From the Cancer and Polio Research Fund Laboratories, School of Biological Sciences, University of Liverpool, P. O. Box 147, Liverpool L69 7ZB, United Kingdom
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ABSTRACT |
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Elevated intracellular levels of S100A4, an
S100-related calcium-binding protein, induce metastatic capability in
benign mammary tumor-derived epithelial cells and in transgenic mice
bearing oncogene-induced benign mammary tumors. The
S100A4(p9Ka) gene in rat mammary epithelial
cells expressing low levels of S100A4 yields a reduced number of
fragments upon digestion with the methylation-sensitive restriction
enzyme, HpaII, compared with the gene from high
S100A4-expressing cells. Genomic sequencing of two potential regulatory
elements in the S100A4 gene, an intronic enhancer and TATA
box region, revealed that in low S100A4-expressing cells, most cytosine
bases exhibited high levels of resistance to conversion to thymine by sodium bisulfite. In derivative cell lines, which express high levels
of S100A4, only a small number of cytosine bases were resistant to
treatment with sodium bisulfite. In contrast, cytosine bases in the DNA
surrounding an upstream regulatory region, which binds inhibitory GC
factor in the low-expressing cell lines, are sensitive to conversion to
thymine by sodium bisulfite in both low- and high-expressing cell
lines. The results suggest that the rat S100A4 gene is
maintained in a different state in the low-expressing cell lines and
that this state might be a consequence of the pattern of methylation in
this regulated gene that does not contain a CpG island.
Altered DNA methylation has been associated with carcinogenesis in
a number of experimental systems (1, 2). Hypomethylation of DNA in
tumor cells might result in the elevated expression of tumor-promoting
genes, whereas hypermethylation of DNA might be involved in reducing
the expression of tumor-suppressing genes (1). Furthermore, it has been
suggested that 5-methylcytosine might contribute to mutations in DNA as
a result of its chemical conversion to thymine (1). Understanding the
methylation status of genes thought to be involved in the development
of cancer is therefore of some importance.
S100A4(p9Ka), a small calcium-binding protein, is present at a
relatively low level in a number of normal differentiated cell types
(3). Cultured rat or mouse mammary epithelial cells exhibit a low level
of S100A4 and its mRNA, whereas closely related metastatic epithelial cells (4, 5) and derivative elongated cells (6-8) exhibit
high levels of S100A4 and its mRNA. Experimental elevation of the
level of S100A4(p9Ka) in benign tumor-derived mammary epithelial cells
induces the metastatic phenotype in the cells (9). In transgenic mice,
multiple rat (10) or mouse (11) S100A4 transgenes confer the
metastatic phenotype on oncogene-induced (10) or spontaneous (11)
benign mammary tumors, respectively. In view of the metastasis-inducing
properties of S100A4 in epithelial cells, it is important to understand
mechanisms whereby its level is kept low in these cells.
The rat S100A4(p9Ka) gene contains a
cis-acting sequence that binds a GC factor-like repressor
protein and exerts a repressive effect on the transcription of the
S100A4(p9Ka) gene in low S100A4(p9Ka)-expressing rat benign mammary tumor cells but not in high-expressing metastatic or
myoepithelial-like rat mammary epithelial cells (12). Hypermethylation is also believed to play a role in the transcriptional silencing of the
mouse (mts1) and human (CAPL) S100A4
genes (13-15). To find out whether DNA methylation modulates
transcription of the S100A4 gene in rat cells, the pattern
and degree of methylation of genomic DNA from high or low
S100A4(p9Ka)-expressing rat mammary tumor-derived cell lines have been
investigated using sodium bisulfite-mediated genomic sequencing.
Cell Culture--
The benign rat mammary epithelial cell line,
Rama 37 (17), which expresses a low level of S100A4(p9Ka) (7), a
derivative benign myoepithelial-like cell convert, Rama 37-E8 (17), and the metastatic Rama 800 cell lines (18), both of which express high
levels of S100A4 and its mRNA (4, 7), were grown as described
previously (19). In order to reduce any alterations due to long term
culture, the Rama 800 cells are used at passage numbers between 11 and
14; the Rama 37 cells are used between passages 28 to 32, and the Rama
37-E8 cells, which have been derived from Rama 37 cells, are used at a
passage number of 3-8 after cloning, which corresponds to an
equivalent passage number of 30-38 for the Rama 37 cells (17). When
experiments with demethylating agents were conducted, cells were
cultured in medium containing 2 µM 5-azacytidine
(5-aza-C),1 10 µM S-adenosyl-L-homocysteine
(SAH), or 10 µM 6-azacytidine (6-aza-C) for 1 week. For
measurement of DNA methyltransferase activity, the cells were treated
with either 2 µM 5-aza-C, 10 µM SAH, or 10 µM 6-aza-C for 24 h.
Isolation of Genomic DNA from Cultured Cells--
Cells were
grown to near confluence in five, 15-cm diameter culture dishes, washed
twice with phosphate-buffered saline, and then drained. After scraping
the cells from each dish into 1 ml of lysis buffer (0.3 M
sodium acetate, 10 mM Tris-HCl, pH 7.5, 1 mM
EDTA, pH 8.0, 1% (w/v) SDS, 0.2 mg/ml proteinase K (freshly added) and
10 µg/ml DNase-free RNase), the cells were incubated at 56 °C for
2-3 h. After extraction with phenol/chloroform and chloroform, the
upper aqueous phase was removed, and nucleic acids were precipitated
with isopropyl alcohol for 10 min at room temperature. The resulting
pellet of DNA was washed with 70% (v/v) ethanol, air-dried, and
dissolved in double-distilled water (ddH2O).
