From the Department of Biological Chemistry, The Johns Hopkins University School of Medicine, Baltimore, Maryland 21205-2185
Received for publication, January 20, 2003, and in revised form, February 3, 2003
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ABSTRACT |
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One of the "signature" phenotypes of highly
malignant, poorly differentiated tumors, including hepatomas, is their
remarkable propensity to utilize glucose at a much higher rate than
normal cells, a property frequently dependent on the marked
overexpression of type II hexokinase (HKII). As the expression of the
gene for this enzyme is nearly silent in liver tissue, we tested the
possibility that DNA methylation/demethylation events may be involved
in its regulation. Initial studies employing methylation restriction endonuclease analysis provided evidence for differential methylation patterns for the HKII gene in normal hepatocytes and hepatoma cells,
the latter represented by a highly glycolytic model cell line (AS-30D).
Subsequently, sequencing following sodium bisulfite treatment revealed
18 methylated CpG sites within a CpG island ( One of the most common biochemical phenotypes of highly
malignant, poorly differentiated cancer cells is their capacity to metabolize glucose at elevated rates (1-3). This aberrant metabolism serves well the goal of the cancer cell to proliferate both by maintaining a constant supply of energy even when oxygen levels decrease and by providing enhanced levels of biosynthetic precursors. Thus, the transformation/progression process that ultimately leads to
the high glycolytic tumor phenotype provides the tumor with a metabolic
advantage over its normal tissue of origin.
Significantly, we have demonstrated in earlier studies the essential
role that hexokinase plays in sustaining the high glycolytic tumor
phenotype (4, 5), particularly the type II isoform that becomes
markedly elevated in rapidly growing, highly malignant hepatomas (6,
7). These experimental observations are dramatic considering that liver
normally expresses glucokinase (type IV "high
Km" hexokinase), whereas the type II "low
Km" form is nearly silent (7). In contrast, within
a poorly differentiated hepatoma, the expression of
HKII1 may be elevated more
than 100-fold (6), whereas the type IV enzyme is undetectable (6, 8).
Thus, in the transformation/progression process the genetic machinery
has been directed to completely down-regulate the expression of type IV
hexokinase and markedly up-regulate that of HKII. The major advantages
of doing this are 2-fold (9), one of which is to enhance the glycolytic
rate. This role is served optimally by HKII as it has a high affinity for ATP and binds to outer mitochondrial membrane porin (10) where it
has more ready access to ATP for phosphorylating glucose (11) and is
less sensitive to both product inhibition (4) and proteolytic
degradation (12). The second advantage is that, by binding to the
mitochondria, HKII acts as an antiapoptotic factor (13), thus
protecting the cancer cells against death signals and promoting their immortality.
In a program designed to elucidate the molecular basis for the marked
activation of HKII in rapidly growing hepatomas, we have employed the
AS-30D cell line growing in ascites form in the peritoneal cavity of
rats. This is a hepatocellular carcinoma line derived originally from a
solid liver tumor induced by feeding rats the carcinogen
dimethylaminoazobenzene (14, 15). This cell line exhibits the high
glycolytic phenotype characteristic of aggressive tumors (4) and
contains markedly elevated levels of both HKII mRNA (7) and the
expressed enzyme bound to the outer mitochondrial membrane (4, 11).
From this cell line we have isolated the HKII promoter (4.3 kb) and
shown that it is quite promiscuous in its activation response to a
number of physiological agents or conditions (7, 16-18). These include hypoxia, glucose, dibutyryl cAMP, a phorbol ester, mutated p53, and the
opposing hormones insulin and glucagon. Furthermore, fluorescence in situ hybridization analysis showed that the HKII gene is
located on a single rat chromosome where it is amplified at least
5-fold without noticeable chromosomal aberrations or rearrangements
(19). Finally, we have sequenced the normal rat liver promoter and
found that it is about 99% identical to the AS-30D hepatoma promoter (GenBankTM accession number AY082375), rendering it
unlikely that liver versus hepatoma differences in HKII
expression are related to differences in the nucleotide sequence of the
two promoters.
Although the above studies demonstrated that a combination of gene
amplification and transcriptional events contribute significantly to
the marked expression of HKII in highly glycolytic hepatoma cells, they
fail to explain why the expression of the enzyme is nearly silent in
normal liver (7). These findings, and the recent progress in the study
of the role of epigenetic factors in the silencing and activation of
genes (20-22), led us to generate a working hypothesis. Stated simply,
our hypothesis envisions that methylation/demethylation events may be
involved in regulating HKII gene expression in hepatocytes and highly
malignant hepatomas. The results of experiments described below provide
substantial support for this working hypothesis.
