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J. Biol. Chem., Vol. 275, Issue 32, 24893-24899, August 11, 2000
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,,§¶
From the Kanematsu Laboratories, Royal Prince Alfred
Hospital, Missenden Road, Camperdown, New South
Wales 2050, Australia, § Commonwealth Scientific and
Industrial Research Organisation Molecular Science, P. O. Box 184, North Ryde, New South Wales 1670, Australia, and the
¶ Department of Medicine, University of Sydney, Sydney, New South
Wales 2050, Australia
Received for publication, August 16, 1999, and in revised form, April 19, 2000
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ABSTRACT |
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 TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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What defines the boundaries between methylated
and unmethylated domains in the genome is unclear. In this study we
used bisulfite genomic sequencing to map the boundaries of methylation
that flank the 5'- and 3'-ends of the CpG island spanning the promoter
region of the glutathione S-transferase (GSTP1)
gene. We show that GSTP1 is expressed in a wide range of
tissues including brain, lung, skeletal muscle, spleen, pancreas, bone
marrow, prostate, heart, and blood and that this expression is
associated with the CpG island being unmethylated. In these normal
tissues a marked boundary was found to separate the methylated and
unmethylated regions of the gene at the 5'-flank of the CpG island, and
this boundary correlated with an (ATAAA)19-24 repeated
sequence. In contrast, the 3'-end of the CpG island was not marked by a
sharp transition in methylation but by a gradual change in methylation
density over about 500 base pairs. In normal tissue the sequences on
either side of the 5'-boundary appear to lie in separate domains in
which CpG methylation is independently controlled. These separate
methylation domains are lost in all prostate cancer where
GSTP1 expression is silenced and methylation extends
throughout the island and spans across both the 5'- and 3'-boundary regions.
Almost all organisms with a genome of greater than 109
base pairs methylate their DNA, generally at the 5'-position of
cytosine in CpG dinucleotides. In vertebrate genomes CpG sites occur
about one-fifth of that expected on the basis of base composition. This has been attributed to the inherent mutability of 5-methyl cytosine to
thymine (1). However, CpG-rich clusters, termed CpG islands, are found
interspersed in large regions of the CpG-depleted sequence (2). The
maintenance of these CpG islands is indicative of a functional role,
and it is now well established that they commonly span gene regulatory
or gene promoter regions (3). It has been suggested that CpG islands
facilitate the expression of housekeeping genes by influencing
nucleosomal positioning and that conditions that alter the formation of
this array, such as methylation, may indirectly affect CpG
island-dependent gene expression (4). Unlike CpG sites in
the remainder of the genome, CpG islands are nearly always maintained
in an unmethylated state (5). Methylation of CpG islands can occur,
however, for example on the inactive X chromosome, in promoters of
imprinted genes and in aberrant methylation associated with oncogenesis
(6). In all of these cases methylation of CpG islands spanning the gene
promoter regions is strongly associated with transcriptional silencing
(7).
As yet there is no clear understanding of what permits CpG islands to
be preferentially unmethylated, what defines the boundary of the
unmethylated region, and what leads to the aberrant methylation of CpG
islands commonly seen in cancer. It has been suggested that maintenance
of the unmethylated CpG islands is dependent on continued active
transcription and/or on the binding of specific proteins that may
protect them from methylation (8). Analysis of the CpG island of the
adenine phosphoribosyl transferase gene in transgenic mice, using
mutated promoters, has shown that Sp1 sites are important for
maintaining this CpG island in its unmethylated state (9, 10). Thus
loss of specific proteins or interference with their binding or loss of
active transcription may all contribute to CpG island promoter
methylation in cancer.
The GSTP1 gene that encodes glutathione
S-transferase- In this paper we have extended the bisulfite sequence analysis of the
methylation profile of the GSTP1 CpG island/promoter to
include the flanking regions where the normal and prostate cancer
methylation profiles diverge. This has allowed identification of a
defined "boundary" region of methylation at the 5'-flank, which is
marked by a repeating ATAAA sequence.
