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Molecular Biology and Genetics |
Department of Surgery, Sections of Urology [H. K., Y. S., C. S., D. F. J.] and Pathology [A. C., A. F.], University of Wisconsin Medical School, Environmental Toxicology [C. R. R., D. F. J.], and University of Wisconsin Comprehensive Cancer Center [L. F. M., C. C., C. R. R., D. F. J.], Madison, Wisconsin 53792, and the Brady Urological Institute, Johns Hopkins Hospital, Baltimore, Maryland 21287 [G. S. B.]
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ABSTRACT |
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 Top ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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In an alternate approach to this problem, we have recently demonstrated that AR methylation at several methylation-sensitive enzyme restriction sites correlates with a loss of expression in AR-negative prostate cancer cell lines (9) . DNA hypermethylation is a well-described epigenetic mechanism that involves the addition of methyl groups to deoxycytosines in the palindromic dinucleotide CpG. Dense clusters of CpGs, termed CpG islands, are found at the 5' end of roughly 60% of genes and typically encompass the transcriptional promoter. Methylation of these CpG islands may function to inactivate gene transcription (10) . CpG island methylation plays a critical role during development by establishing haploid gene dosage and in the long-term repression of selected genes. Aberrant methylation has been implicated in the silencing of autosomal tumor and growth suppressor genes in cancer in a manner analogous to mutation or deletion (10) .
The mechanisms by which DNA methylation exerts its repressive function have not been completely defined. Islands of methylated DNA exist in an inactive or condensed chromatin structure in association with MeCP2, a protein that selectively binds to methylated DNA sequences, and with histone deacetylase (11) . Deacetylation appears to lead to the compaction of chromatin by favoring interactions between adjacent nucleosomes. The association between local methylation and higher order structure that results in a closed chromatin structure is complex (12) . Not all potentially methylatable CpGs in an island are required for gene silencing, and, furthermore, methylated DNA segments can confer silencing at a distance in cis (13) . The majority of studies that associate gene inactivation with methylation rely on an analysis at one or several CpG sites, typically using methylation-sensitive restriction enzymes. This often produces conflicting or contradictory results (14 , 15) . Recently, new techniques using sodium bisulfite treatment have been described that permit the examination of methylation at each CpG site within a CpG island (16) . Fine methylation mapping of the RB, GSTP-1, and MGMT genes has identified regions of increased methylation density that correlate with known transcription binding sites (14 , 17 , 18) . Other studies confirm that methylation of specific sequences may exert a local repressive effect on transcription by the direct inhibition of transcription factor binding (19 , 20) . Therefore, determining gene promoter sites that are methylated and associated with the loss of gene transcription may identify sequences important or critical to transcription.
Males have one copy of the AR located at Xq11–12. In females, most genes located on the X chromosome contain CpG islands that undergo methylation on one copy to establish normal gene dosage. The 3-kb AR promoter CpG island extends from roughly -500 through exon 1 and fulfills the criteria for a CpG island (21) . Methylation of the AR promoter during X inactivation has been used previously to study clonality in female tumors (22 , 23) . These studies have focused on methylation at one or several CpG sites located in exon 1 of the AR in cells with two X chromosomes.
The inactivation of single copy X-linked genes in males is capable of causing total loss of gene expression. Deletion or mutation may lead to the inactivation of X-linked disease genes in inherited diseases in males. One example is the X-linked lymphoproliferative syndrome that predisposes individuals to lymphomas (24) . An alternate mechanism for the loss of X-linked gene transcription is aberrant methylation. FMR-1 gene expression is lost via hypermethylation and results in a syndrome that includes mental retardation in affected males (25) . Thus far, methylation of specific X-linked genes has not been demonstrated to be associated with the development of sporadic cancers in males.
To further define the spatial relationships between AR CpG island methylation and gene silencing, we performed a detailed methylation analysis of prostate cancer cell lines and tumors. We found regions of consistent methylation in AR expression-negative prostate tumor cells that delineate critical sites for gene transcription. Furthermore, these hot spots identify markers for AR gene silencing. Aberrant methylation of the AR was not detected in human normal prostate or primary prostate cancer tissues. Methylation was found in hormone-insensitive prostate cancers and was associated with a loss of AR expression. Many different mechanisms contribute to overcoming the androgen dependence of advanced prostate cancer. AR methylation may represent a phenotype associated with the development of hormone independence in a subset of prostate cancer that does not express the AR. This finding is of therapeutic interest because we have demonstrated previously that AR gene methylation is reversible when prostate cancer cells are exposed to the demethylating agents 5-azacytidine and 5' aza-2-deoxycytidine (9) .