Animals and Cells--
Rats (Sprague-Dawley, female) were
obtained from Charles River Breeding Laboratories. Their care and
experimental use was approved by and conducted in accordance with the
guidelines of The Johns Hopkins University Animal Care and Use
Committee. Rat hepatocytes, freshly prepared by the collagenase
perfusion method (23), were kindly provided by Dr. Anna Mae Diehl,
Department of Medicine, The Johns Hopkins University School of
Medicine. The normal rat liver (clone 9) cells (American Type Tissue
Culture Collection) were grown in 90% DMEM/Ham's F-12 (1:1) with 15 mM HEPES, pH 7.5, L-glutamine, and 10% fetal
bovine serum at 37 °C in a humidified atmosphere with 5%
CO2. The clone 9 cells were maintained in the exponential
growth phase at all times with subculture every 48 h at 1:5
dilution. AS-30D hepatoma cells were grown in the peritoneal cavity of
female Sprague-Dawley rats (100-150 g) and were harvested from the
ascites fluid 6-7 days post-transplantation as described earlier
(6).
Primers--
The primers (Table I)
used in sodium bisulfite sequencing and RT-PCR experiments were
synthesized by Invitrogen.
Methylation-sensitive Restriction Endonuclease (MSRE)
Analysis--
The genomic DNA was obtained from freshly isolated rat
hepatocytes and AS-30D hepatoma cells using a genomic DNA isolation kit
(Qiagen) according to the manufacturer's protocol. The rat hepatocyte
and hepatoma genomic DNA (30 µg) were digested to completion with
different methylation-sensitive restriction enzymes, BstUI, HhaI, HpaII, EagI, and ClaI
(New England Biolabs), according to the manufacturer's protocol. The
digested DNA was fractionated on a 1% agarose gel and subsequently
depurinated (0.25 N HCl) for 20 min, denatured (1.5 M NaCl, 0.5 M NaOH) for 30 min, and neutralized
(0.5 M Tris-Cl, pH 8.0, 1.5 M NaCl) for 30 min.
The gel was subsequently soaked in 10× SSC (1.5 M NaCl,
0.15 M sodium citrate, pH 7.0) for 30 min and transferred
overnight onto a nylon membrane using TurboblotterTM rapid
downward transfer system (Schleicher & Schuell). On the following day,
the DNA was fixed onto the membrane by UV cross-linking and hybridized
with the 4.3-kb HKII promoter and associated first exon and first
intron (7).
The full-length HKII promoter (7) was used to prepare the probe using
Gene Images random prime labeling module (Amersham Biosciences) according to the manufacturer's protocol. The blot was
prehybridized with buffer (5× SSC, 0.1% w/v SDS, 5% w/v dextran sulfate) at 60 °C for 30 min. The blot was hybridized with a
heat-denatured labeled HKII probe (10 ng/ml) at 60 °C for 16-18 h.
After stringency washes at 60 °C (once with 1× SSC, 0.1% SDS; once
with 0.5× SSC, 0.1% SDS), the methylation-sensitive restriction
fragments of the HKII promoter were detected using Gene
Images CDP-Star detection module according to the manufacturer's protocol.
Sodium Bisulfite Conversion--
Sodium bisulfite deaminates
unmethylated cytosine to uracil in single-stranded DNA under conditions
where the 5-methylcytosine remains nonreactive. Thus, all cytosine
residues remaining after PCR amplification and sequencing represent
cytosines that were methylated in the original DNA sequence.
The genomic DNA was isolated from freshly isolated rat hepatocyte and
AS-30D hepatoma cells using a genomic DNA isolation kit (Qiagen)
according to the manufacturer's protocol. DNA (10 µg) was digested
to completion by BglII (New England Biolabs) at 37 °C and
purified using a Wizard DNA Clean-Up System (Promega). The bisulfite
reaction was carried out for 16-18 h at 50 °C, pH 5.0, on 1 µg of
BglII-digested genomic DNA from either rat hepatocytes or
AS-30D cells using CpGenomeTM DNA modification kit
(Intergen) according to the manufacturer's instructions. The modified
DNA was finally eluted in 50 µl of TE (10 mM Tris, 0.1 mM EDTA, pH 7.5) and stored at PCR Amplification of Sodium Bisulfite-modified DNA and
Primers--
PCR amplifications were performed using the
HotStarTaqTM PCR kit (Qiagen). Sodium bisulfite-treated DNA
(100 ng) was amplified in a 50-µl reaction mix containing 200 µM each of the four dNTPs, 30 pmol of each primer, 1.5 mM MgCl2, 1× PCR buffer, 1× Q solution, and
2.5 units of HotStarTaq DNA polymerase (Qiagen). All reagents used were
those supplied with the kit. The sequences of strand-specific primers
containing the modified cytosine bases together with the annealing
temperature used for the amplification of sodium bisulfite-treated DNA
are summarized in Table I. The general hotstart thermal cycler program
used for all the reactions was as follows: 95 °C for 15 min × 1 cycle; 94 °C for 1 min, 48 or 50 °C for 1 min, 72 °C for 1 min × 40 cycles; 72 °C for 10 min × 1 cycle.