Isolation of Cloned cDNA for Rat Calcyclin--
Pools of
cloned cDNAs from a library (8) corresponding to the mRNA of
the myoepithelial-like, benign mammary tumor-derived cell line, Rama 29 (19), were screened by hybrid-selected translation followed by
two-dimensional gel electrophoresis of the translation products (8) to
identify the protein product of the selected mRNA. Individual
clones of a positive pool were similarly screened. One cloned DNA,
hybridizing to an mRNA that encoded a protein that migrated close
to S100A4(p9Ka) upon two-dimensional gel electrophoresis (20),
corresponded to part of the mRNA for rat calcyclin (21).
Nucleic Acid Hybridization--
50 µg of genomic DNA
were digested with restriction enzymes, MspI or
HpaII, (100 units), and 10-µg samples of digested DNA were
subjected to electrophoresis on 0.8% (w/v) agarose gels. 10 ng of
either MspI or HpaII-digested cloned
S100A4(p9Ka) DNA provided a positive
hybridization control. The fractionated DNA was depurinated,
neutralized, blotted, and fixed onto Hybond-N membranes, according to
the manufacturer's instructions (Amersham Corp., Little Chalfont, UK).
The UV-treated membranes were incubated with 2.5 ng/ml of either a
denatured NcoI-BamHI fragment of the S100A4(p9Ka) gene (Fig. 1) or a cloned cDNA
corresponding to rat calcyclin mRNA radioactively labeled (22) with
[32P]dCTP (approximately 109 cpm/µg of
DNA). The incubation conditions for hybridization and washing of the
filters were as described previously (9).
For Northern hybridization, 10 µg of total RNA was subjected to
electrophoresis on 0.8% (w/v) formaldehyde gels (23), transferred onto
Hybond-N membranes, and hybridized with a radioactively labeled (22)
cDNA of rat S100A4(p9Ka) probe (specific
activity of 5 × 107-1 × 108
cpm/µg of DNA). The membranes were washed twice for 20 min with 0.1×
SSC (15 mM sodium chloride, 1.5 mM sodium
citrate), 0.1% (w/v) SDS at 65 °C, and subjected to
autoradiography. Equal loading of RNA samples onto each lane was
ensured by hybridizing the filter to a probe for the constitutively
expressed rat non-muscle actin mRNA (24).
Sensitivity of Deoxycytidines to Sodium Bisulfite--
The
method was an adaptation (25, 26). Briefly, genomic DNA from either
high or low S100A4(p9Ka)-expressing cell lines was isolated and treated
with sodium bisulfite solution, under conditions in which cytosine
bases, but not methylcytosine bases, are converted to uracil. Regions
of the relevant genomic DNA are then amplified using strand-specific
primers, and the resulting PCR products are cloned. DNA sequencing of
both strands of the resulting PCR products is used to identify the
presence of cytosine or methylcytosine in the specific region of the
original DNA sample. Genomic DNA was isolated from cultured Rama
37CL-A3, Rama 37-E8, and Rama 800 cells as described above. 10 µg of
the genomic DNA was digested with EcoRI to reduce its
viscosity. The digested DNA was extracted twice with a
phenol/chloroform/isoamyl alcohol (25:24:1, v/v) mixture and
precipitated with ethanol. Samples (2 µg) of the DNA were denatured
in 100 µl of 0.2 N NaOH at 37 °C for 20 min. 800 µl
of 5 M sodium bisulfite solution (2.5 M sodium
metabisulfite, 125 mM hydroquinone, pH 5.0) was added to the denatured DNA template, vortexed, and centrifuged briefly before
being overlaid with 120 µl of mineral oil. The mixture was incubated
in a water bath at 50 °C for 3 h. The reaction mixture was
carefully removed from beneath the mineral oil, and the DNA was
extracted using "glass milk" powder (Bio 101). After 30 min on ice
and centrifuging, the pellet was washed according to the manufacturer
(Bio 101). DNA was extracted with 100 µl of 0.2 N NaOH
solution, allowed to stand at room temperature for 10 min for
desulfonation, and then precipitated with 0.3 volumes of 10 M ammonium acetate, pH 7.0, and 2.5 volumes of ethanol. The
DNA was dissolved in 50 µl of ddH2O. PCR was used for
amplifying the appropriate region of the
S100A4(p9Ka) gene for DNA sequencing. Primer
pairs (Table I) with flanking
EcoRI recognition sequences (not shown) to aid subsequent
cloning were specific for the bisulfite-modified DNA. The sense strands
of the regulatory regions surrounding the GC factor recognition
sequence (nucleotides DNA Methyltransferase Assay--
In order to find out whether
there was an alteration in the DNA methyltransferase activity between
the high-expressing and low-expressing S100A4(p9Ka) cell lines, the
ability of cell extracts to incorporate methyl-3H from
S-adenosyl-L-[methyl-3H]methionine
into poly(dI-dC) was measured (27). Cultured cells were diluted to a
concentration of 3 × 105/ml, plated into 9-cm dishes
overnight, washed twice with phosphate-buffered saline, and resuspended
in 500 µl of 50 mM Tris-HCl, pH 7.8, 1 mM
EDTA, 1 mM dithiothreitol, 0.1% (w/v) sodium azide, 6 mg/ml phenylmethylsulfonyl fluoride, 10% (w/v) glycerol, 1% (w/v)
Tween 80 (27). The suspension was passed twice through a 22-gauge needle and subjected to three freeze-thaw cycles of Analysis of the Pattern of Methylation of the S100A4(p9Ka) Gene in
Genomic DNA of Cell Lines Using a Methylation-sensitive Restriction
Enzyme--
Genomic DNAs isolated from the low-expressing Rama 37CL-A3
cells, the myoepithelial-like Rama 37-E8, and the metastatic epithelial cell line, Rama 800, share a common MspI-digestion pattern
with the cloned S100A4(p9Ka) DNA (Fig. 2)
detected by a probe (Fig. 1)
corresponding to the upstream and first exon region of the rat
S100A4(p9Ka) gene. This pattern consists of three
major bands of about 0.25, 0.4, and 0.9 kbp (Fig.
