Molecular Cancer Research 3:110-118 (2005)
© 2005 American Association for Cancer Research
Signaling and Regulation
A Nonclassic CCAAT Enhancer Element Binding Protein Binding Site Contributes to 
-Methylacyl-CoA Racemase Expression in Prostate Cancer
Shan Zha1 and
William B. Isaacs1,2
1 Brady Urological Institute and 2 Johns Hopkins Oncology Center, Johns Hopkins Medical Institutions, Baltimore, Maryland
Requests for reprints: William B. Isaacs, Department of Urology, Johns Hopkins Hospital, Marburg 115, 600 North Wolfe Street, Baltimore, MD 21287. Phone: 410-955-2518; Fax: 410-955-0833. E-mail: wisaacs{at}jhmi.edu
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Abstract
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-Methylacyl-CoA racemase (AMACR), an enzyme involved in branched-chain fatty acid ß-oxidation that is normally expressed at high levels in human liver, is specifically and consistently overexpressed at both mRNA and protein levels in human prostate cancer and potentially other cancer types. To characterize the mechanisms underlying transcriptional regulation of AMACR at the genetic and epigenetic levels, we performed a series of methylation and reporter assays in prostate cancer tissues and cell lines. The results ruled out altered methylation patterns as the cause of overexpression in prostate cancer cells. However, promoter deletion analysis identified an 8-bp nonclassic CCAAT enhancer element located 
80 bp upstream of the transcriptional initiation site that is responsible for AMACR expression in both prostate cancer cell lines and cell lines of liver origin. Deletion or mutation of this element completely abolished AMACR promoter activity. Ectopic expression of CCAAT/enhancer binding protein ß increased luciferase activity driven by a wild-type AMACR promoter sequence but not by the sequence in which the putative CCAAT/enhancer binding protein binding element had been mutated. These results implicate a nonclassic CCAAT enhancer element in the AMACR gene promoter as playing a critical role in the regulation of AMACR gene expression.
Key Words: -methylacyl-CoA racemase • CCAAT/enhancer binding protein • prostate cancer • methylation
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Introduction
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-Methylacyl-CoA racemase (AMACR) is an enzyme involved in ß-oxidation of branched-chain fatty acids and bile acid intermediates and is expressed most predominantly in normal liver and kidney, with more limited expression in the intestinal tract (1). Normal prostate epithelial and stromal cells typically show little expression of AMACR (2-4). In contrast, >95% of the primary prostate cancer cases show strong expression of AMACR protein, with >70% of the precancerous lesions, high-grade prostatic intraepithelial hyperplasia, expressing AMACR at intermediate levels (3). The remarkable consistence and specificity of AMACR overexpression in prostate cancer has made it a promising new diagnostic marker for this disease (3-5). Recent studies also imply that elevated AMACR expression is functionally important for optimal growth of prostate cancer cells in vitro, suggesting novel therapeutic strategies based on this gene and/or pathway (6).
Despite this interest, little is known about the transcriptional regulation of AMACR in normal tissue as well as in prostate cancer. In previous studies, we showed that despite higher expression of AMACR in androgen receptor–positive prostate cancer cell lines AMACR mRNA and protein levels are regulated independently of androgen receptor signaling (6). A more recent study suggested that exposure to biological substrates of AMACR, phytanic acid and its -oxidation product, pristanic acid, stabilizes the AMACR protein in a prostate cancer cell line without affecting its transcription (7). In another study, enhancer of zeste homologue 2, a polycomb protein that has been suggested to be up-regulated in hormone refractory and metastatic prostate cancer, has been suggested to repress AMACR expression based on a microarray profiling after ectopic expression of enhancer of zeste homologue 2 in a prostate cancer cell line (8). Although it is possible that protein stability might contribute to the enhanced AMACR protein level seen in clinical samples, the fact that overexpression of AMACR was first detected by cDNA microarray studies and quantitative PCR analyses revealed large increases at the mRNA level clearly indicated that a significant part of AMACR overexpression could be attributed to increased mRNA level. To begin to understand the transcriptional regulation of the AMACR gene, we explored methylation and transactivation as potential causes for increased AMACR mRNA level. Our data rule out differential methylation as a mechanism to explain the cancer-associated gene up-regulation. Promoter deletion analysis focused our attention instead on a nonclassic CCAAT enhancer element located 80 bp from the transcriptional initiation site as the cis-element responsible for the regulation of expression of AMACR in prostate cancer as well as in liver cell lines.