DNA Samples--
DNA from three primary prostate cancer samples
and corresponding normal DNA from the diseased prostate were isolated
as follows. Tissue samples were isolated from patients undergoing
radical prostatectomy for prostate cancer. Tissue slices, about 4-mm
thick, were snap frozen in liquid nitrogen, and histology was performed on adjacent slices to identify regions of tumor and normal tissue. Tissue samples were isolated from the frozen slice using a punch and
ground into a powder under liquid nitrogen using a mortar and pestle.
DNA was isolated using TrizolTM reagent (Life Technologies,
Inc.) according to the manufacturer's protocols. DNA was further
treated with RNase A and then proteinase K before phenol extraction and
ethanol precipitation. DNA from the prostate tumor cell lines LNCaP and
PC3 was prepared as described previously (14). DNA from the following
normal tissues were obtained at autopsy from a 74-year-old male: brain
(cerebellum), lung, skeletal muscle, spleen, liver, pancreas, prostate,
heart, bone marrow, and blood. All tissues were examined by the
pathologist and deemed disease-free.
Bisulfite Conversion--
Sodium bisulfite converts cytosine
residues to uracil residues in single-stranded DNA under conditions
whereby 5-methylcytosine remains nonreactive. All cytosine residues
remaining in the target sequence after PCR amplification represent
previously methylated cytosines. The bisulfite reaction was carried out
on 1-2 µg of HindIII-digested patient DNA for 16 h
at 55 °C under conditions described by Clark et al. (15)
and Clark and Frommer (16). The samples were purified using Wizard DNA
Clean-Up System desalting columns (Promega), eluted in 50 µl of
H2O, and incubated with 5 µl of 3 M NaOH for
15 min at 37 °C. The solutions were neutralized by the addition of
NH4OAc, pH 7, to 3 M, and the DNA was ethanol precipitated, dried, resuspended in 10 µl of 10 mM
Tris-HCl (pH 8), 0.1 mM EDTA in the case of tissue samples
and 50 µl for cell line DNA and stored at PCR Amplification and Primers--
PCR amplifications were
performed in 50-µl reaction mixtures containing 2 µl of
bisulfite-treated genomic DNA, 200 µM of each of the four
dNTPs, 6 ng/µl of each of the primers, 1-2 mM
MgCl2, 2 units of AmpliTaq DNA polymerase (Perkin-Elmer),
and reaction buffer consisting of 67 mM Tris, 16.6 mM ammonium sulfate, 1.7 mg/ml bovine serum albumin, and 10 mM Sequence Analysis--
The PCR fragments (I-IV) were cloned
using the pCR-ScriptTM Amp SK(+) cloning kit (Stratagene)
according to the manufacturers instructions. Individual clones were
either sequenced manually using Sequenase version 2.0 DNA sequencing
kit (USB) or using PRISMTM DyeDeoxy Terminator Cycle
Sequencing Kit (PE/ABI) with AmpliTaq DNA polymerase and the automated
373A DNA Sequencer (ABI).
For automated direct PCR sequencing and quantitative Genescan analysis,
PCR products were reamplified using a Biotinylated/M13-tailed primer
mixture containing GST11-M13 and GST12 primers (see Table I). The direct PCR sequencing reactions
were performed using a PRISM Sequenase Dye Primer Sequencing Kit
(PE/ABI) on an automated 373A DNA Sequencer (ABI). Details of the
Genescan analysis are described by Millar et al. (14). The
percent methylation is calculated using peak height of C
versus peak height of C plus peak height of T for each
position.