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MATERIALS AND METHODS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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DNA Extraction, Sodium Bisulfite Reaction, and PCR Amplification.
Genomic DNA was extracted using standard methods. One µg of genomic
DNA was treated with sodium bisulfite at 50°C for 18 h using a
CpGenome amplification kit (Oncor, Gaithersburg, MD). Five separate
regions within the AR CpG island were amplified using 100 ng of sodium
bisulfite-modified DNA in 20 µl of reaction buffer containing 1x PCR
buffer, 1.5 µM MgCl2, 125
µM deoxynucleotide triphosphates, 10 pmol of primers, and
1 unit of AmpliTaq Gold (Perkin-Elmer, Branchberg, NJ). All PCR primers
were designed for top strand amplification (Table 1)
. PCR conditions were as follows: 94°C for 9 min, followed by 35
cycles of 94°C for 50 s, 50°C–57°C for 1 min, and 72°C
for 50 s. An additional extension at 72°C for 4–20 min was then
performed for all reactions.
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To assess the methylation status of individual alleles, PCR fragments
surrounding the transcription start site (region 2) were subcloned
using the TOPO TA vector system (Invitrogen, Carlsbad, CA).
After transformation using Escherichia coli, we randomly
selected 10–25 individual E. coli colonies for each cell
line assessed. Plasmid DNA was isolated (QIA Miniprep kit; QIAGEN). Ten
to 20 plasmids were then sequenced using region 2 primers (Table 1)
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Ms-SNuPE Assay.
We surveyed methylation status at 14 CpG sites (Table 1)
in human
prostate samples and metastatic cancer samples using the Ms-SNuPE assay
(27)
. The reaction was performed using approximately 20 ng
of PCR products generated for each region as described above. Products
were placed in a 20-µl reaction buffer containing 1x PCR buffer, 10
pmol of each of the Ms-SNUPE primers, 1.5 µM
MgCl2, 1 µCi of either
[32P]dCTP or [32P]TTP,
and 1 unit of AmpliTaq Gold. Ms-SNUPE primers were designed for the top
strand. Reaction conditions included denaturing at 94°C for 10 min,
annealing at 55°C for 1 min, and single base extension at 72°C for
1 min. Denatured samples were placed in loading dye (95% formamide, 5
mM EDTA, 0.2% XC, and 0.2% bromphenol blue)
and electrophoresed on a 15% denatured polyacrylamide gel (7
M urea). Radioactive intensity was measured using a
PhosphorImager (Molecular Dynamics, Sunnyvale, CA). Reactions were
performed in triplicate. Controls included known proportions of
unmethylated and fully methylated plasmids.
Immunohistochemistry.
Ten metastatic tumor samples were available for immunohistochemical
analysis. Deparaffinized slides underwent microwave treatment in 1
mM EDTA (pH 8.0) for 20 s and were then placed in 1x
xylene cyanol wash solution (Ventana Biotek Systems, Tucson,
AZ). An automated immunostainer (Ventana GenII) was used to incubate
slides for 30 min at 37°C with a mouse monoclonal anti-AR antibody
(Ab-1, clone AR-441; Neomarkers, Fremont, CA) or mouse IgG as a
negative control (4 µg/ml). The Ventana biotin-avidin-horseradish
peroxidase detection system with diaminobenzidine substrate was used.
Slides were counterstained with hematoxylin, dehydrated, and
coverslipped. A known positive control of normal prostate tissue was
treated identically. Adjacent sections from each metastasis also
stained positively with fatty acid
synthetase.4
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RESULTS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). We initially mapped the prostate cancer cell lines in region 2 to
determine whether any consensus regions of methylation correlated with
loss of AR expression. Region 2 extended from -200 to +115 and
contained 21 CpG dinucleotide sites surrounding the consensus
transcription start site (Table 2)
. This core promoter contains several cis-acting elements
important to AR transcription (28)
and contains the
minimal AR promoter (29)
. LNCaP and PC3, which express the
AR (30)
, were unmethylated within this region as evidenced
by a lack of cytosine dinucleotides after sodium bisulfite sequencing.
During sodium bisulfite treatment and PCR, unmethylated cytosines are
converted via uridine to thymidine. In female DNA, which contains one
transcriptionally inactive X chromosome (and one active X chromosome),
all CpG dinucleotides were partially methylated as predicted (data not
shown). In contrast, cell lines not that do not express the AR, Du145,
PPC-1, DuPro, and TSU-PR1, showed marked methylation across CpG island
region 2. Cell line Du145 demonstrated complete, dense methylation
within all CpGs.