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Results
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CpG Island Adjacent to AMACR Transcriptional Initiation Sites Is Not Methylated in Either Normal Prostate or Prostate Cancer Cells
Somatic changes in CpG dinucleotide methylation patterns occur commonly during oncogenesis. In prostate cancer, glutathione S-transferase 
is the most consistently down-regulated gene yet identified, with promoter hypermethylation being observed in >95% of cases (9). In addition to gene silencing by promoter hypermethylation, gene activation associated with promoter hypomethylation has been shown for several cancer-associated genes (10, 11). Given the consistent nature of AMACR overexpression in prostate cancer and the pericentromeric genomic localization of AMACR (5p13.2-q11.1), we suspected that changes in DNA methylation, particularly hypomethylation, could be a potential cause of AMACR dysregulation. Using the GrailEXP software package (http://grail.lsd.ornl.gov/grailexp/, Oak Ridge National Laboratory, Oak Ridge, TN), a CpG island stretching from 95 to 450 nucleotides downstream of the transcriptional initiation site was identified in exon 1 of the human AMACR gene with a GC percentage of 69.1 and a CG/GC ratio of 0.83 (Fig. 1C). To explore if this potential CpG island is functionally important for regulation of AMACR expression, we first tested if global demethylation affects the expression of AMACR in prostate cancer cell lines, especially the ones with relatively low expression levels of AMACR mRNA (Fig. 1A). We treated prostate cancer cell lines, PC-3 and DU145, with different concentrations of the methyltransferase inhibitor 5-deoxyazacytidine (0.25-4 µmol/L) for 5 consecutive days, with medium changed every other day. AMACR expression was measured by quantitative reverse transcription-PCR and presented as the AMACR mRNA level per ß-actin transcript (Fig. 1B). In DU145 cells, global demethylation induced the expression of AMACR in a dose-dependent fashion. Although secondary effects downstream of 5-deoxyazacytidine-induced changes in other gene expression levels could not be excluded, these data suggested that methylation might play a role in suppression AMACR expression in this cell line. On the other hand, demethylation with 5-deoxyazacytidine had no effect of AMACR mRNA level in PC-3 cells (Fig. 1B), despite the apparent cellular senescence evidenced by morphologic changes (data not shown).
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FIGURE 1. Expression of AMACR and methylation analysis of AMACR promoter. A. Western blot shows the expression of AMACR protein in prostate cancer cell lines (LNCaP, PC-3, LAPC4, CWr22-Rv1, and DU145), liver cell line (HepG2), and embryonic kidney cell line (HEK293). Note: On longer exposure, DU145 has a faint band representing AMACR protein. B. Quantitative reverse transcription-PCR analysis of AMACR mRNA in human prostate cancer cell lines DU145 and PC-3 after the cells had been treated with different concentrations of methyltransferase inhibitor 5-deoxyazacytidine (0, 0.25, 0.5, 1,2, and 4 µmol/L) for 5 days. Normalized against ß-actin mRNA level. C. Schematic illustration of the only CpG island identified in the human AMACR gene (in exon 1).  , CpG dinucleotide. Bottom, methylation status of each CpG dinucleotide in this region from four paired human prostate tissues (normal or cancerous) identified through bisulfite sequencing. Five clones from each sample were sequenced and methylation percentage at each site is represented by different shades of gray: white, no methylation; light gray, 20-40% (1/5-2/5); dark gray, –60% to 80% (3/5-4/5); black, all methylated. Western blot, expression of AMACR protein in these samples.