2A). The band of 0.9 kbp contains the two DNA fragments of 850 and 860 bp, and the additional two bands correspond to the DNA fragments of 419 and 277 bp, of the
expected size of fragments generated by complete digestion of the
cloned S100A4 gene with MspI (Fig. 1), according
to the previously reported nucleotide sequences of the rat
S100A4(p9Ka) gene (10, 18). Digestion of DNA from
the Rama 37-E8 and Rama 800 cell lines with the methylation-sensitive
isoschizomer, HpaII, produces the same digestion pattern as
the cloned fragment of upstream genomic DNA (Fig. 2A),
suggesting that the region of the S100A4(p9Ka)
gene detected by the probe is unmethylated in CCGG sequences in these
two cell lines. In contrast, genomic DNA from
low-S100A4(p9Ka)-expressing Rama 37CL-A3 cells yielded a unique high
molecular weight band of approximately 2.3 kbp with HpaII
(Fig. 2, lane 2), and the band of DNA corresponding to the 419-bp fragment was absent. The result suggests that the
MspI site at
Unlike S100A4(p9Ka), which is expressed at a low level in Rama 37CL-A3
cells, but at a high level in Rama 37-E8 and Rama 800 cells, S100A6
(calcyclin) mRNA is normally expressed at the same level in all
three cell lines (7). A radiolabeled calcyclin cDNA probe
corresponding to the 3' non-coding and exon 2 regions of rat calcyclin
gene was hybridized to genomic DNAs isolated from the Rama 37CL-A3 and
Rama 800 cells, which were digested with the methylation-sensitive
restriction enzyme HpaII. The result showed that both Rama
37CL-A3 and Rama 800 cells share the same HpaII digestion
pattern (Fig. 2B).
The Effects of Demethylating Agents on the Level of the Endogenous
DNA Methyltransferase and on S100A4(p9Ka) mRNA in the Rama
Cells--
Rama 37CL-A3 and Rama 800 cells were cultured in the
absence of, or in the presence of, either 2 µM 5-aza-C or
10 µM SAH or 10 µM inactive analogue,
6-aza-C, for 24 h (see "Experimental Procedures"). In the
absence of inhibitors, the specific activity of DNA methyltransferase
(DNA-MT) in Rama 37CL-A3 cells is about 1.5 times higher than that in
Rama 800 cells (Fig. 3). DNA-MT activity
fell by an average 98 and 90% in Rama 37CL-A3 and Rama 800 cells,
respectively, after treatment with 2 µM 5-aza-C or 10 µM SAH (Fig. 3). In contrast, treatment with the inactive
analogue, 6-aza-C, had no significant effect on DNA-MT activity (Fig.
3).
In order to determine the effect of these various compounds on S100A4
mRNA levels, Rama 37CL-A3 cells were treated in culture with either
5-aza-C or SAH or the non-methylation inhibitor, 6-aza-C. The cells
were tested for the removal of methyl groups by the methylation
inhibitors by carrying out bisulfite treatment, strand-specific amplification, and sequencing of the TATA box region of the
S100A4(p9Ka) gene. Rama 37CL-A3 cells treated
with the methylation inhibitors showed the methylation pattern of the
Rama 800 cells.
Total RNA was isolated from the treated cells, and the levels of
S100A4(p9Ka) mRNA and of a normalization control mRNA,
Sensitivity of Deoxycytidine to Sodium Bisulfite in the
S100A4(p9Ka) Gene and Its Upstream Region--
An examination of the
nucleotide sequence between 2,696 base pairs upstream and 2,474 bp
downstream of the start site of transcription of the rat
S100A4(p9Ka) gene revealed that there are only 44 CpG dinucleotides in this region. Although these CpG dinucleotides are
clustered, particularly in the regions
The TATA box region contains DNA between
In the TATA box region of the low S100A4(p9Ka)-expressing Rama 37CL-A3
cells, 21/27 of the cytosine residues in the non-coding strand
(excluding primer sequences) were protected from bisulfite modification
(Fig. 5). At three further positions, protection of cytosine in the
amplification product of the 5' primer was apparent but was not
confirmed as amplified as G by the 3' primer, and at two further
positions, amplification of G by the 3' primer, suggesting protected C,
was not confirmed by a C in the product of the 5' primer. The most
likely explanation for these 5 minor discrepancies during PCR
amplification is the preferential amplification of DNA strands of DNA
from a template heterogeneous at these particular sites.