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CpG Methylation of Individual Alleles in the Core Promoter Region
(Region 2).
The direct sequence data demonstrated that the hypermethylation in
region 2 around the transcription start site was inversely correlated
with the AR expression. However, incomplete methylation was detected in
all cell lines that do not express the AR (Du145, DuPro, PPC-1,
and TSU-PR1) and may have been due to either variations in methylation
at individual CpG sites or the presence of unmethylated alleles. We
assessed the methylation status of the core promoter region (region 2)
for AR-negative cell lines in 10–20 individual alleles (Fig. 2)
. PP analysis of the methylation patterns on individual alleles in
normal female tissue demonstrated that 8 of 18 alleles were completely
unmethylated, consistent with the predicted pattern. This data further
confirmed a minimal PCR bias in this region. Methylation density on
individual X strands varied (50% of potential sites were
methylated), but CpGs between -131 and -121 and +44 and +54 contained
consistent methylation on at least one site in all methylated alleles.
CpG site +12 was also consistently methylated. Methylation at all CpG
sites was not required for gene silencing of the AR.
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. All alleles of PPC-1 also contained
methylation. Only several sites were methylated in several alleles, but
methylation consistently involved +44 to +54 and -131 to -121.
Methylation of these AR-negative cell lines involved all alleles, and
several regions adjacent to the transcription start site were
consistently involved. A second group of cell lines, DuPro and TSU-PR1, demonstrated methylation in only 40–70% of the alleles. The remaining alleles in region 2 were without any detectable methylation. All alleles contained lower amounts of methylation when compared with normal female tissues and other AR-negative lines, PPC-1 and Du145. Methylation was found to preferentially encompass the identified hot spots. Therefore, both variations in methylation at specific sites and the presence of unmethylated alleles may contribute to the partial methylation found in these lines.
Mapping of CpG Methylation in Other CpG Island Promoter Regions.
The methylation status of individual CpGs was determined by direct
sequencing in four other regions of the CpG island (Table 2)
. Region 1,
extending from -526 to -259 (eight CpG sites), was tested to map
methylation at the 5' edge of the CpG island. We found methylation in
all prostate cancer cell lines, in normal prostate, and in normal renal
tissues and lymphocytes. Methylation occurred frequently at the island
edge and did not correlate with AR expression. In regions 3 (+130 to
+446), 4 (+498 to +668; data not shown), and 5 (+920 to +1408), we also
found no consistent sites of methylation that differentiated
AR-expressing cell lines from non-AR-expressing cell lines. LNCaP was
found to be completely unmethylated in these three regions. PC3, a cell
line that expresses AR at low levels, contained several scattered and
minimally methylated sites (10–40%), including several that
encompassed the translation start site. Transcriptionally inactive cell
lines, including Du145, contained methylated and unmethylated CpGs in
these 3' regions. Complete methylation was noted in TSU-PR1 at sites
+1216 and +1218, which may indicate the presence of another
transcriptionally important downstream region.
AR Methylation in Normal Prostate Tissues, Primary Prostate
Cancers, and Metastatic Cancers.
We examined methylation status in 10 paired normal prostate and primary
(localized) prostate cancers derived from radical prostatectomy
specimens. Fifteen metastatic lesions were also harvested from 12
patients who had died from hormone-independent prostate cancer. We
initially surveyed 14 CpG sites using a quantitative methylation assay,
Ms-SNuPE (27)
. CpG sites examined by Ms-SNuPE (listed in
Table 1
) included the preferentially methylated sites identified from
fine-mapping of prostate cancer cell lines. All normal prostate and
primary prostate cancers did not contain methylation in the AR core
promoter region or around the translation start site. All of these
tissues expressed the AR (data not shown. The Ms-SNuPE analysis of 15
metastatic samples detected methylation of the CpG promoter in 1 lymph
node (Fig. 3
, M1) and 1 cranial (Fig. 3
,
M2) metastasis from different patients. Sample M1 contained
70% methylation at two CpG sites in region 2 (-177 and -131), with
lower levels of methylation found at other sites. Sample M2 contained
methylation (35%) at CpG sites +1257 and +1408 downstream from the
translation start site. No significant methylation was detected in
region 2 sites tested in this sample.
