ABCB1 downstream promoter methylation that vary significantly from levels in MCF-7CC cells (Po0.05). (d–f) The average methylation for each of 66 CpG sites within the ABCB1 downstream promoter for all bacterial clones from MCF-7CC,MCF-7EPI and MCF-7TAX-2 cells selected to dose level 8 (d), 9 (e)and12(f) is depicted. Aza, 5-aza-20-deoxycytidine; BSP, bisulfite sequencing PCR.

Figure 2 Examination of ABCB1 gene copy number and ABCB1 transcript stability in MCF-7CC, MCF-7EPI and MCF-7TAX-2 cells (selected to dose level 12). ABCB1 gene copy number was assessed by Q-PCR using ABCB1-specific primers and genomic DNA isolated from each cell line (a), and by FISH using an orange-labeled ABCB1/ABCB4 specific probe (RP11-457B15) and a chromosome-7 centromeric probe (CEP7) labeled with spectrum green (b–f). A minimum of 100 cells were scored for the number of spots hybridizing to the CEP7 probe (b) and the ABCB1 probe (c) using the Metafer4/Metacyte v3.3.101 software program. Representative FISH images for MCF-7CC (d), MCF-7EPI (e) and MCF-7TAX-2 (f) cells are depicted. The stability of ABCB1 transcripts was also assessed in MCF-7EPI (g) and MCF-7TAX-2 (h) cells relative to that in MCF-7CC cells (all selected to dose level 9). This involved measuring ABCB1 mRNA levels by Q-PCR following treatment with an appropriate concentration of actinomycin-D to inhibit 495% of RNA synthesis by Q-PCR. FISH, fluorescence in situ hybridization; Q-PCR, quantitative reverse transcription PCR.

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MCF-7CC

MCF-7 dose 8 EPI MCF-7TAX-2 dose 8

MCF-7EPI dose 9 MCF-7TAX-2 dose 9

MCF-7TAX-2 dose 12 MCF-7EPI dose 12

Figure 4 The mean methylation status of individual CpG sites (66) for each bacterial clone was assessed by BSP for MCF-7CC cells (a); MCF-7EPI cells at selection doses 8 (b), 9 (c)and12(d); and MCF-7TAX-2 cells at selection doses 8 (e), 9 (f)and12(g). BSP, bisulfite sequencing PCR. Circles represent each CpG site within the ABCB1 promoter. Open circles indicate unmethylated CpG sites whereas filled circles indicate methylated CpG sites.

MCF-7TAX-2 cells selected to dose-8, respectively (Figures 5c ABCB1 promoter methylation for MCF-7EPI cells was and d). In contrast, the pattern of methylation across all obtained when the analysis was restricted to selection doses

CpG sites within MCF-7CC cells selected to doses 1 and 12 8 and 9, suggesting a particular importance of promoter were quite similar (Figure 5a), as were the methyl- hypomethylation in the onset of ABCB1 gene expression ation pattern across all CpG sites for MCF-7CC, MCF-7EPI and and drug resistance in these cells. MCF-7TAX-2 cells selected at dose-8 (Figure 3d). Decreased methylation of the ABCB1 downstream promoter and Correlations between ABCB1 gene expression and promoter increased ABCB1 expression was induced by Aza treatment of methylation MCF-7CC dose 12 cells Pearson correlation coefficients (r) were calculated for the Treatment of MCF-7CC cells selected to dose-12 with 1 mM relationship between ABCB1 gene expression and ABCB1 Aza for 96 h decreased the average percentage methylation downstream promoter methylation in the MCF-7TAX-2 and as compared with that in untreated MCF-7CC cells MCF-7EPI cell lines at various selection doses. MCF-7TAX-2 (Figure 3a), which approached significance (P ¼ 0.064). cells selected to doses 8, 9 and 12 had a Pearson correlation Similar to the drug-resistant cell lines, both highly and coefficient for these two parameters of À0.97 (Table 2). MCF- lowly methylated populations were observed in equivalent

7EPI cells selected to doses 8, 9 and 12 had a Pearson proportions by BSP analysis of bacterial clones housing correlation coefficient of À0.27 (Table 2), likely due to the bisulfite-treated, PCR-amplified DNA from Aza-treated considerably higher ABCB1 mRNA expression at dose-12 MCF-7CC cells (Figure 3b). When segregated into hypo- (compared with dose-9), but similar reductions in the level methylated and hypermethylated populations, the average of ABCB1 promoter methylation at these two doses. A strong percentage methylation of the hypomethylated group negative correlation between ABCB1 gene expression and (36.2%) of Aza-treated cells was significantly different from

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Table 1 Comparison of average methylation, ABCB1 mRNA expression and usage of the upstream ABCB1 promoter in clonal isolates of MCF-7TAX and MCF-7CC cells (selected to dose level 12) relative to their respective cell populations