In contrast to the results with the low S100A4(p9Ka)-expressing Rama
37CL-A3 cells, less protection was observed in the two cell lines that
contain high levels of S100A4(p9Ka) and its mRNA, Rama 37-E8 and
Rama 800. In the Rama 800 cells (Fig. 5),
no cytosine residues were protected, and in the Rama 37-E8 cells, a
single cytosine residue in a CCC cluster was protected (Fig.
5) and confirmed by a G in the product of the 3' primer. Protection of
this single base is not associated with the expression of S100A4(p9Ka) mRNA, since both the Rama 37-E8 and Rama 800 cells express similar, high, levels of S100A4(p9Ka) mRNA.
The cytosine residues in the intron region of the
S100A4(p9Ka) gene were found to be completely
protected in the coding strand of genomic DNA of the Rama 37CL-A3 cells
(Fig. 6, A and C),
and precisely complementary sequences were obtained from the products of the 5' and 3' amplification primers (Fig. 6, A and
B). In contrast, in both Rama 37-E8 and Rama 800 cells, only
one of these cytosines was protected from sodium bisulfite-induced
conversion to thymine (Fig. 6, A, B, and
C) in this region of the S100A4(p9Ka)
gene. This cytosine is located in a CpNpG trinucleotide.
In the Rama 37CL-A3 cells, the protected cytosines that were observed
in the TATA box and intron regions of the
S100A4(p9Ka) gene occurred as single cytosines or
as cytosine clusters as well as in CpG and CpNpG di- and trinucleotides
(Figs. 5 and 6).
In the upstream regulatory region of DNA, which surrounds the GC factor
recognition sequence, only one protected cytosine residue (at position
The rat S100A4(p9Ka) gene (12, 20) does not
contain a CpG island located between 2,696 base pairs upstream and
2,474 base pairs downstream of the start site of transcription, in
common with the human (30) and mouse (14) S100A4 genes.
However, the rat S100A4(p9Ka) gene shows a
10-20-fold increase in expression between low-expressing benign rat
mammary epithelial (Rama 37CL-A3) and high-expressing derivative
elongated, myoepithelial-like (Rama 37-E8), or metastatic (Rama 800)
epithelial mammary tumor cells (4, 6, 7). Using
MspI/HpaII digestion and Southern blotting analyses, different patterns of methylation of the
S100A4(p9Ka) gene were revealed between the
low-expressing Rama 37CL-A3 cell line on the one hand and the high
expressing, Rama 37-E8 and Rama 800 cell lines on the other. Treatment
of the Rama 37CL-A3 cells with either of two demethylating agents,
5-aza-C or SAH, significantly raises the level of p9Ka mRNA in this
cell line, strongly suggesting that methylation of cytosine bases is,
at least in part, responsible for repressing the production of
S100A4(p9Ka) mRNA. The fact that the methylation inhibitors do not
increase significantly the level of S100A4(p9Ka) mRNA in the
high-expressing Rama 800 cells suggests that in the Rama 37CL-A3 cells
the methylation inhibitors are affecting directly the methylation of
the S100A4 gene and are not acting indirectly, for example,
by affecting the expression of another gene product.
Mapping by genomic sequencing of individual protected cytosine
bases indicated that cytosines in the TATA box and putative enhancer-containing DNA regions of the
S100A4(p9Ka) gene were highly protected from the
action of sodium bisulfite only in the low S100A4(p9Ka)-expressing cell
line but not in the two high S100A4(p9Ka)-expressing cell lines. In
these regions of the S100A4(p9Ka) gene,
resistance to conversion of cytosine bases to uracil by sodium
bisulfite was found infrequently in CpG dinucleotides due to their
paucity (no CpG in the TATA box region, 2 CpGs in the intron region).
In contrast, protection of cytosine bases from reaction with bisulfite
is more commonly observed in CpNpG trinucleotides (8 in the TATA box
region and 6 in the intron region). Methylation of CpNpG trinucleotides
has been shown to occur in mammalian cells using the bisulfite
modification procedure, but generally only a proportion of the
molecules examined were methylated (31). In the present experiments,
all randomly picked clones gave the same result.
In the Rama 37CL-A3 cells, protection from sodium bisulfite is also
evident in sequences CpCpCpApA, CpCpTpA, CpCpCpTpG, CpCpCpApT, CpTpCpTpT, CpApA, CpApCpApA, CpTpC, CpTpT, and CpTpA (16 such sequences
in the TATA box region and 8 in the intron region). It is possible that
the protection afforded to the cytosine bases is due to their
methylation. A low level of similar unconverted cytosines has been
reported recently (31); however, it was not possible to distinguish a
low background of unreacted cytosine bases from a low level of
non-symmetrical methylation in the plasmid-based experimental system
employed. Although in genes that possess discrete CpG islands, it is
well established that cytosine methylation occurs predominantly in CpG
dinucleotides; few genes that do not possess CpG islands have so far
been studied. In the genome as a whole, methylation of CpA, CpT, and
CpC dinucleotides has been detected by nearest-neighbor analysis (32).
Although a greater proportion of CpG dinucleotides are methylated
due to the low overall occurrence of this dinucleotide in the genome,
over 50% of the methylation of the genome as a whole occurs in CpA,
CpT, and CpC dinucleotides (32). However, it is not known whether these
methylated dinucleotides are associated with any particular genes.