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. Direct sequencing of three metastatic samples confirmed
substantial methylation in M1 in AR regions 1 and 2 adjacent to the
transcription start site (Table 2)
. This methylation (from -177 to
-121 and +44 to +108) encompassed the two consensus methylation
regions and was similar to methylation seen in AR-negative prostate
cancer cell lines. Sample M2 contained 30% methylation extending
downstream from +1257 through +1408. M3 demonstrated no methylation and
expressed the AR (data not shown). Direct sequencing confirmed the
Ms-SNuPE results. To correlate AR expression with methylation, an immunohistochemistry assay for AR expression was performed in metastatic lesions from which tissue sections could be obtained. AR staining was found to be heterogeneous and present in most samples, including sample M3 (data not shown). Four of 10 samples were found to contain no staining in 50% or more of cells, including samples M1 (75–90% negative) and M2 (50% negative). In summary, methylation of the AR promoter occurred in two of four samples containing a significant loss of AR expression.
Methylation of Non-CpG Sequences.
There have been several reports documenting methylation occurring on
the outer cytosine of CpNpG sequences (17
, 18)
. We
surveyed C to T conversion in more than 4000 cytosines not followed by
guanine in region 2 (data not shown). We found four methylation
variants: (a) two at CTG; (b) one on the outer
cytosine of CpNpCpG; and (c) one not associated with
guanine. The CpNpG methylation variants were observed only on the
methylated alleles and were reproducible. If this is due to incomplete
sodium bisulfite treatment, the frequency of error is <0.1% with our
assay.
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DISCUSSION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Regional variations in methylation density occur across the CpG island in AR-negative prostate cancer cell lines. However, it is significant that we were able to identify several consensus regions of methylation adjacent to the AR transcription start site where methylation correlated with loss of AR expression. These methylation hot spots putatively identify promoter regions critical to transcription. In vitro methylation of an identified hot spot in a reporter construct containing the p16/cdk2 gene led to a subsequent decrease in reporter expression (33) . Consensus methylation sequences may encompass known transcription factor binding sites, such as AP-2, and methylation may directly block transcription factor binding (19 , 20) . In the AR, consensus regions of methylation occur within the AR minimal promoter as determined by deletion studies (29) . Although investigated widely, the specific cis regulatory elements within this region have not been clearly defined (28 , 29 , 34) . We have identified one hot spot in the AR promoter occurring at -132 to -122 adjacent to a purine-rich region found to positively regulate AR promoter activity by the functional analysis of deletion mutants (28) . A second consensus region of methylation was found between +44 and +54. This fragment did not correlate with any previously identified cis-acting elements. In several cell lines and in one metastasis (M2) lacking AR expression, dense methylation downstream from the translation start site may indicate another potential consensus region of methylation. This region was not fully characterized in our study. Methylation of these hot spots may additionally reflect a repressive chromatin structure and nucleosome positioning (35) . We suggest that methylation of these hot spots may function in a manner similar to an inactivating mutation in the AR gene.
The analysis of methylation by fine-mapping prostate cancer cells in vitro and in vivo demonstrated a region of relative lack of methylation that directly encompassed the transcription start site (-56 to +20). This was confirmed by an analysis of individual alleles. Transcriptional inactivation associated with methylation does not clearly require every CpG to be methylated in this region. Undermethylation may reflect the binding of proteins to this region, thus protecting the DNA from the action of DNA methyltransferase. This region contains a Sp1 site (-42), and Sp1 sites are generally devoid of methylation in otherwise hypermethylated genes such as the X-linked gene HPRT (36) . Sp1 transcription factors may also attract factors that induce the removal of methylated cytosines (37 , 38) . The finding of regional hypomethylation also emphasizes the necessity of examining all or multiple CpG sites when assessing genes for hypermethylation.
To further address the question of methylation heterogeneity, we isolated and sequenced individual copies of AR DNA. In normal female tissues and prostate cancer cells, we confirmed that methylation occurs consistently within the two identified hot spots. Complete methylation of all CpGs in the minimal promoter was not observed, although one cell line (Du145) contained methylation on >90% of the CpGs in this region. Methylation density, as well as location, appears to be important for gene silencing. Methylation on as few as one or two critical sites is sufficient to inactivate transcription of the Epstein-Barr virus (39) . With the exception of the identified hot spots, we noted a marked variation in the methylation of individual CpGs between alleles, even in normal female tissues. It was recently reported that T cells from normal individuals demonstrate a significant amount of diversity in the methylation of specific Notl loci, a methylation-sensitive restriction enzyme site typically found within CpG islands (40) . Epigenotypes from peripheral blood also contain significant heterogeneity across the FMR1 locus (25) . Variability in methylation found at most sites in the AR further confirms a lack of faithful inheritance of specific methylation patterns during DNA replication, even among molecules from clonal cellular populations. This heterogeneity reflects the mechanisms regulating maintenance methylation and demethylation.