Population and mammalian Average Fold change in ABCB1 Fold change in upstream promoter cell clones (numbered) methylation (%) expression relative to population usage relative to population

MCF-7CC population 67.27±1.39 1 1 MCF-7CC 1 61.63±2.39 0.25 (À4.00) 1.78 MCF-7CC 2 58.44±3.19 0.35 (À2.89) 0.41 (À2.46) MCF-7CC 3 64.85±3.18 1.17 1.64 MCF-7CC 4 63.86±2.74 0.26 (À3.82) 0.66 (À1.52) MCF-7CC 5 65.18±2.84 0.93 (À1.07) 3.71 MCF-7CC 6 58.67±3.47 0.51 (À1.96) 0.80 (À1.24) MCF-7CC 7 65.73±2.84 0.26 (À3.85) 0.27 (À3.76) MCF-7TAX-2 population 41.49±1.24 1 1 MCF-7TAX-2 1 40.76±1.43 1.05 1.12 MCF-7TAX-2 2 41.71±1.73 1.16 0.79 (À1.27) MCF-7TAX-2 3 40.95±2.16 0.84 (À1.19) 0.66 (À1.52) MCF-7TAX-2 4 39.56±1.57 0.72 (À1.39) 0.45 (À2.20) MCF-7TAX-2 5 41.48±2.08 0.66 (À1.52) 0.57 (À1.77) MCF-7TAX-2 6 41.02±1.72 0.45 (À2.22) 0.41 (À2.46) MCF-7TAX-2 7 42.59±1.72 0.50 (À2.00) 0.49 (À2.05) MCF-7TAX-2 8 37.02±1.87 0.66 (À1.52) 0.62 (À1.62) MCF-7TAX-2 9 39.71±1.21 0.54 (À1.85) 0.52 (À1.92)

Negative numbers in parentheses indicate fold downregulation.

that of untreated MCF-7CC cells, whereas the methylated Most changes in the methylation of ABCB1 CpG sites that population was not (Figure 3c). In addition to reduced occurred between doses 1 and 12 in MCF-7CC cells were methylation, Aza-treated MCF-7CC cells exhibited increased small increases in methylation (Figure 5e). In contrast, expression of ABCB1 by 7.0±1.4-fold over that in untreated mainly large reductions in methylation were observed across

MCF-7CC cells (Figure 6a). Treatment of MCF-7CC cells several CpG sites when MCF-7CC cells were treated with Aza selected to dose-12 with Aza and the histone deacetylase (Figure 5f), or when MCF-7 cells acquired resistance to inhibitor TSA had a synergistic effect on ABCB1 expression, epirubicin (Figure 5g) or to paclitaxel (Figure 5h). Only CpG with the combined treatment increasing ABCB1 expression sites 13 and 47 within the ABCB1 promoter of MCF-7EPI cells by 31.8±8.2-fold (Figure 6a). Treatment of MCF-7CC cells and site 45 within MCF-7TAX-2 cells increased methylation with TSA alone had only a slight effect on ABCB1 (Figures 5g and h, respectively). Small increases in methyla- expression, increasing it by 1.8±0.4-fold over that in tion in response to Aza treatment occurred at CpG sites 3, untreated MCF-7CC cells (Figure 6b). 22, 49–51, 54 and 57 of the MCF-7CC promoter (Figure 5f). CpG sites that exhibited the most striking reductions in

Identification of key CpG sites within the ABCB1 downstream methylation within MCF-7EPI were sites 1, 4, 30, 54, 57, promoter that undergo strong hypomethylation in drug-resistant 60–64 and 66, while in MCF-7TAX-2 cells it was sites 6–8, 21, cells 30, 32, 49, 53–57, 59, 62, 63, 65 and 66. Dramatic reductions

To determine whether distinct or broad-based reductions in were seen in response to Aza treatment of MCF-7CC cells at CpG site methylation take place with the ABCB1 promoter sites 5, 8, 9, 13, 15, 20 and all but two sites spanning CpG of drug-resistant cells, the change in methylation of each sites 27–49. Important elements within the ABCB1 promoter individual CpG site was quantified in MCF-7EPI cells and are highlighted in Table 3 and proteins that have been MCF-7TAX-2 cells at dose-12 relative to dose-8 (Figures 5g and implicated directly or indirectly in ABCB1 downstream h, respectively). Similar comparisons were made between promoter function are shown in Table 4 (along with the

MCF-7CC cells at dose-12 relative to dose-1 (Figure 5e). This corresponding CpG sites contained within the regions). was accomplished by subtracting the average percentage In addition to distinct changes in methylation induced by methylation of the lower dose from the higher dose. selection with paclitaxel or epirubicin, common reductions Methylation changes at individual sites were also character- in methylation took place upon selection for epirubicin or ized in Aza-treated MCF-7CC cells (dose-12) as compared paclitaxel resistance. For example, most sites within the with that in untreated cells by subtracting the percentage 30-end of the promoter (CpG sites 49–66) exhibited dramatic methylation in untreated cells from that in Aza-treated cells reductions in methylation, while this region was largely