Northern Blot Analysis--
Multiple tissue Northern blots I and
II were purchased from CLONTECH and probed with a
PCR fragment generated (see Table I) from exon 7 of the
GSTP1 transcript according to the manufacturer's instructions. The blots were then stripped and reprobed with the Like many "housekeeping" genes the GSTP1 gene
contains a typical CpG island that extends from ~400 base pairs
upstream to 800 base pairs downstream of the transcription initiation
site (Fig. 1A). We have
previously shown that the core promoter region is unmethylated in
normal prostate cells but becomes methylated in prostate cancer. To
understand the mechanism responsible for abnormal methylation of this
CpG island in prostate cancer tissues we have now: 1) extended the
methylation analysis of the GSTP1 gene across the entire CpG
island in a range of normal tissues, particularly in relation to the
extent of the unmethylated domain and the nature of its boundaries and
2) compared the methylation profile of the GSTP1 gene in
these domains to the methylation profile in prostate cancer cells.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
(GST-)1 contains a typical
CpG island promoter region and is widely expressed in most tissues,
with the exception of liver (11). It has recently been demonstrated
that GSTP1 becomes methylated in a high proportion of
prostate cancers and that this methylation is accompanied by gene
silencing (12, 13, 14). Detailed bisulfite sequencing analysis of the
CpG island spanning the core promoter region of the GSTP1
gene showed that methylation is extensive at essentially all CpG sites
in prostate cancer DNA (14). In contrast, the CpG island is completely
unmethylated in normal prostate tissue. However methylation was found
to occur outside the CpG island in the CpG-depleted 3'-region of the
GSTP1 gene in both normal and cancer tissue (14).
MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
20 °C. PCR
amplification reactions were performed within 24 h of bisulfite conversion.
-mercaptoethanol in buffer (10 mM
Tris-HCl (pH 8.0), 0.1 mM EDTA). The strand-specific nested primers used for amplification of bisulfite-treated DNA are indicated in Table I.
PCR primers used on bisulfite-treated DNA
-actin internal probe supplied to determine the loading levels of
each mRNA species. Blots were exposed and quantified using a
Molecular Dynamics PhosphorImager and ImageQuant software.
RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
View larger version (36K):
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Fig. 1.
Map of the GSTP1 gene.
A, C+G density plot of the GSTP1 gene with
positions of CpG dinucleotides above the line. The scale is marked in
base pairs (bp). The transcription start site is shown by an
arrow. B, the relative position and sizes of the
seven exons of the GSTP1 gene are shown. The
arrows represent the location of the two Alu repeats (21).
The four separate sequence regions that amplified and CpG sites
flanking each PCR region are shown. C, the sequence of the
5'-upstream PCR region is shown with the primer sequences
shaded and the position of CpG sites numbered
with respect to the start of transcription above the sequence. PCR1
contains the Alu repeat-(441-717) encompassing CpG sites 56 to
44.
Expression and Methylation Profile of the GSTP1 Promoter in Normal
Tissues--
The GST- protein is known to be widely expressed in
most tissues (11), but it is not known if the methylation pattern is equivalent in these tissues. Therefore, we examined the expression and
methylation profile of the GSTP1 gene in a number of normal tissues including heart, brain placenta, lung, liver skeletal muscle,
kidney, pancreas, spleen, thymus, prostate, testis, ovary, small
intestine, colon, and peripheral blood. Fig.
2 shows the tissue distribution of
expression of GSTP1 by hybridization to multiple tissue
mRNA Northern blots. Extensive expression was seen in almost all
tissues, in agreement with data obtained from immunohistochemical
analysis and studies of GST enzyme expression (11). However,
lowered GSTP1 expression was observed in the liver; this is
also consistent with previous data on protein levels (17, 18).
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We first examined the methylation profile of the CpG-rich promoter and
upstream flanking region. This region spanned 66 CpG sites (56 CpG to
+10 CpG relative to the transcription start site) and was analyzed by
PCR amplification of two separate regions (PCRI and -II, Fig.