Similar, diverse, non-symmetric sites of methylation have been
described previously as "densely methylated islands" at origins of
replication associated with ribosomal protein S14 and the
dhfr locus in Chinese hamster ovary cells (33). However, it
has been suggested that these densely methylated islands of methylated cytosine residues might be artifactual, arising from incomplete denaturation of the DNA prior to treatment with sodium bisulfite, at
least at the dhfr locus (34). In the present experiments, it
is unlikely that the unusual pattern of methylation arises from
incomplete denaturation of the DNA. Neither of two closely related cell
lines, Rama 37-E8 nor Rama 800, showed the same pattern of protection
of cytosine bases as Rama 37CL-A3 cells, even though treatment of their
DNA with sodium bisulfite was carried out under identical conditions
and at the same time as the DNA from the Rama 37CL-A3 cells. The lack
of protection of cytosines in the upstream region of the DNA from the
Rama 37CL-A3 cells further argues against a generalized technical
failure to denature the DNA prior to reaction with sodium bisulfite.
However, it is possible that the DNA in the TATA box and intron regions
of the S100A4(p9Ka) gene in the Rama 37CL-A3
cells is in some way different from that isolated from the Rama 37-E8
and Rama 800 cells, such that it is not completely denatured under
conditions that denature the DNA from Rama 37-E8 and Rama 800 cells
(24). Based on the derepressive effect of methylation inhibitors on
S100A4(p9Ka) mRNA levels and the results of the digestions with
MspI and HpaII, the reduced accessibility of
cytosines to bisulfate in the Rama 37CL-A3 DNA is likely to be
associated with methylation. However, it cannot be ruled out that the
reduced accessibility of cytosine bases to sodium bisulfite in the Rama
37CL-A3 cells is a consequence of CpG methylation outside the regions
of the genome analyzed in the present experiments.
The region surrounding the upstream cis-acting inhibitory
sequence of the S100A4(p9Ka) gene (12) exhibits
little or no protection of cytosine bases in all three cell lines. In
the Rama 37CL-A3 cells, there is a clear contrast between the density
of protected cytosine bases observed in this region (0/39) and the
density of protected cytosine bases in the TATA box (21/27) and intron regions (16/16) of the rat S100A4(p9Ka) gene. It
is possible that the unprotected nature of this region of the gene in
all three cell lines is associated with the binding of the inhibitory
GC factor (12). In the Rama 37-E8 and Rama 800 cells, the level of GC
factor mRNA is lower than in the Rama 37CL-A3 cells (12).
Treatment of Rama 37CL-A3 cells with demethylating agents, but not an
inactive analogue, reduces the number of methylcytosine residues in the
S100A4(p9Ka) gene region of the Rama 37CL-A3
cells and causes an approximately 5-6-fold increase in the steady
state level of S100A4(p9Ka). However, although the endogenous
S100A4(p9Ka) mRNA in the Rama 37CL-A3 cells can be increased to a
certain extent, neither of the two demethylating agents was able to
increase the level of this mRNA to more than half that of its
relative level in the Rama 800 cells. Since the inhibitors have no
effect on S100A4(p9Ka) mRNA level in at least one high-expressing
cell line (Rama 800), it is possible that this lack of full induction
represents the presence of inhibitory levels of GC factor which serve
to limit the expression of the S100A4(p9Ka) gene
in Rama 37CL-A3 cells, and suggests that methylation of positive
regulatory elements cooperates with an inhibitory factor to completely
inhibit the production of S100A4 mRNA.
Altered levels of DNA methyltransferases and/or patterns of methylation
are thought to contribute to the formation of cancer. Since it is
likely that cytosine methylation of the
S100A4(p9Ka) gene plays a role in the suppression
of transcription of rat and mouse S100A4(p9Ka), and in view of the
metastasis-inducing properties of human S100A4 (35), it is possible
that inadvertent hypomethylation of the
S100A4(p9Ka) gene in human breast epithelial
cells might contribute to the malignant progression of some breast cancers.
INTRODUCTION
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Abstract
Introduction
References
EXPERIMENTAL PROCEDURES
1, 404 to
1,227) (12) and the first intron
(nucleotides +135 to +312) (16) relative to the start site of
transcription of the S100A4(p9Ka) gene and the
antisense strand of the TATA box region (nucleotides +36 to
113) were
amplified. 50-µl PCR mixtures contained 100 ng of bisulfite-treated
DNA, 1× PCR buffer (20 mM Tris-HCl, pH 8.0, 50 mM KCl), 20 pmol of each primer, 2.5 mM
MgCl2 (empirically determined), 0.05% (w/v) detergent
W-I, 0.2 mM of each dNTP, and 2.5 units of Taq
DNA polymerase. The reactions were incubated in a programmable heating
block for l cycle at 95 °C for 5 min and for 30 cycles at 95 °C
for 30 s, a suitable annealing temperature for each primer pair
(Table I) for 60 s and 72 °C for 90 s. Controls, in which
either DNA or enzyme was omitted, did not yield PCR product. The PCR
mixture was extracted, precipitated with ethanol, washed, and dried.