(Figure 5f). Thus negative values indicate reductions in the unchanged upon Aza treatment of MCF-7CC cells. This average percentage methylation, while positive values region containing CpG sites 49–66 includes the translation indicate increased methylation. start site (Table 3) and an element important for negative

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100 MCF-7 50 CC Dose 1 MCF-7CC [Dose 12 relative to Dose 1] 90 Dose 12 80 25 70 60 50 0 40 30 -25 20 2 4 6 8 1012141618202224262830323436384042444648505254565860626466 10 -50 CpG Site

Average Methylation (%) Methylation Average 0 0 10203040506070 Change (%) Methylation CpG Site -75

50 MCF-7 (Dose 12) Aza-treated MCF-7 (Dose 12) Cells [Relative to Untreated cells] 100 CC Control CC 90 Aza-treated 25 80 CpG Site 70 2 4 6 8 1012141618202224262830323436384042444648505254565860 62 64 66 60 0 50 40 30 -25 20 10 -50 Average Methylation (%) Methylation Average 0 Change (%) Methylation 0 10203040506070 -75 CpG Site

100 MCF-7 Dose 8 50 EPI MCF-7EPI [Dose 12 Relative to Dose 8] 90 Dose 9 80 Dose 12 25 70 CpG Site 60 2 4 6 8 1012141618202224262830323436384042444648505254565860626466 50 0 40 30 -25 20 10 -50

Average Methylation (%) Methylation Average 0 Methylation Change (%) Methylation 0 10203040506070 -75 CpG Site 50 MCF-7TAX-2 [Dose 12 Relative to Dose 8] 100 MCF-7TAX-2 Dose 8 90 25 Dose 9 CpG Site 80 Dose 12 70 2 4 6 8 1012141618202224262830323436384042444648505254565860626466 60 0 50 40 -25 30 20 10 -50 Methylation Change (%) Methylation Average Methylation (%) Methylation Average 0 0 10203040506070 -75 CpG Site

Figure 5 The mean percent methylation of each of the 66 CpG sites within the ABCB1 downstream promoter of MCF-7CC, MCF-7EPI and MCF-7TAX-2 cells at various selection doses, and of MCF-7CC cells with or without Aza treatment. The mean percent methylation value for each CpG site within the ABCB1 downstream promoter was determined in BSP experiments (nX16 except for Aza-treated MCF-7CC cells, where n ¼ 10). (a–d) Plots depict the percent methylation of each of the 66 CpG sites for MCF-7CC cells at selection doses 1 and 12 (a), and for MCF-7EPI or MCF-7TAX-2 cells at selection doses 8, 9 and 12 (c and d, respectively). The effect of Aza treatment on MCF-7CC cells (dose-12) on the percent methylation of each CpG site is also depicted (b). The percent change in the methylation of each site upon ‘selection’ of MCF-7CC cells from dose level 1 to dose level 12 is shown (e), as well as the effect of Aza treatment on MCF-7CC (dose-12) cells relative to untreated cells (f). The change in methylation of each CpG 0 site as MCF-7EPI (g) or MCF-7TAX-2 (h) cells were selected from dose level 8 to dose level 12 is also displayed. Aza, 5-aza-2 -deoxycytidine; BSP, bisulfite sequencing PCR.

regulation by p53 (Table 4). Other regional reductions in methylation that were similar upon selection for drug resistance and upon Aza treatment occurred around sites Table 2 Correlations between ABCB1 gene expression and 19–22, which correspond to the consensus Inr site and the promoter methylation for MCF-7TAX-2 and MCF-7EPI cell lines for doses representative of before and after the acquisition of drug transcription start site (Table 3), and sites 32–44, involved in resistance regulation by p53 (Table 4).

Cell line and dose Pearson’s correlation coefficient (r2) Quantification of transcripts from upstream and downstream ABCB1 promoters in MCF-7CC, MCF-7EPI and MCF-7TAX-2 cells MCF-7TAX-2 doses 8, 9 and 12 À0.97 The expression of transcripts from both upstream and MCF-7EPI doses 8, 9 and 12 À0.27 downstream ABCB1 promoters was investigated in MCF-7EPI doses 8 and 9 À1.0 MCF-7CC, MCF-7EPI and MCF-7TAX-2 cells (selected to dose

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Amplification Plot Amplification Plot 1.000 E+1 1.000 E+1