1B). The methylation profiles were analyzed by either direct
PCR sequencing or cloning and sequencing. The core promoter region PCR
fragment (PCRII; spanning CpGs 28 to +10) was analyzed by direct
sequencing of the PCR product from each tissue sample. Direct
sequencing gives an average of the methylation level at any one CpG
site in the mixture of molecules amplified, as described by Millar
et al. (14). The 5'-upstream PCR fragment (PCRI; spanning
CpGs 56 to 30) contained a number of polymorphisms and therefore
could not be used reliably for direct sequence analysis; consequently
levels of methylation for the 5'-upstream region were determined by
cloning the PCR product and sequencing individual clones. The
polymorphisms found in the 5'-upstream region include variation in the
number and fidelity of (ATAAA)19-24 repeats as well as
sequence polymorphisms that include CpG site 48 and 33 (14). An
example of the methylation profile obtained for the clones generated
from the PCR fragment amplified from normal prostate DNA is shown in
Fig. 3. As can be seen the methylation
profile is heterogenous; in fact a heterogenous methylation profile was
common to all the normal tissue samples tested. The methylation data
generated from the clonal analysis was averaged for each CpG site and
as such corresponds to the direct PCR sequencing analysis.
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Analysis of the extent of methylation at individual CpG sites in the
normal tissues, prostate, blood, brain, spleen, smooth muscle, lung,
bone marrow, pancreas, and heart showed no methylation at all in the
core promoter region (CpGs 28 to +10) (Fig.
4B). The exception was in
normal liver DNA where significant methylation was seen in a cluster of
sites, from CpG site 7 to CpG site +7 encompassing the transcription
start site. A similar methylation profile was observed in two other
samples of normal liver DNA that were sequenced (data not shown).
Interestingly, liver was the only normal tissue examined that showed
reduced GSTP1 expression, indicating a correlation between
the methylation of these sites and expression.
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In the 5'-upstream region (CpGs 56 to 30), a marked changed was
observed in the methylation pattern of all the normal tissues examined
(Fig. 4A). There was essentially no methylation in all the
normal tissues up to and including CpG site 43; in contrast further
upstream, from CpG site 44 and beyond, there was extensive methylation at most CpG sites. However, considerable variability was
noted between different tissues in the level and pattern of methylation. Some sites (CpG sites 56, 55, and 53) were heavily methylated (75-100%) in nearly all tissues, whereas CpG site 52 and
48 was notable in that it was commonly unmethylated or
undermethylated in some tissues. Also the six CpG sites, 44 to 49,
were not methylated in smooth muscle cell DNA. Interestingly the abrupt transition from the DNA domain that is extensively methylated in all
normal tissues to that which is unmethylated, between CpG sites 44
and 43, corresponds to the location of an (ATAAA)19-24 repeat sequence (Fig. 1C). Moreover, this pattern of
methylation was found to occur in both GSTP1 alleles (data
not shown). Immediately upstream of the (ATAAA)19-24
repeat are two members of the Alu interspersed repeat sequence family
(Fig. 1). The CpG sites (spanning CpGs 56 to 44) in the Alu
sequence immediately adjacent to the (ATAAA)19-24 repeat
were hypermethylated in all normal tissues examined (Fig.
4A). The (ATAAA)19-24 repeat thus corresponds
to a distinct boundary that separates the methylated Alu repeat DNA at
the 5'-flank of the CpG island from the unmethylated DNA within the CpG island.
In contrast there is no clear sequence boundary at the 3'-end of the
GSTP1 CpG island. Moreover the 3'-flank of the CpG island does not contain any Alu repeats or any other obvious repeat sequences. We previously noted that in normal prostate tissue the CpG island, up to CpG site 33 as well as sites 52 and 53, was unmethylated whereas
the 3'-end of the GSTP1 gene from CpG site 69 to 103 was extensively methylated (14). To locate the exact methylation boundary
at the 3'-end of CpG island, we have now sequenced a further 1 kilobase, from CpG site 13 in intron 1 to CpG site 67 in intron 4 (PCRIII and -IV, Fig. 1B), from four normal prostate tissue
samples. As shown in Fig. 5, there is no
methylation in the CpG sites within the CpG island; however, there is
considerable heterogeneity in the methylation profile at the 3'-flank
of the CpG island and more CpG sites become methylated further
downstream from the CpG island region. Interesting, the first site that
is methylated is CpG 31, which is located at the junction of intron 1 and exon 2. The profile of each of the four normal prostate samples is
different but all lack a distinct methylation boundary gradually
increasing in methylation density over a 500-base pair region (see Fig.