The analysis of methylation in specific alleles revealed that two AR-negative prostate cancer cell lines, TSU-PR1 and DuPro, contained unmethylated alleles in the minimal promoter region of the CpG island. Therefore, methylation may not directly regulate gene silencing in one or both of these cell lines. However, sequencing of each allele was not tested in all regions of the CpG island. Complete methylation may occur in other downstream regions containing important elements for transcription. For example, we noted full methylation of sites +1216 and +1218 in TSU-PR1 and dense methylation of other downstream sites in one metastasis (M2). Our previous data demonstrating that the demethylating agent 5' aza-2-deoxycytidine induces AR reexpression in these two cell lines support the presence of other methylated regions that are of importance to AR transcription (9) . Methylated DNA associates with repressive chromatin characterized by the presence of underacetylated histones. We are able to induce expression of the AR in all AR-methylated cell lines by sequentially exposing prostate cancer cells to 5' aza-2-deoxycytidine and the histone deacetylation inhibitor trichostatin A.5
Regional methylation within the AR CpG island was associated with
silencing of the AR gene both in vitro and in
vivo. In the prostate cancer samples examined, methylation
occurred only in advanced hormone-independent disease from patients who
had died of prostate cancer. A significant loss of AR expression
(
50%) was found in 4 of 10 specimens tested. This rate is similar to
that seen in previous studies (2, 3, 4)
. In certain tissues,
such as bone metastases, the loss of AR expression may be greater
(3)
. Methylation clearly encompassed the previously
identified hot spots in the transcription start site in one metastatic
sample (M1). A second metastatic tumor (M2) contained methylation
extending downstream from the translation start site. AR loss of
expression due to methylation in this region alone did not clearly lead
to a loss of expression on the basis of our data, but we cannot rule
out the presence of another critical hot spot in the 3' region of the
CpG island. It has been observed, based on methylation patterns in the
E-cadherin and the Von Hippel-Lindau (VHL) genes,
that during carcinogenesis, methylation propagates from 5' and 3'
"de novo methylation centers" at the edges of the CpG
island (41)
. Associated with this progressive methylation
was the loss of gene transcription. Therefore, incomplete methylation
may serve as a marker for gene inactivation in some cases.
The loss of AR expression may represent an important phenotype in prostate cancer progression. Growth under these AR-independent conditions appears to be dependent on the acquisition of autocrine pathways, such as activation of epidermal growth factor receptor or transforming growth factor ß (42 , 43) , or the activation of alternate pathways including HER-2/neu tyrosine kinase (44) . The loss of AR expression associated with methylation of the gene may represent a tumor with a ‘hypermethylator phenotype’ that has been postulated to occur in colon cancer and other cancers (45) . Alternatively, inactivation of the AR may be a selective alteration important for hormone-independent growth in some prostate cancers. Expressed AR may function as a growth suppressor in PC3 and LNCaP (containing a mutant AR) cancer cells (46 , 47) . A similar "squelching" phenomenon has been proposed to explain the observation that estrogen receptor reexpression inhibits estrogen receptor-negative breast cancer cells (48) . These observations suggest that the loss of AR expression, including that associated with AR methylation, may be an important step in the progression of a subset of prostate tumors.
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FOOTNOTES |
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1 This work was supported by NIH CA76184-01 and a
University of Wisconsin Howard Hughes Faculty Development Award. 
2 To whom requests for reprints should be
addressed, at K6/527 Clinical Science Center, 600 Highland Avenue,
Madison, WI 53792. Phone: (608) 265-8828; Fax: (608) 263-0454, E-mail: jarrard{at}surgery.wisc.edu
3 The abbreviations used are: AR, androgen
receptor; Ms-SNuPE, methylation-sensitive single nucleotide primer
extension.
4 G. Steven Bova, personal communication.
Received 11/ 1/99. Accepted 4/21/00.
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REFERENCES |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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(GSTP1) gene in prostate cancer. Oncogene, 18: 1313-1324, 1999.[Medline]
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3-kb CG-rich region (each CpG is indicated by a
vertical tic) extending across the transcription start
site (right angle arrow at +1) and through exon 1
(black bar). Regions examined by mapping methylation are
shown (designated as Regions 1–5).
Methylation-sensitive restriction enzyme sites are noted as follows:
Smal, S; BssHll,
B; and Hpall, H.
UTR, untranslated region. ATG,
translation start site.
; methylated CpG
dinucleotide, •.