1.000 Aza & TSA 1.000

1.000 E-1 1.000 E-1

Rn 1.000 E-2 Untreated Rn 1.000 E-2 Untreated Δ Aza Δ TSA 1.000 E-3 1.000 E-3

1.000 E-4 1.000 E-4

1.000 E-5 1.000 E-5 0 5 10 15 20 25 30 35 40 0 5 10 15 20 25 30 35 40 Cycle Cycle

Figure 6 The effects of DNA methylase or histone deacetylase inhibitors on ABCB1 expression and drug resistance in MCF-7CC (dose-12) cells. (a) The effect of Aza alone or in combination with TSA treatment on ABCB1 expression in MCF-7CC (dose 12) cells was assessed by Q-PCR. Cells were treated with 1 mM Aza and/or 50 ng mlÀ1 TSA, with drug and media replacement every 24 h. (b) The effect of TSA treatment alone on ABCB1 0 expression in MCF-7CC (dose-12) cells was also assessed by Q-PCR (c). Aza, 5-aza-2 -deoxycytidine; Q-PCR, quantitative reverse transcription PCR; TSA, trichostatin-A.

Table 3 Promoter elements involved in transcriptional promoter and a second with a hypomethylated active regulation of ABCB1 downstream promoter). To assess this possibility, we isolated

single clones from MCF-7CC and MCF-7TAX-2 cells and Name DNA region CpG sites References examined both their expression of ABCB1 and methylation of their ABCB1 promoters. No significant differences in a32 Transcription start site +1 and +5 18 promoter methylation were observed in clones of MCF-7 Consensus Inr site À6 to +11 18,19 33 CC and MCF-7 cells relative to the entire cell population Translation start site +136 64a34 TAX-2 MDR1 enhancesome À136 to À76 10–12 35,36 (Table 1). Furthermore, all MCF-7TAX-2 biological clones did GC element À56 to À43 13a14a15 37 express ABCB1 mRNA at levels akin to that of the GC-rich element À110 to À103 11 and 12 38 population, with the greatest difference being 2.22-fold. 39 Inverted CCAAT box À82 to À73 None All MCF-7TAX-2 clones also used both promoters at compar- Heat-shock element À152 to À178 8a9a 34,40 able levels to the population, with the greatest difference Heat-shock-like element À99 to À66 None 34 being a 2.45 difference in upstream promoter usage (Table

1). MCF-7CC clones also behaved similarly to the popula- aAdjacent CpG site near to the element. tion, with both consistent exhibition of low ABCB1 expression and usage of the upstream promoter (Table 1). level 12) by Q-PCR experiments using primers that detect transcripts from the upstream promoter (‘upstream’ primers) or from both upstream and downstream promoters (‘total’ Discussion primers). MCF-7CC cells expressed very low amount of ABCB1 transcript, which appeared to be derived solely from Changes in the expression of the ABCB1 drug transporter the upstream promoter (since the level of total transcripts often occur in tumors and cultured tumor cells upon and the level of transcripts from the upstream promoter acquisition of resistance to chemotherapy agents. The exact were identical; Figure 7a). In contrast, the amplification mechanism responsible for increased ABCB1 expression in plots for both MCF-7EPI and MCF-7TAX-2 cells revealed drug-resistant cancer cells remains unclear. This study used a significantly higher ABCB1 expression in these cells, with unique panel of progressively drug-resistant breast tumor transcripts emanating from both upstream and downstream cells to assess the temporal and causal relationships between ABCB1 promoters (Figures 7b and c, respectively). exposure to specific chemotherapeutics, changes in ABCB1 promoter methylation and acquisition of drug resistance. Methylation levels and promoter usage in clonal isolates of We have previously shown that a specific threshold drug MCF-7CC and MCF-7TAX-2 cells were comparable to that of dose must be reached in order to acquire drug resistance and the population that multiple molecular mechanisms appear to be associated Two populations with dissimilarly methylated ABCB1 with the acquisition of drug resistance, including decreased downstream promoters were evident by BSP analysis of drug accumulation through overexpression of the Abcb1 13,14 bacterial clones housing DNA from MCF-7TAX-2 cells. ABCB1 drug transporter in MCF-7EPI and MCF-7TAX-2 cells transcription via both upstream and downstream ABCB1 (unpublished observations). promoter was also evident. This raised the possibility that We first examined the possibilities that differences in two MCF-7TAX-2 cell populations exist (one with a methy- ABCB1 gene amplification, ABCB1 transcript stability or lated downstream promoter and usage of the upstream ABCB1 mRNA translation may account for the higher

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Table 4 Proteins and nearest CpG sites directly or indirectly implicated in the transcriptional regulation of the ABCB1 gene via elements within the ABCB1 downstream promoter