The DNA was dissolved in 17 µl of ddH2O, digested with 10 units of EcoRI at 37 °C for 2 h, and heated at
65 °C for 10 min. The mixture was subjected to electrophoresis on a
0.8% (w/v) low-melting agarose gel, and the band of amplified DNA
fragment was excised and ligated to EcoRI-cut pBluescript II
(Stratagene). The ligated material was transformed into competent
XL1-Blue Escherichia coli cells and a blue/white screening
procedure was applied. Plasmid DNA was prepared from randomly picked,
white bacterial colonies (23), and the nucleotide sequence of the
inserts was determined manually. For each bisulfite reaction, up to
five independent colonies corresponding to the cloned upper strand of
the amplified DNA, and up to five independent colonies corresponding to
the cloned lower strand of the amplified DNA were sequenced. The
results of the multiple sequencing reactions were the same for each
strand. Protection of cytosines was recorded when protected C on one
strand corresponded to a G and not an A on the opposite strand of the PCR product.
Primers used for the strand-specific amplification of specific regions
of the S100A4(p9Ka) gene following treatment of genomic DNA with sodium
bisulfite
70 and 37° C,
and the protein concentration was determined (28). 5 µg of cellular
protein was mixed with 0.5 µg of poly(dI-dC) and 3 µCi of
S-adenosyl-L-[methyl-3H]methionine
(American Radiolabeled Chemicals Inc.) in a total volume of 20 µl.
The reaction was incubated at 37° C for 2 h, stopped with 300 µl of 1% (w/v) SDS, 2 mM EDTA, 3% (w/v)
4-aminosalicylate, 5% (v/v) butanol, 0.25 mg/ml calf thymus DNA, 1 mg/ml proteinase K, and incubated for 30 min at 37° C. This mixture
was extracted once with a phenol/chloroform/isoamyl alcohol (25:24:1,
v/v) mixture, and the DNA was precipitated with ethanol. The resulting
pellet was dissolved in 40 µl of 0.3 N NaOH and incubated
at 37° C for 45 min. This solution was then spotted onto a Whatman
filter, dried, and washed with 5% (w/v) trichloroacetic acid and 70%
(v/v) ethanol. The radioactivity incorporated into the poly(dI-dC) was determined using the 3H protocol of a scintillation
spectrometer (Packard).
RESULTS
2,146 bp and some upstream sites (Fig. 1) are
methylated in the Rama 37CL-A3 cells and cannot be cut by the
methylation-sensitive restriction enzyme HpaII, whereas
sites at
1,450 and
1,727 are unmethylated in this cell line due to
the presence of the 277-bp band in the HpaII-digested DNA.
The results also strongly suggest that the MspI site at
600 is not methylated in the Rama 37CL-A3 cells. The results suggest
that MspI sites flanking the potentially regulatory GC
factor recognition sequence (10) are not methylated in the Rama 37CL-A3
cells.
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Fig. 1.
Sites for restriction enzyme MspI
in the upstream region of the rat S100A4(p9Ka)
gene. The locations of MspI recognition sequences in
the rat S100A4 gene (12, 20) are shown as vertical
lines above the representation of the gene with the sizes in bp of
the expected bands from complete digestion. The
NcoI-BamHI DNA fragment used as a probe to detect
digested DNA in the Southern blotting experiments and the locations of
the regulatory sites are also shown.
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Fig. 2.
Analysis of the methylation pattern of the
S100A4(p9Ka) gene in the genomic DNA of Rama
37CL-A3, Rama 37-E8, and Rama 800 cells by Southern blotting and
hybridization. A, 10 µg of genomic DNA from Rama
37CL-A3 cells (lanes 1 and 2), Rama 37-E8 cells
(lanes 3 and 4), Rama 800 cells (lanes
5 and 6) or 10 pg of a cloned
S100A4(p9Ka) gene fragment comprising 2,696 bp of
upstream DNA, the entire first exon, the first intron, and 15 bp of the
second exon of the rat S100A4(p9Ka) gene
(lanes 7 and 8) were digested with restriction
enzymes MspI (lanes 1, 3, 5, and 7) or
HpaII (lanes 2, 4, 6, and 8) and
subjected to 0.8% (w/v) agarose gel electrophoresis, Southern
blotting, and hybridization with a 32P-labeled
NcoI-BamHI fragment (Fig. 1) and autoradiography
against film for 36 h at 70 °C with an intensifying screen.
The resulting autoradiograph is shown. Horizontal arrows
correspond to the bands of hybridization observed with the cloned
S100A4(p9Ka) gene (lanes 7 and
8) with approximate sizes in kbp. These bands correspond to
digestion products of 850, 860, 419, and 277 bp (Fig. 1). B,
10 µg of genomic DNA from Rama 37CL-A3 cells (lane 1) or
Rama 800 cells (lane 2) was digested with HpaII
and transferred to a Nylon filter that was incubated with a
32P-labeled cloned calcyclin cDNA probe.
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Fig. 3.
Inhibitory effect of demethylating agents on
DNA-MT activity in Rama 37CL-A3 and Rama 800 cells. Rama 37CL-A3
and Rama 800 cells were cultured with or without inhibitors of
methylation, 5-aza-C (2 µM) or SAH (10 µM)
or the non-methylation inhibitory 6-aza-C (10 µM), as
described under "Experimental Procedures." Total cellular protein
was extracted from the untreated or treated cells, and the activity of
DNA methyltransferase (DNA-MT) was determined as the incorporation of
C[3H]3 into poly(dI-dC) substrate per 5 µg
of whole cell protein. The values are the averages of three separate
measurements ± S.D.
-actin, were determined using Northern blotting and hybridization
techniques. A 0.7-kb band of radioactivity that corresponded to the
size of S100A4(p9Ka) mRNA (8) was detected in RNA isolated from
untreated Rama 37CL-A3 cells, indicating a low level of this mRNA
in these cells. In the RNA from the Rama 800 cells, which naturally
display a high level of expression of S100A4(p9Ka) (4), there was a strong band of hybridization at 0.7 kbp, indicating abundant levels of
S100A4(p9Ka) mRNA (Fig. 4).