7).
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Methylation Profile in Prostate Cancer DNA--
We have previously
shown that the core promoter region (CpGs 28 to +10) and all sites
examined in the body and 3'-end of the gene are extensively methylated
in prostate cancer DNA (14). We have now extended this analysis to
examine the extent of methylation in the region upstream of the core
promoter in the region (CpGs 56 to 30) where we have found a marked
boundary of methylation in normal tissue. We examined the methylation
state in two prostate cancer cell lines, PC-3, which expresses
GSTP1 and LNCaP, a GSTP1 nonexpressing cell line.
Using reverse transcriptase-PCR we have shown that the treatment of
LNCaP cells with 5-azacytidine, a demethylating agent, reactivates
GSTP1 mRNA expression (data not shown). In addition, we
analyzed prostate tumor DNA from three patients (BC, CC, and DC) and
DNA isolated from a histologically normal region of the prostate of one
of these patients (BN).
For LNCaP cells that do not express GSTP1 the extensive high
level of methylation seen in the core promoter region was found to
continue through the upstream region to and beyond the ATAAA boundary
(Fig. 6A). The methylation
profile of DNA from PC3 cells that express GSTP1 was
distinctly different from that of LNCaP cells. There was very little
methylation in the upstream region except beyond the boundary region
from (CpG 46 to 56). The low methylation observed is consistent
with the generally lower methylation level seen in the core promoter
(Fig. 6B). Immunohistochemical analysis of both cell lines
(data not shown) demonstrated that in the fully methylated LNCaP cells
the GST- protein was not expressed. PC-3 cells on the other hand
harbored two distinct classes of cells. The majority of the cells
expressed abundant GST- protein, whereas in a subset of cells the
GST- protein could not be detected. This is consistent with the
methylation patterns observed, that is the patterns represent an
average of the methylation profile of the pooled DNA that contain a
mixture of methylated and unmethylated molecules.
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In all three primary prostate cancer specimens (BC, CC, and DC),
extensive methylation was seen downstream of the ATAAA boundary (43
CpG to +10 CpG) (Fig. 6, A and B). The
methylation pattern was heterogenous at any one CpG site and varied
between patient samples. The levels of methylation across the promoter
region varied from 25 to 75% methylation for each CpG site, as
determined by direct PCR sequencing (Fig. 6B). However, when
we examined clones derived from the core promoter PCRII fragment we
found that two distinct classes of molecules existed, those that were completely unmethylated and clones that showed extensive methylation through this region (data not shown). Because the cancer samples are
not homogeneous and contain a mixture of normal and cancerous prostate
epithelial cells, as well as stromal elements, it is likely that the
unmethylated clones derive from contaminating normal cells. Therefore
in the analysis of the clonal data from PCRI, presented in Fig.
6A, we have only presented the frequency of methylation of
individual CpG sites among the population of methylated molecules. This
explains why the methylation pattern in the cancer samples appears to
be more intense from CpG 43 to 30 than from CpG 28 to CpG +10. As
we have reported for GSTP1 and other genes methylated in
cancer (14, 19, 20), individual differences in methylation of specific
CpG sites, e.g. CpG site 36, were evident between the
different patients. Beyond the boundary region (CpG 43), the extent
and pattern of methylation was essentially similar in the three cancer
samples and in the two normal prostate DNA samples studied, normal
prostate and BN (Fig. 4A and 6A).