Name Repressor/activator DNA region CpG sites References

Sp1 Activator À56 to À43 13a14a15 37,38,41 Sp3 Activator À56 to À43 13a14a15 35 Egr-1 Activator À56 to À43 13a14a15 38 NF-Y Activator À70 to À100 12a37 Ets-1 Activator À69 to À63 13a42 Hypoxia-inducible factor-1 (HIF-1) Activator À49 to À45 15a35 Inverted multiple start site element downstream (iMED) Activator À105 to À100 12 43 Heat-shock factor-1 (HSF-1) Activator À178 to À152 8a9a40 AP-1 Activator À121 to À115 11a36 YB-1 Activator (controversial) À82 to À73 None 39,44 NF-R1 Repressor À123 to À115, À56 to À45 11a,15 45 NF-R2 Activator À119 to À111 11 45 NF-R3 Activator À52 to À41 15 45 p53 Repressor À72 to À40, 13–15, 46 Activator +128 to +700 27–66 MDR1 promoter enhancing factor-1 (MEF-1) Activator À118 to À111 11 47 T-cell factor/lymphoid enhancer factor (TEF/LEF) Activator À1813 to À261 1–5 35 Glucocorticoid receptor (GR) Repressor À149 to À147 9a36 IL-6-induced CAAT enhancer-binding protein (CEBPb) Activator À147 to À139 9 48 Unidentified repressor Repressor À110 11 38 Ras/Raf Activator À69 to À41 13–15 36 NFkB/c-Fos complex Repressor À118 to À111 11 49

Abbreviations: IL, interleukin; NFkB, nuclear factor-kB. aAdjacent CpG sites.

Amplification Plot Amplification Plot Amplification Plot 1.000 E+1 1.000 E+1 1.000 E+1

Total Total 1.000 1.000 1.000

1.000 E-1 1.000 E-1 1.000 E-1 Upstream Upstream

1.000 E-2 1.000 E-2 Rn Rn Rn 1.000 E-2 Δ Δ Δ

1.000 E-3 1.000 E-3 1.000 E-3

1.000 E-4 1.000 E-4 1.000 E-4 MCF-7CC MCF-7EPI MCF-7TAX-2 (Dose12) (Dose12) (Dose12) 1.000 E-5 1.000 E-5 1.000 E-5 0 5 10 15 20 25 30 35 40 0 5 10 15 20 25 30 35 40 0 5 10 15 20 25 30 35 40 Cycle Cycle Cycle

Figure 7 Expression of ABCB1 transcripts from the upstream and downstream promoters in MCF-7CC, MCF-7TAX-2 and MCF-7EPI cells. Representative Q-PCR amplification plots are shown for MCF-7CC (a), MCF-7EPI (b) and MCF-7TAX-2 (c) cells selected to dose-12 using primers specific for all ABCB1 transcripts (total) and primers specific for transcripts derived solely from the upstream promoter (upstream). Q-PCR, quantitative reverse transcription PCR.

ABCB1 transcript and Abcb1 protein levels in MCF-7EPI and MCF-7TAX-2 cells (and particularly in MCF-7EPI cells) was MCF-7TAX-2 cells (relative to MCF-7CC cells). Previously, we confirmed at selection dose-12 by immunoprecipitation and demonstrated that ABCB1 transcript expression was drama- immunoblotting experiments13 and in this study by flow tically increased in MCF-7EPI and MCF-7TAX-2 cells at cytometry and immunofluorescence microscopy (Figure 1). threshold dose-9 as compared with MCF-7CC cells. Expres- The degree of Abcb1 protein expression was consistent with sion of ABCB1 increased with increasing selection dose. No that previously observed at the mRNA transcript level (MCF- 13 increase in ABCB1 expression was observed in these cells at 7EPI4MCF-7TAX-2bMCF-7CC). This was an important selection doses 1–8 (prior to acquisition of drug resis- observation as increased expression of ABCB1 at the tance).13 Higher expression of Abcb1 protein levels in transcript level is not always accompanied by elevated

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protein levels. Despite overexpression of ABCB1 mRNA, two CpG sites (Figures 3e and 5c). Broad promoter demethyla- independent studies identified a block in the translation of tion of these sites was maintained at the highest selection ABCB1 mRNA within K562 cells.18,19 Although the possibi- dose (dose-12; Figure 5c). Similar to our observations in lity exists within our MCF-7 cell lines that all transcripts MCF-7EPI cells, CpG sites within the ABCB1 downstream produced are not translated into protein, the high Abcb1 promoter of MCF-7TAX-2 cells selected to dose-8 were mostly protein expression levels within MCF-7EPI and MCF-7TAX-2 methylated in the clones selected for analysis (Figures 3d indicates that a translational blockade was not present in and 5d). However, ABCB1 promoter methylation was these drug-resistant cell lines. significantly decreased in MCF-7TAX-2 cells at selection Gene amplification is also a potential mechanism for dose-9 and further decreased at selection dose-12 both in increased ABCB1 transcription, given previously published terms of average promoter methylation (Figure 3a) and findings using a variety of drug-resistant cancer cell lines.20–23 methylation across specific CpG sites (Figures 3e and f, and