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To examine the issue of possible promoter methylation changes in clinical prostate cancer tissue, where the up-regulation of AMACR has been most consistently associated with oncogenesis, we analyzed the methylation status of the exon 1 CpG island in the DNA from four pairs of matched prostate normal/tumor tissues using bisulfite sequencing (Fig. 1C). The results indicated that the majority of the CpG dinucleotides in this region are not methylated in either normal or cancerous samples. When particular CpGs are observed to be methylated in a given normal sample, the percentage of methylation in the corresponding tumor sample is quite similar, indicating no cancer-associated changes in DNA methylation pattern. Hence, the data do not support CpG island hypomethylation as a primary cause for AMACR overexpression in clinical prostate cancer tissues, although DNA methylation might play a role in restricting AMACR expression in some prostate state cancer cell lines, such as DU145. Indeed, bisulfite sequencing in this latter line revealed partial methylation of CpG dinucleotides in exon 1 (most CpG sites were methylated at 40-60%, although methylation at >50% of the CpG residues in any individual cloned promoter was not observed; data not shown).
5' Proximal Sequence Regulates the Expression of AMACR in Prostate Cancer Cell Lines and Liver Cell Line HepG2
We next focused our efforts on identifying possible cis-acting regulatory elements located upstream of the AMACR gene. To test if reporter-based assays are valid tools to study the transcriptional activity of AMACR, we first asked if the activity of AMACR upstream sequence-driven luciferase activities correlate with the expression level of the endogenous AMACR gene in each particular cell lines. The luciferase reporter driven by 1,711 bp upstream of the transcription start site and the complete 5' untranslated region from human AMACR was transfected into a panel of prostate cancer, liver, and kidney cell lines. In each case, empty pGL3Basic (pGL3b) vector was introduced into parallel wells as control for the variability between cell lines. The relative luciferase activity from AMACR promoter-driven plasmid was divided by the relative luciferase activity from empty pGL3b vector from the same cell lines and plotted in Fig. 2A. Prostate cancer cell line LAPC4 has the highest expression of endogenous AMACR, and it also has the highest luciferase ratio, whereas the PC-3 cell line with low AMACR expression displayed a low luciferase activity. It is interesting to note that DU145 presented an exception to this trend with moderate activity from luciferase reporter (data not shown) but relatively low endogenous expression, which is consistent with suppressed AMACR expression associated with partial promoter methylation in DU145. Furthermore, a panel of serial deletion constructs (Fig. 2C) was introduced into the same cells lines, and the activities from different constructs were plotted as a ratio to empty pGL3b as in Fig. 2A. Sequence located between 114 and 40 bp upstream of transcriptional initiation sites contributes to >80% of the reporter activity in all three high AMACR expression cell lines (Fig. 2B). To further narrow the area, reporter constructs containing 90 bp upstream of the AMACR transcription initiation site were generated and tested in LAPC4 and HepG2. The results mapped the cis-element required for transactivation to a 50-bp region between –90 and –40. It is worth noting that the +27 reporter (containing only the sequence from 27 bp downstream of transcriptional initiation site to the initiating methionine) has no activity when compared with the pGL3b empty vector, but the reporter containing the 40-bp upstream sequence displayed constant basal activity, albeit significantly lower than other longer reporter constructs, suggesting the presence of minimal promoter sequence between –40 and +27. No apparent TATA box was found in this area.
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FIGURE 2. Analyses of AMACR upstream sequences by luciferase reporter assays. A. Relative activity of 1,711-bp human AMACR-driven luciferase reporter in different cell lines. Luciferase activities in each cell line were divided by the activity from blank pGL3b promoter in the same cell line to normalize the cell line derived variances in luciferase assays. B. Luciferase reporter activities of different length AMACR promoter reporters were plotted as the distance to the transcriptional initiation site for each cell line. Activities are illustrated as AMACR upstream sequence-driven luciferase activities in each cell line divided by the activity of blank pGL3b promoter in the same cell line. C. Schematic illustration of the series of promoter deletion constructs used in Fig. 1D. D. Activity (i.e., relative luciferase units = reporter-driven firefly luciferase signal / thymidine kinase promoter-driven Renilla luciferase signal) of AMACR promoter deletion reporter constructs in LAPC4 and HepG2 cells. LAPC4 cells have relatively low relative luciferase units due to relatively higher activity of thymidine kinase promoter in this cell line in comparison with HepG2 cells. E. Schematic illustration of the minimal promoter reporter series and internal deletion reporter constructs used in Fig. 1F. F. Activity (relative luciferase units) of minimal promoters and internal deletion reporters in LAPC4 and HepG2 cells.