Semi-quantitative analysis of the hybridization of the Northern blots
showed that untreated Rama 800 cells contained 12-13-fold more
S100A4(p9Ka) mRNA than the Rama 37CL-A3 cells (Table
II). Treatment of the Rama 800 cells with
5-aza-C, 6-aza-C, or SAH did not significantly affect this relative
level of S100A4(p9Ka) mRNA in the Rama 800 cells (Table II). In
contrast, treatment of the Rama 37CL-A3 cells with either of the
methylation inhibitors 5-aza-C or SAH raised the normalized levels of
S100A4(p9Ka) mRNA in the treated cells 4-6-fold over the
non-treated Rama 37CL-A3 cells (Table II). Treating the Rama 37CL-A3
cells with the inactive agent, 6-aza-C, failed to elevate the level of
S100A4(p9Ka) mRNA (Fig. 4 and Table II). The results suggest that
methylation of DNA in the Rama 37CL-A3 cells plays a role in
restricting the level of S100A4(p9Ka) mRNA in these cells.
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Fig. 4.
The influence of treatment with demethylating
agents on the level of S100A4(p9Ka) mRNA in Rama 37CL-A3
cells. Rama 37CL-A3 and Rama 800 cells were treated with
demethylating agents, either 5-aza-C or SAH, or control 6-aza-C, as
described under "Experimental Procedures." Samples of RNA (10 µg)
isolated from Rama 37CL-A3 cells (lane 1) or Rama 37CL-A3
cells treated with 6-aza-C (lane 2), or 5-aza-C (lane
3), or SAH (lane 4), or Rama 800 cells (lane
5), or Rama 800 cells treated with 6-aza-C (lane 6), or
5-aza-C (lane 7), or SAH (lane 8) were subjected
to formaldehyde-containing 0.8% (w/v) agarose gel electrophoresis. RNA
was transferred to a nylon filter and incubated with an S100A4(p9Ka)
cDNA probe (upper panel) radioactively labeled with
32P. The autoradiograph of a washed filter is shown after
exposure for 26 h. Molecular sizes are shown in kb. A -actin
probe (lower panel) was used to ensure uniform loading of
samples.
Levels of S100A4(p9Ka) mRNA in cell line, Rama 37CL-A3 treated with
demethylating agents, relative to those in the high
S100A4(p9Ka)-expressing cell line, Rama 800
2,496 to
2,190,
1,730 to
1,616, and +106 to +2,600, the frequencies in these regions of the
gene are, respectively, only 3, 4, and 0.9% of the total cytosines,
considerably lower than the expected random frequency of about 6%.
Thus the rat S100A4(p9Ka) gene does not contain
CpG islands associated with the upstream, intronic, or exonic
sequences. In order to investigate any relationship between DNA
methylation and transcriptional regulation in the rat
S100A4(p9Ka) gene, three regions of the gene
containing previously identified cis-acting regulatory
elements were examined for the sensitivity of cytosine residues to
conversion to thymine by sodium bisulfite in DNA from the low- (Rama
37CL-A3) and high-expressing (Rama 37-E8 and Rama 800) cell lines.
These three regions were designated "TATA box," "intron," and
"upstream regulatory" regions.
113 and +36 base pairs (12,
20) relative to the start site of transcription. The intron region,
between +135 and +312 bp, contains a positive cis-acting
regulatory element found in the first intron of the mouse (16) and rat
(29) S100A4 genes. The upstream regulatory region
corresponds to DNA between
1,404 bp and
1,227 bp 5' of the
transcriptional start site and contains the repressive GC-factor regulatory sequence element that inhibits transcription of S100A4(p9Ka) mRNA in the low S100A4(p9Ka)-expressing Rama 37CL-A3 cells
(12).
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Fig. 5.
Genomic sequence mapping of protected
cytosine bases in the TATA box region of the
S100A4(p9Ka) gene. 2 µg of
EcoRI-digested genomic DNA for each cell line were subjected
to bisulfite modification, and the antisense strand of the TATA box
region of the S100A4(p9Ka) gene was amplified by
PCR using a pair of strand-specific primers (Table I), as described
under "Experimental Procedures." The amplified product from treated
DNA of cell lines, Rama 37CL-A3 (i), Rama 800 (ii), or Rama 37-E8 (iii) was cloned into
EcoRI-digested pBluescript II vector, and sequencing gels
arising from nucleotide sequencing of the strands amplified by the 5'
(A) and 3' (B) strand-specific PCR primers are
shown. The sequences above and below the
horizontal broken lines are derived from the pBluescript II
vector. A summary map of protected cytosines (C) in the TATA
box region of the S100A4(p9Ka) gene of the Rama
37CL-A3 cells between 113 and +36 shows the nucleotide sequence of
the antisense strand in which the 5' strand-specific amplification
primer is solid underlined, and the position of the 3'
primer is also indicated by dashed underline.
represents
protected cytosine bases confirmed by a guanine in the product of the
3' primer;
represents cytosine bases not confirmed by a guanine in
the product of the 3' primer;
represents guanine in the product of
the 3' primer that did not correspond to protected C in the product of
the 5' primer;
represents a confirmed protected cytosine that was
observed in genomic DNA of both the Rama 37CL-A3 and Rama 37-E8 cells
but not in the DNA from Rama 800 cells.