No ABCB1 gene amplification could be detected in MCF-7EPI 5d). Demethylation of the ABCB1 promoter in MCF-7EPI and MCF-7TAX-2 cells (relative to MCF-7CC cells) when and MCF-7TAX-2 cells as cells were selected from dose-8 to assessed by Q-PCR experiments using genomic DNA and dose-12, was confirmed by pyrosequencing experiments ABCB1-specific primers (Figure 2). This lack of ABCB1 (Table 1).26 amplification was further confirmed by FISH studies Further proof of a causal relationship between the (Figure 2). Interestingly, we have identified gene amplifica- epigenetic modifications of the ABCB1 downstream promo- tion as a primary mechanism of ABCB1 induction in MCF-7 ter and ABCB1 gene expression was provided in this study cells that we selected for resistance to docetaxel (MCF-7TXT through experiments involving application of DNA methyl- cells).16 Since gene amplification was not found to transferase or histone deacetylase inhibitors (Aza and TSA, contribute to elevated ABCB1 expression in MCF-7EPI and respectively). Reduced DNA methylation induced by Aza MCF-7TAX-2 cells selected in an identical manner, each treatment of MCF-7CC cells selected to dose-12 (Figures 3a–c selection and/or chemotherapy drug used during selection and 5b and f) resulted in a moderate induction (seven-fold) may result in a different mechanism of ABCB1 upregulation. of ABCB1 expression, while addition of both Aza and TSA Furthermore, it appears that the same class of chemother- increased ABCB1 mRNA levels by B32-fold (Figure 3g). This apeutic agents (for example, the taxanes) need not induce synergy between DNA methyltransferase inhibitors and ABCB1 expression by the same mechanism(s). histone deacetylase inhibitors in stimulating ABCB1 mRNA Another possible mechanism for elevated ABCB1 expres- expression is supported by another study, which showed sion within drug-resistant cancer cells is ABCB1 mRNA that TSA or Aza treatment alone had little effect or weakly stabilization. To evaluate the role of this mechanism, the induced ABCB1 expression, respectively, in CEM-CCRF half-life of ABCB1 transcripts was determined through RNA cells.27 In contrast, treatment of these cells with both Aza decay experiments using actinomycin-D to prevent synth- and TSA induced a 10-fold higher level of ABCB1 expression esis of new transcripts. Since actinomycin-D is a substrate for than with Aza treatment alone. The study further showed the Abcb1 drug transporter,24 the concentration necessary that Aza-induced demethylation of the ABCB1 promoter to inhibit 95% of RNA synthesis was determined for each resulted in the release of methyl-CpG-binding protein-2 cell line individually prior to use in RNA decay experiments. from the promoter, promoter acetylation and partial The stability of the ABCB1 mRNA was not increased within transcriptional activation at the downstream ABCB1 pro- 27 MCF-7EPI or MCF-7TAX-2 cells upon the acquisition of drug moter. Interestingly, another study showed that acetyla- resistance (selection dose-9; Figures 2g and h). Conse- tion of the ABCB1 downstream promoter was dependent quently, alterations in the stability of ABCB1 transcripts upon its methylation state. Partial hypomethylation of could not account for the higher levels of ABCB1 mRNA and the ABCB1 promoter in CEM-Bcl2 cells induced ABCB1 protein found in drug-resistant MCF-7EPI and MCF-7TAX-2 transcription and daunorubicin-induced H3 acetylation of cells relative to MCF-7CC cells. the ABCB1 promoter preceded such changes in ABCB1 Due to recent reports of decreased methylation of the expression.12 downstream ABCB1 promoter in association with drug The effect of Aza treatment, TSA treatment and combined resistance,4–7,10,25 we next investigated this mechanism as Aza and TSA treatment on epirubicin or paclitaxel sensitivity the possible cause of increased ABCB1 expression in our was assessed using clonogenic assays. Under identical drug-resistant cell lines. Most of the variation in the degree conditions as those, which induced demethylation of of promoter methylation within drug-sensitive cell lines the ABCB1 promoter and increased ABCB1 expression in

(including MCF-7DOX-2, MCF-7EPI and MCF-7TAX-2 cells MCF-7CC cells, addition of 1 mM Aza to cultures for 96 h selected to dose-8 and MCF-7CC cells ‘selected’ to dose-12) consistently prevented colony formation in semi-solid occurred within the first 30 CpG sites (Figures 3d and 5a), medium, even after 14 days post administration. Similarly, which is consistent with the bisulfite sequencing data for no colony formation was observed when a combination of MCF-7 cells reported by David et al.8 Significantly decreased Aza and TSA was administered to cells. Unfortunately, this methylation of the ABCB1 downstream promoter was prevented us from assessing whether reduced ABCB1 observed in MCF-7EPI cells at the onset of drug resistance promoter methylation (with and without histone acetyla- (selection dose-9) both in terms of average promoter tion) was sufficient to induce resistance to epirubicin or to methylation (Figure 3a) and methylation across specific paclitaxel. Thus, while the two agents could induce