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Analyses of the 50 bp between –90 and –40 using MatInspector 2.2 on Transfact Web site (http://www.gene-regulation.com/; ref. 12) revealed potential binding sites for Sp1 family members, CCAAT/enhancer binding protein (C/EBP) and activator protein-2. To further narrow down the area, we divided this 50-bp region into three smaller regions (–90 to –73, –73 to –53, and –53 to –40; Fig 3A). Reporter constructs composed of these mini-activation sequences were generated with PCR and ligated into luciferase reporters. The results in LAPC4 and HepG2 cells indicated that DNA fragments between –90 and –73 contain the cis-element conferring most of the transcriptional activity, because >80% of the reporter expression was lost in the –73 to –53 reporter (Fig. 3B). To test if –90 to –73 fragment is responsible for transcriptional activation even when the distal sequences are present, internal deletion reporters were also made by removing –114 to –90, –114 to –73, or –114 to –53 from the –1,048-bp reporter construct as illustrated in Fig. 2E. The results further confirmed that the 17-bp sequence located from –90 to –73 indeed contained the cis-element(s) responsible for AMACR transactivation.
A Nonclassic CCAAT Enhancer Element Contributes to AMACR Transactivation
Having such a short sequence identified, we next attempted to find the transcription factor(s) that binds to this sequence and contributes to AMACR expression in both liver and prostate cancer cells. Despite extensive bioinformatic analyses, we could not match this sequence to any well-defined consensus binding site for known transcriptional factors. However, when low stringency searches were applied, nonclassic binding sites for activator protein-2, Sp1, and C/EBP could all be found within these 17 bp. When tested by ectopic expression of transcriptional factors with the luciferase reporter system, C/EBPß but not activator protein-2 or Sp-1 caused a mild increase of luciferase activity (Fig. 3E; data for activator protein-2 and Sp1 not shown). To further test if C/EBP binding is important for transactivation, we generated four additional luciferase reporters in the –1,048
(114-53) background. The 3XRac2wt reporter construct contained three tandem copies of the nonclassic C/EBP motif oriented as in the endogenous AMACR promoter (–95 to –66). The 4XRac2mC reporter contains four inserted tandem copies of same sequence in the same orientation but with mutations in the core C/EBP binding motif, rendering it unable to bind C/EBP family members. A reporter construct containing three wild-type tandem repeats in the opposite orientation was also made to test the effect of this variable on transactivation (3XRac2wtinv). As a control for the sequence between –66 and –73 bp, 3XRac3, containing three tandem copies of –73 to –44 of the AMACR promoter, was also constructed (Fig. 3A). When transfected into HepG2 and LAPC4, the backbone reporter [–1,048(–114 to –53)] showed no significant activity as expected. The reporter containing wild-type C/EBP binding motif in either plus or minus orientation induced significant reporter activity in both cells lines, whereas the reporter-containing mutations in the C/EBP binding motif did not reveal any significant luciferase activity above the background (Fig. 3B). The result verified that C/EBP binding is required for AMACR transactivation in reporter assays.
In addition, the effect of ectopically expressed C/EBPß on these different reporters was tested. As expected, only the reporter with tandem repeats of the wild-type C/EBP binding motif, but not the mutated motif, could be induced by C/EBPß (Fig. 3E). The expression of C/EBPß in these experiments was verified by Western blot (Fig. 3D). Finally, when either C/EBP, C/EBPß, or C/EBP
was overexpressed in DU145 cell lines, a slight increase of endogenous AMACR expression was seen in Western blot (Fig. 3F), further supporting the notion that C/EBP family members regulate AMACR expression. It is worth noting that the C/EBPß antibody used has a weak cross-reactivity with C/EBP on the Western blot.