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DISCUSSION |
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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It is well established that CpG islands remain unmethylated in normal cells and are generally associated with transcriptionally active genes (5). However, in cancer hypermethylation of CpG islands is a common aberration and this is often associated with gene silencing (6). What has not yet been established is what defines the unmethylated CpG island domain, that is are there distinct sequence boundaries between methylated and unmethylated sequences or do the methylation profiles merge, and moreover how are these boundaries disrupted or bypassed in a cancer cell. To define the sequence boundaries between the methylated and unmethylated domains of a CpG island, we have finely mapped the methylation profile of the upstream and downstream ends of the GSTP1 CpG island in a number of normal tissues including prostate tissue. The GSTP1 gene was chosen because it has a typical CpG island spanning the promoter and exons 1-3 of the gene, it is known to be expressed in a range of normal tissues, and it is frequently hypermethylated in prostate cancer.
A schematic representation of the methylation profiles across the
GSTP1 CpG island found in normal prostate tissues and
corresponding cancer samples is shown in Fig.
7. In the normal prostate samples there
is a marked transition from extensively methylated DNA upstream of the
GSTP1 promoter region to the unmethylated domain of the CpG
island and this coincides with the position of an
(ATAAA)19-24 repeat. The boundary between the methylated
and unmethylated domains in most other tissues examined also correlates
well with the position of the ATAAA repeat sequence, even though in
brain and smooth muscle the 5'-boundary position is less marked. The
boundary separating the methylated and unmethylated DNA domains at the
3'-end of the GSTP1 CpG island is less clearly defined.
There is considerable heterogeneity of methylation through a 500-base
pair region spanning the end of the CpG island in intron 2 and
extending into exons 3 and 4. In prostate cancer the distinct exclusion
of methylation from the CpG island is lost, and the CpG island becomes
methylated in both GSTP1 alleles.
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The nature of the sequences in the boundary region may give a clue as to the mechanism that normally protects the GSTP1 CpG island from methylation. The ATAAA repeat sequence that flanks the 5'-CpG island is present in about 20 copies and may itself act as a barrier to the methylation of the GSTP1 island in normal cells or could just be fortuitously located 5' or 3' to the "real" barrier sequence. The sequence immediately adjacent and extending 5' from the ATAAA repeat is a member of the Alu family of interspersed repeated DNA sequences, most closely matching subfamily Sx. The 3'-end of the Alu sequence corresponds exactly with the start of the ATAAA repeat. Indeed the ATAAA repeat may be an expansion of the residual poly(A) tail of the Alu element. It has been noted previously that unmethylated CpG islands are often flanked by methylated Alu sequences (21). In particular Graff et al. (21) showed that Alu sequences upstream of the E-cadherin and VHL genes were methylated in normal tissues, whereas adjacent CpG island sequences were not; however, the precise junction of the methylated and unmethylated domains was not determined in this study.
CpG islands are not always flanked by Alu sequences, however, as noted for the 3'-end of the GSTP1 island. Indeed the 3'-boundary of the GSTP1 CpG island, which is diffuse in nature, does not correspond to any identifiable sequence motif. However the start of methylation does correlate with transition from high CpG density of the island to more sparsely spaced CpG sites in the body of the gene. Moreover some of the first CpG site that was found to be methylated (CpG site 31, 1546 base) is located at the junction between intron 1 and exon 2. This location is similar to another boundary of methylation we have previously identified at the 3'-end of the HIC1 CpG island, which also occurs at an exon/intron boundary (22). As for the GSTP1 CpG island the HIC1 intron sequences are unmethylated, whereas adjacent exon sequences show substantial, but not complete methylation.