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significant ABCB1 transcript expression, this may or may considerably closer in proximity.8 Such significant reduction not have been sufficient to induce drug resistance. The lack in the methylation of a series of closely spaced CpG sites of colony formation is likely related to the cytotoxicity of near the site of transcript initiation may induce strong Aza and/or the nonspecific nature of this demethylating changes in chromatin structure, enabling binding of agent, which may have affected a variety of cellular transcription factors, RNA polymerase-II and other proteins processes, including cell adhesion or cell proliferation. involved in ABCB1 transcription initiation. These regional Consistent with these hypotheses, dramatic reductions in differences in DNA methylation status may suggest that long-term survival were observed in a clonogenic assay every CpG site is not equal in importance, but instead there when HeLa cells were treated with 1 mM Aza for 48 h.28 Aza may be critical areas involved in regulating ABCB1 gene treatment of these cells also resulted in strong growth expression by DNA methylation. inhibition due to arrest of the cell cycle in G2. Cell viability Dramatic methylation changes distinct to each drug- was retained at Aza concentrations at or below 100 nM,28 but resistant cell line were also observed (Figures 5g and h). The this would be expected to result in considerably reduced CpG sites strikingly hypomethylated in MCF-7EPI cells ABCB1 promoter hypomethylation and Abcb1 expression, included sites 1, 4, 30 and 66. Both site-1 and site-4 are making induction of drug resistance less likely. within the binding region of T-cell factor (TEF)/lymphoid The effect of TSA treatment alone on epirubicin or enhancer factor (LEF) (Table 4). Interestingly methylation at paclitaxel sensitivity in MCF-7CC cells (at the concentration site-13 was substantially increased and is located near the we used above to measure its effects on ABCB1 gene binding site of many transcription factors, is also located expression) was also assessed using clonogenic assays. TSA within the site implicated in regulation by p53, which acts to treatment of MCF-7CC cells had no effect on paclitaxel repress transcription (Table 4). Thus increased methylation at sensitivity. It did, however, increase sensitivity to epirubicin. this site could block repression by p53 thereby inducing Since TSA treatment alone had no effect on ABCB1 transcription. Substantial methylation changes were observed expression in our study, this enhanced sensitivity could in sites 10–13 within MCF-7EPI cells, which correspond to not be attributed to changes in Abcb1 expression or CpG sites that may be important in ABCB1 downstream function. Likely, TSA had effects on the acetylation and promoter regulation by multiple factors (Table 4). None expression of other genes that affect cellular sensitivity to of these sites exhibited large changes in methylation in epirubicin. MCF-7CC cells upon Aza treatment. The greatest methyl- In addition to broad changes in methylation, dramatic ation changes within the ABCB1 downstream promoter of reductions in the methylation of specific CpG sites were MCF-7TAX-2 cells occurred at CpG sites 6–8, 21, 32, 49 and 65, observed in MCF-7EPI and MCF-7TAX-2 cells upon acquisition some of which also exhibited striking changes upon Aza of drug resistance (Figures 5g and h, respectively). In treatment of MCF-7CC cells. The translation start site is located contrast, increased methylation at various CpG sites were very close to site-64, while site-8 is adjacent to the region observed upon ‘selection’ of MCF-7 cells in the absence of regulated by heat-shock factor-1 (HSF-1) (Table 4), suggesting drug (Figure 5e), suggesting that the reductions in ABCB1 it may be involved in the regulation of ABCB1 transcription downstream promoter methylation were due to selection for within paclitaxel-resistant cells. It is possible that the dramatic drug resistance and not propagation in long-term culture. reductions in methylation of CpG sites unique to MCF-7EPI When comparing drug-sensitive (dose-8) and drug-resistant cells play a particular role in ABCB1 induction, since MCF-7EPI (dose-12) MCF-7EPI and MCF-7TAX-2 cells, similar wave-like cells have considerably higher ABCB1 expression than patterns of methylation changes were observed across CpG MCF-7TAX or Aza-treated MCF-7CC cells. sites. There were particular ‘valleys’ of reduced CpG site Interestingly, two populations of differentially methylated methylation, followed or preceded by areas with little ABCB1 promoters were identified in bacterial clones hous- change. A region with strong hypomethylation induced by ing bisulfite-treated, PCR-amplified DNA from MCF-7TAX-2 selection for drug resistance spans CpG sites 49–66. Shared or MCF-7EPI cells at selection doses 9 and higher (Figures 3 valleys of hypomethylation in drug-resistant and Aza- and 4). Although this could be indicative of two different treated cells span CpG sites 19–22, which correspond to members of the cell population (high and very low ABCB1 the consensus Inr site, a site adjacent to the transcription expressors), this is not supported by examination of Abcb1 start site (Table 3), and CpG sites 32–44 implicated in the protein levels by confocal microscopy and flow cytometry regulation of ABCB1 expression by p53 (Table 4). Aza (Figure 1). Since Q-PCR experiments demonstrated that both treatment did not result in dramatic reduction in the upstream and downstream ABCB1 promoters are active in methylation of CpG sites 49–66 (Figure 5f). Since the region MCF-7EPI and MCF-7TAX-2 cells with heterogeneously methy- spanning these sites includes the translation start site (Table lated ABCB1 promoters (as opposed to the homogenously