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Discussion
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AMACR plays a required role in the complete ß-oxidation of branched-chain fatty acids and bile acid synthesis (1). Its expression in normal liver cells well suites its function (1, 13, 14). Its highly consistent overexpression in human prostate cancer is rather unexpected (3-5). Understanding the underlying mechanism for AMACR overexpression is important for resolving the metabolic alterations that accompany prostate epithelial cell transformation and any potential use of the AMACR promoter as a prostate cancer targeting tool. In this study, we perfomed promoter methylation analyses and a series of luciferase reporter assays to define transcriptional regulatory mechanisms for AMACR. These latter assays led to the identification of a nonclassic C/EBP binding site located 80 bp upstream of the transcriptional initiation site that conferred the transactivation of AMACR in prostate cancer cell lines and liver cell line in vitro. The C/EBP family of transcriptional factors consists of at least six members of the basic leucine zipper DNA binding proteins that bind to the same CCAAT palindromic DNA sequence through homodimerization or heterodimerization with family members. Previous studies suggested that C/EBP family transcription factors play important role in adipocyte differentiation (15). Sequential expression of C/EBPß and C/EBP followed by C/EBP is sufficient to induce adipocyte differentiation of 3T3-L1 preadipocytes in the absence of exogenous hormonal stimuli (15). In concert with this observation, a series of genes involved in fatty acid synthesis and degradation have been identified as C/EBP target genes in both human and rodent cell lines. In this sense, it is not surprising that a C/EBP binding element contributes to AMACR transactivation.
C/EBP and C/EBPß have also been widely studied in tumorigenesis. C/EBP expression is associated with increased expression of p21 cell cycle inhibitory protein (16), cell cycle arrest after viral infection (17), and terminal differentiation (18). Loss-of-function mutations in C/EBP are pro-proliferative and have been reported in acute myeloblastic leukemia (19). On the other hand, C/EBPß expression has been strongly linked to differentiation and proliferation of the mammary gland in response to hormonal stimuli (20). At least three different isoforms of C/EBPß have been characterized. They all share the same carboxyl terminus but initiate from different translation initiation codons at the amino terminus. The expression level and ratio within different C/EBPß isoforms has been correlated with pathologic progression of breast cancer in some cases (21). In contrast, little is known of the expression and regulation of C/EBP family members in normal prostate or during prostate oncogenesis, although C/EBP was first cloned from prostate (22). Given the unique metabolic properties of the normal prostate and the metabolic changes that occur during prostate oncogenesis, a better understanding of the expression and alternation of C/EBP family members, and other transcription factors involved in fatty acid metabolic regulation, is warranted and would further extend our understanding of cancer-associated metabolic changes.
As their name implies, the classic consensus sequence for C/EBP family members is the CCAAT box. However, there are many examples where C/EBP family members are capable of binding and activating nonclassic sites, such as the multiple C/EBP binding sites identified in p21 promoter (23). It has been suggested that in some cases palindromic sequences of CAA(N)nTTG or even CA(N)nTG are sufficient to recruit C/EBP binding. The sequence we identified in this study (GTGCGCAGA) shares some similarity with the nonclassic CCAAT C/EBP binding element at the promoter of the EBV lytic cycle transactivator protein ZTA (ATGACATCA) (24).
It is worth noting that the effect of overexpression of C/EBP on endogenous AMACR gene expression in DU145 was less significant than that seen for the luciferase reporter assays. One of the possible explanations is the methylation status of the AMACR promoter in DU145 cells. Although the AMACR promoter was not significantly methylated in either normal prostate or prostate cancer tissues we analyzed here, bisulfite sequence analysis revealed partial methylation of CpG dinucleotides in exon 1 of the AMACR gene in DU145 cells, and the expression AMACR could be induced in these cells by global demethylation with 5-deoxyazacytidine. Unfortunately, due to difficulties in performing reliable luciferase assay in PC-3 cells in our hands and the relatively high endogenous expression of C/EBP family members in CWr22-RV1 cells, DU145 cells were the only prostate cancer cells that we could perform matched luciferase reporter and overexpression assays to test the effect of increased C/EBP expression on AMACR promoter activity.