As discussed above, the boundaries of CpG islands do not appear to harbor common sequence elements at their flanking ends. Indeed the ATAAA repeat sequence boundary found directly 5' to the GSTP1 CpG island appears to be unique as we have not detected such a structure in other CpG island genes analyzed. However a similar pentanucleotide repeat element has been identified downstream of an Alu sequence in the 3'-untranslated region of a zinc finger cDNA sequence (23). The ATAAA repeat could be fortuitously located 5' or 3' to a real barrier sequence, which protects CpG islands from methylation. Alternatively the ATAAA repeat may itself act as a distinct barrier to CpG island methylation of the GSTP1 gene in normal cells but is inert in prostate cancer cells. The repeat sequence may provide binding sites for specific protein factors, in particular members of the high mobility group-I(Y) family of mammalian nonhistone proteins. These have been demonstrated to bind specifically to the minor groove of A-T-rich sequences and to function as gene transcriptional regulatory proteins (24). Moreover elevated high mobility group-I(Y) gene expression has been associated with progressive neoplastic transformation (25). Tamimi et al. (26) have shown that in prostate cancer high expression of high mobility group-I(Y) was observed in prostate tumors with higher Gleason grades. It will therefore be of interest to determine if the increased level of high mobility group-I(Y) expression plays a role in the initiation of hypermethylation of the GSTP1 CpG island in prostate cancer.
Whether hypermethylation in the tumor cell is initiated from the ends
of the CpG island or at "hypermethylation centers" within the CpG
island is unclear. From studies of transfected DNA, Graff et
al. (21) have suggested that methylation may progressively encroach from methylated Alu sequence regions flanking CpG islands. Others have suggested that methylation is initiated at "centers" within the islands and progressively spreads (27). The GSTP1 gene from all the separate prostate cancer tissues was found to be
extensively methylated throughout the entire CpG island. However, the
DNA samples studied were isolated from cells many generations after the
initiation of the methylation process. It is therefore difficult to
infer how the process may have begun. In the case of the PC3 (Fig. 5)
and DU145 (data not shown) prostate cancer cell lines, methylation is
found in the core CpG-rich promoter region but does not extend through
the 5'-flanking region. This pattern of methylation within the island
is also seen in DNA from liver, where GST- is expressed at low
levels. This would suggest that in these cells at least methylation
could have been initiated at sites within the CpG island or spread from
the 3'-methylated flanking sequences.
The susceptibility to initial methylation events in cancer may be
determined by transcriptional activity (or lack of) or by the loss of
or interference to binding of specific transcription factors such as
Sp1 (9, 10). Interestingly it has been noted that strong expression of
GST- protein in normal prostate epithelium is limited to the basal
cells and that many differentiated secretory epithelial cells do not
stain with anti-GST- antibodies (12). Thus methylation of the GSTP1
gene in prostate cancer cells may be preceded by a loss of expression
of the gene in normal epithelium. These observations indicate that
early GSTP1 gene inactivation in prostate cancer cells may
be a mechanism that predisposes the CpG island promoter region to the
de novo methylation pathway resulting in the spread of
methylation throughout the island in both alleles.
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ACKNOWLEDGEMENTS |
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We thank Dr. P Katelaris, S. Danieletto, and A. Lochhead for support in obtaining prostate tissue samples. DNA from normal tissue samples were kindly provided by Christoff Grunau, Yena.
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FOOTNOTES |
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The on-line version of this article (available at
http://www.jbc.org) contains primers, CpG sites, and exon sequences.
To whom correspondence should be addressed: CSIRO, Molecular
Science, P. O. Box 184, North Ryde, NSW 1670, Australia. Tel.: 612 9490 5148; Fax: 612 9490 5005; E-mail:
susan.clark@molsci.csiro.au.
Published, JBC Papers in Press, April 21, 2000, DOI 10.1074/jbc.M906538199
* This work was supported by Grant RG/44/96 from the New South Wales Cancer Council and National Health and Medical Research Council Grants 82294 and 82348.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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ABBREVIATIONS |
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The abbreviations used are: GST, glutathione S-transferase; PCR, polymerase chain reaction.
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REFERENCES |
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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