3) and an important element for transcriptional activation methylated promoters of MCF-7CC cells possessing very low by p53 (Table 4), this may account for the lower levels of ABCB1 expression via the upstream promoter; Figure 7), it is

ABCB1 expression in Aza-treated MCF-7CC cells as compared likely that the entire population of MCF-7TAX-2 and MCF- with that in MCF-7TAX-2 and MCF-7EPI cells. 7EPI cells express ABCB1. Therefore, the ABCB1 downstream It is interesting to note that the first 30 CpG sites promoter may exhibit allelic differences in methylation within the ABCB1 downstream promoter are more widely within a given drug-resistant cell. Hypomethylation of the distributed than the remaining 36 CpG sites, which are downstream promoter could induce its availability in alleles

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with this modification, while alleles that maintain methy- tion can regulate allele-specific promoter usage in a variety lated downstream promoters may have adapted to activate of genes. Aberrant DNA methylation patterns are being transcription from the upstream promoter. To address this investigated as molecular markers to customize individual possibility, the methylation levels of the ABCB1 downstream chemotherapeutic treatments in breast cancers,31 and this promoter and the usage of the ABCB1 upstream promoter knowledge of ABCB1 methylation changes in association with were investigated in clonal isolates of MCF-7CC and MCF- drug resistance may help to improve our ability to distin- 7TAX-2 cells (selected to dose 12). No significant differences guish between drug-sensitive and drug-resistant tumors. in ABCB1 promoter methylation were observed amongst clonal isolates of MCF-7CC or MCF-7TAX-2 cells (Table 1). All MCF-7TAX-2 and MCF-7CC clones expressed the ABCB1 Conflict of interest transcript and used the upstream and downstream promoters at comparable levels to the population (Table 1). Similar The authors declare no conflict of interest. findings would be expected for the clonal isolates derived from drug-resistant MCF-7 sub-lines that displayed hypo- EPI Acknowledgments and hypermethylated populations. Interestingly, Aza-induced demethylation of MCF-7CC cells also produced hypo- We thank Matthew L Poulin and Liying Yan of EpigenDx and hypermethylated ABCB1 downstream promoters as (Worchester, MA, USA) for primer design and performing pyrose- determined by bisulfite sequencing analysis of bacterial quencing experiments assessing ABCB1 promoter methylation in clones housing DNA from Aza-treated cells. This suggested clonal isolates of MCF-7CC and MCF-7TAX-2 cells. We also thank that some alleles are resistant to Aza- or drug-induced Dr Gilbert Coˆte´, Bernice Suitor and Anne McBain of the Sudbury promoter hypomethylation (Figures 3b and c). Taken Regional Hospital for providing technical support for the FISH together, our findings suggest that the heterogeneously experiments. Funding: This project was funded by the Canadian methylated ABCB1 downstream promoters within drug- Breast Cancer Foundation—Ontario Region. This work was also supported, in part, by funds from the Canadian Institutes of Health resistant cells by BSP analysis is due to differentially Research (Grant MOP-89993), the Northern Cancer Research methylated promoters in ABCB1 alleles (and thus differen- Foundation and core support funds from Cancer Care Ontario. tial promoter usage) that occur in the same cells. This result is also supported by our recently published findings References indicating that hypermethylation of the downstream ABCB1 downstream promoter upon acquisition of docetaxel 1 Baker EK, El-Osta A. The rise of DNA methylation and the importance of resistance in MCF-7 cells results in exclusive usage of the chromatin on multidrug resistance in cancer. Exp Cell Res 2003; 290: upstream promoter for ABCB1 transcription.16 There have 177–194. also been previous reports of allele-specific gene expression 2 Lehnert M. Clinical multidrug resistance in cancer: a multifactorial 29 problem. Eur J Cancer 1996; 32A: 912–920. linked to allele-specific methylation of genes, although 3 Gottesman MM, Ambudkar SV. Overview: ABC transporters and human this has not been reported for the ABCB1 gene. However, disease. 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