In summary, we have identified a nonclassic C/EBP binding motif in the promoter of the AMACR gene, which seems to play a critical role in its transcriptional regulation. Further study of C/EBP family members and their interactions in prostate cancer in particular are called for to expand our understanding of lipid metabolism in the normal prostate and the mechanisms responsible for dysregulation during prostate tumorigenesis. As recent data suggest that AMACR overexpression is beneficial for the optimal proliferation of prostate cancer cell line in vitro (6), it will be interesting to know what the exact role of the C/EBP family members in establishing the expression pattern is and what the other C/EBP target genes are in prostate and prostate cancer during prostate tumorigenesis. Furthermore, as AMACR is only one of a series of enzymes required for branched-chain fatty acid ß-oxidation, it is important to know if and how the other enzymes in the pathway are coordinately regulated. Indeed, at least one of the enzymes acting downstream of AMACR in peroxisomal ß-oxidation, D-bifunctional protein, is also overexpressed in prostate cancer (25). Alternatively, the search for other C/EBP target genes in prostate cancer might reveal alternative pathways beyond branched-chain fatty acid ß-oxidation, which may contribute to the metabolic pattern changes occurring during prostate tumorigenesis.
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Materials and Methods
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Cell Lines, Chemicals, Plasmids, and Antibodies
To extract DNA for bisulfite sequencing, fresh tissues were harvested as described previously from patients undergoing prostatectomy at Johns Hopkins Hospital for the treatment of prostate cancer (26). The human prostate cancer cell lines (LNCaP, PC-3, DU145, and CWR22-Rv1), human embryonic kidney cell line (HEK293), and human hepatocellular carcinoma cell line (HepG2) were acquired from the American Type Culture Collection (Manassas, VA) and cultured in RPMI 1640 (Invitrogen, Carlsbad, CA) or DMEM (Invitrogen) in case of HEK293 and HepG2 with 10% fetal bovine serum (Life Technologies, Grand Island, NY). The human prostate cancer cell line LAPC-4 (27) was obtained from Dr. John Isaacs (Johns Hopkins University) and was maintained in Iscove's modified DMEM (Invitrogen) with 10% fetal bovine serum and 1 nmol/L R1881 (methyltrienolone, New England Nuclear, Boston, MA).
Control plasmids pGL3b and pRL-TK were purchased from Promega (Madison, WI) and used for both luciferase assay optimization and normalization. Empty pcDNA3.1(+ and –) vectors were purchased from Invitrogen and used to compensate for unequal DNA/cytomegalovirus promoter levels in transfection/luciferase assays. The largest AMACR promoter-driven luciferase reporter, containing the seamless connection of 1,711-bp AMACR upstream sequence and 88-bp 5' untranslated region to firefly luciferase gene in which the initiating methionine for luciferase coincides with the initiating methionine for AMACR with no vector sequence in between, were generated as described before through PCR and cloning (6). The series of deletion reporter constructs (–1,048 EcoRI, –359 PstI, –202 SacI, and –114 SmaI) were generated by restriction enzyme digestion, polishing, and re-ligation. The small deletion reporter constructs (–90, –73, –53, –40, and +27) were generated by PCR amplification with linker sequences at both sides. Taking advantage of blunt ends generated by cutting with SmaI (–114), the PCR amplified proximal sequence with linker could be polished and inserted after cutting with SmaI. Finally, the tandem repeats were made by ligating synthetic oligonucleotides together with linker sequences and subcloning into the desired backbone sequences. Oligonucleotide sequences used are listed in Fig. 3C with an additional GTAC linker at 5'. All reporter constructs obtained were confirmed to have the designed sequence and orientation by automated DNA sequencing. Reporter constructs used are also graphically illustrated in Figs. 2C and 3A and C. Rat C/EBP (with only five nucleotides different from human C/EBP) and human C/EBPß expression vectors are generous gift from Drs. Gary Hayward and Daniel Lane (Johns Hopkins University). Human C/EBP expression vector was acquired by direct PCR amplification from human genomic DNA (C/EBP family members are intron-less genes) with linkers on both ends and ligation into pcDNA3.1.