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Fig. 6.
Genomic sequence mapping of protected
cytosine bases in the first intron region of the
S100A4(p9Ka) gene. 2 µg of
EcoRI-digested genomic DNA for each cell line was subjected
to bisulfite modification, and the sense strand in the first intron
region of the S100A4(p9Ka) gene was amplified by
PCR using a pair of strand-specific primers (Table I), as described
under "Experimental Procedures." The amplified products from
treated DNA of cell lines, Rama 37CL-A3 (i), Rama 800 (ii), and Rama 37-E8 (iii) were cloned into
EcoRI-digested pBluescript II vector, and sequencing gels
arising from nucleotide sequencing of the strands amplified by the 5'
(A) and 3' (B) strand-specific PCR primers (Table
I) are shown. The sequences above and below the
horizontal broken lines are derived from the pBluescript II
vector. A summary map of protected cytosines (C) in the
intron region of the S100A4(p9Ka) gene of the
Rama 37CL-A3 cells between +135 and +312 shows the nucleotide sequence
of the sense strand of the intron region in which the 5'
strand-specific amplification primer is solid
underlined and the position of the 3' primer is indicated by
dashed underlines. represents protected cytosine bases
confirmed by a guanine in the product of the 3' primer;
represents
a protected cytosine that was observed in genomic DNA of the Rama
37CL-A3, Rama 37-E8, and Rama 800 cells and that was confirmed by a
guanine in the product of the 3' primer.
1,287) was detected in genomic DNA from either Rama 37-E8 or Rama 800 cells (Fig. 7A) when the
amplified product of the 5' primer was analyzed. In DNA from the Rama
37CL-A3 cell line, analyzed under exactly the same reaction conditions, only the same cytosine at
1,287 and three others out of 35 cytosine bases were apparently protected from reaction with bisulfite when the
amplified product of the 5' primer was analyzed. When the complementary
strand of the PCR product amplified by the 3' primer was sequenced,
guanine bases complementary to the apparently protected cytosine bases
appeared as adenine for all three cell lines, suggesting a lack of
protection in all three cell lines. Thus, in contrast to the results
with the TATA box and intron regions, the upstream regulatory region
from the S100A4(p9Ka) gene from Rama 37CL-A3 cells showed little protection of cytosine bases (Fig. 7C).
These results are consistent with the methylation results obtained from the Southern blot analysis, in which MspI restriction sites
at
600 and
1,450 bp, flanking the
1,404 and
1,227 upstream
region, were also unmethylated in all three cell lines tested (Fig.
1).
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Fig. 7.
Genomic sequence mapping of protected
cytosine bases in the upstream regulatory region of the
S100A4(p9Ka) gene. 2 µg of
EcoRI-digested genomic DNA for each cell line were subjected
to bisulfite modification, and the sense strand of the upstream region
of the S100A4(p9Ka) gene was amplified by PCR
using a pair of strand-specific primers (Table I), as described under
"Experimental Procedures." The amplified product from treated DNA
of cell lines, Rama 37CL-A3 (i), Rama 800 (ii),
or Rama 37-E8 (iii) was cloned into
EcoRI-digested pBluescript II vector, and sequencing gels
arising from nucleotide sequencing of the strands amplified by the 5'
(A) and 3' (B) strand-specific PCR primers are
shown. The sequences above and below the
horizontal broken lines are derived from the pBluescript II
vector. A summary map of protected cytosines (C) in the
upstream region of the S100A4(p9Ka) gene of the
Rama 37CL-A3 cells between 1,404 and
1,227 shows the nucleotide
sequence of the sense strand in which the 5' strand-specific
amplification primer is solid underlined and the
position of the 3' primer is indicated by dashed underlines.
represents protected cytosine bases not confirmed by a guanine in
the product of the 3' primer;
represents a protected cytosine in
the Rama 37CL-A3 cells that was also observed in genomic DNA from the
Rama 800 and Rama 37-E8 cells but that was not confirmed by a guanine
in the product of the 3' primer in any of the three cell types. The
GC-factor recognition sequence is doubly underlined.
DISCUSSION
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ACKNOWLEDGEMENTS |
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We thank the Cancer and Polio Research Fund for providing running expenses to enable this work to be carried out. We thank Maureen Wilde for photography.
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FOOTNOTES |
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* This work was supported by a grant from The Sino-British Friendship Scholarship Scheme administered by the British Council (to D. C.) and by the Cancer and Polio Research Fund.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.
Present address: CRC Laboratories, Dept. of Cancer Medicine,
Imperial College School of Medicine, Charing Cross Campus, St. Dunstan's Rd., London, W6 8RP, UK.
§ To whom correspondence should be addressed: Cancer and Polio Research Fund Laboratories, Life Sciences Bldg., School of Biological Sciences, University of Liverpool, P. O. Box 147, Liverpool, L69 7ZB, UK. Tel.: 44-151-794 4327; Fax: 44-151-794 4349; E-mail brb{at}liv.ac.uk.
The abbreviations used are: 5-aza-C, 5-azacytidine; 6-aza-C, 6-azacytidine; ddH2O, double-distilled water; DNA-MT, DNA methyltransferase; bp, base pair(s); kb(p), kilobase (pair); PCR, polymerase chain reaction; SAH, S-adenosyl-L-homocysteine.
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REFERENCES |
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