Anti-AMACR polyclonal rabbit antibody was a generous gift from Dr. R. Wanders (University of Amsterdam, Amsterdam, the Netherlands). Monoclonal mouse anti--tubulin was obtained from Calbiochem (La Jolla, CA), polyclonal rabbit anti-C/EBPß was Santa Cruz Biotechnology (Santa Cruz, CA), and horseradish peroxidase–conjugated goat anti-mouse and anti-rabbit IgG was Pierce (Rockford, IL).
Protein and mRNA Analyses
For Western blot, cells were washed and lysed (4% SDS, 100 mmol/L Tris-HCl 7.4, 1 mmol/L EDTA). Protein concentration is determined using a bicinchoninic acid assay (Pierce) and 50 µg total protein reduced with ß-mercaptoethanol were subject to electrophoresis (10% SDS-PAGE gel), transferred to nitrocellulose (Bio-Rad, Hercules, CA), and probed with first and secondary antibodies essentially as described by Laemmli (28). For real-time quantitative reverse transcription-PCR analysis, total RNA was extracted with RNeasy (Qiagen, Valencia, CA) after treatment with 5-deoxyazacytidine (Sigma, St. Louis, MO) at different concentrations (0.25-4 µmol/L) for 5 days. Reverse transcription and quantitative amplification of AMACR were carried out as described previously (3) and ß-actin mRNA was measured using Taqman-based real-time PCR kit developed by Applied Biosystems (Foster City, CA). The expression of AMACR was presented as the ratio between AMACR and ß-actin mRNA.
Bisulfite Sequencing
Bisulfite treatment of DNA was done as described previously (29). Briefly, genomic DNA were extracted using DNeasy (Qiagen); 2 µg DNA were subjected to NaOH for 10 minutes, hydroquinone and sodium bisulfite overnight, purification, NaOH neutralization, and final precipitation by ethanol. For PCR, bisulfite-treated DNA (2 µL, 100 ng) was used as template for subsequent PCR reaction. Nested PCRs were carried out with external primer pair (forward 5'-AGYGTTATGGTATTGTAGGGTATT-3' and reverse 5'-ACRTTAACCTAAACCCRTAAATAA-3') first; then, 2 µL of the PCR product were used in the amplification with the inner primer pair (forward 5'-GTGTTATGGTTTTGGTTGAT-3' and reverse 5'-AAATAATCACTAACAATACCCTAAA-3'). The final PCR products were gel purified and cloned into TOPO vectors (Invitrogen). Five individual clones from each sample were sequenced at Johns Hopkins University Sequencing Facility using automated DNA sequencers.
Transfection and Luciferase Assays
Luciferase assays were done essentially as described (6). Briefly, medium density cells in 96-well plates (Isoplate TC, Perkin-Elmer Wallac, Gaithersburg, MD) were transfected with LipofectAMINE 2000 (Invitrogen) at 0.25 µL per 96-well plate well. To compensate for unequal DNA/cytomegalovirus promoter levels between groups, pcDNA empty vector was included as appropriate. pRL-TK (10 ng), which encodes Renilla luciferase, was included in all transfections to normalize the transfection efficiency. Luciferase assays were carried out using nonproprietary substrate-buffer mixture (30) after the cells were washed and lysed (Passive lysis buffer, Promega) on a Wallac 1450 Microbeta Jet luminescence reader. All experiments were repeated at least thrice, and all experimental groups therein were done in replicates of four to six. Firefly luciferase values were normalized to the corresponding Renilla values (shown as relative luciferase units) to control for transfection efficiency.
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Acknowledgements
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We thank David H. Koch for the generous support.
Received October 18, 2004;
revised December 21, 2004;
accepted December 28, 2004.
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Abstract
Introduction