Sequence Analysis--
The PCR fragments amplified from rat
hepatocyte and AS-30D hepatoma modified DNA were cloned using pCR®2.1
TA Cloning® kit (Invitrogen) according to the manufacturer's
instructions. The positive clones were sequenced in the Biosynthesis
and Sequencing Facility, Department of Biological Chemistry, The Johns
Hopkins University School of Medicine.
Analysis of Sodium Bisulfite Modification Efficiency--
To
test the efficiency of bisulfite conversion, the modified DNA was
PCR-amplified using modified primers specific for HKII and digested
with the restriction enzymes ApoI (R DNA Demethylation by DNMT Inhibitors, 5'-Azacytidine and
5'-Aza-2'-deoxycytidine--
Clone 9 hepatocyte cells that
predominantly express high Km glucokinase were used
in this study. These cells were seeded at a density of 5 × 105 cells/100-mm dish and maintained in DMEM/Ham's F-12
(1:1) (Invitrogen) and 10% fetal bovine serum as detailed before. The
test populations of cells were treated with either 2.5 and 5 µM 5'azaC (Sigma) or 2.5 and 5 µM 5'azadC
(Sigma). The cells were harvested after 96 and 120 h of drug
treatment, and total RNA was isolated using RNeasy® kit
(Qiagen) according to the manufacturer's instructions.
RT-PCR Analysis--
RT-PCR was performed using
TITANIUMTM One-Step RT-PCR kit
(Clontech) according to the manufacturer's
protocol. Total RNA (1 µg) from each test sample was used for
multiplex RT-PCR in a 50-µl reaction mixture containing 40 mM Tricine, 20 mM KCl, 3 mM
MgCl2, 0.2 mM dNTPs, 20 units of recombinant
RNase inhibitor (Promega), and 20 pmol each of the HKII-specific
primers: HKRTF (5'-GTGTGCTCCGAGTAAGGGTGAC-3', sense, position 469-490
of HKII cDNA) and HKRTR (5'-CGGTTCGGATGTCATTGAGTG-3', antisense,
position 1023 to 1003 of HKII cDNA), 5 pmol of rat Western Blot Analysis--
Clone 9 cells were seeded at a
density of 2 × 106 cells/150-mm dish and treated with
DNMT inhibitors: 5'azaC (2.5 and 5 µM) and 5'azadC (2.5 and 5 µM) for 120 h as described earlier. Total cell
lysate (100 µg) from each test sample was separated by 10% SDS-PAGE.
Subsequently, the proteins on the gel were transferred in the cold onto
a polyvinylidene difluoride membrane (Bio-Rad) in CAPS buffer (10 mM CAPS, 10% v/v methanol, pH 11) at 100 V/2 h. The
membranes were then blocked for overnight at 4 °C with 5% nonfat
dry milk in TBST (20 mM Tris, 136 mM NaCl,
0.15% Tween 20, pH 7.6), incubated with rabbit anti-HKII polyclonal
antibody (Santa Cruz Biotechnology) at 22 °C for 1 h, followed
by 1 h of incubation with a secondary antibody, horseradish
peroxidase-conjugated anti-rabbit IgG (Amersham Biosciences). Finally,
HKII protein was detected by an ECL system (Amersham Biosciences)
according to the manufacturer's protocol.
Establishment of a Stable CRLdM Cell Line--
The DNA
demethylase (dMTase) cDNA, a kind gift from Dr. M. Szyf (McGill
University, Montreal, Canada), had been cloned previously in the
mammalian expression vector pcDNA 3.1/His (Invitrogen) containing
neomycin for selection of stable transfectants (24). Clone 9 cells were
maintained in DMEM/Ham's F-12 (1:1) containing 10% fetal bovine
serum, as described earlier. The cells were seeded in 6-well plates at
a density of 2 × 105 cells/well. The dMTase
expression construct (2 µg) was transfected per well using
LipofectAMINETM 2000 reagent (Invitrogen) according to the
manufacturer's protocol. After transfection for 48 h, the cells
were split 1:10 in DMEM/Ham's F-12 (1:1) containing 10% fetal bovine
serum and 400 µg/ml G418 (Geneticin®)-selective antibiotic
(Invitrogen). The cells (CRLdM) were selected on G418 for 14 days, and
viable colonies were expanded for further experiments.
Nucleotide Sequence Accession Numbers--
The
GenBankTM accession numbers for the rat HKII promoter
sequence from normal liver and hepatoma cells (AS-30D) are AY082375 and
U19605, respectively.
A CpG Island Is Located within a Region That Includes the
Transcription Start Site of the HKII Promoter, the First Exon, and Part
of the First Intron--
As an initial test of our hypothesis that
methylation/demethylation events may be involved in regulating HKII
gene expression in normal liver and hepatoma cells, we carried out a
search for CpG dinucleotide rich "CpG islands" using computer
algorithm "CpG Island Finder" (25). Such islands frequently contain
methylated cytosines in repressed genes (20-22). Significantly, a high
density of CpG dinucleotides was found in a response element-rich
region straddling the transcription start site (Fig.
1A). This region ( MSRE Analysis Indicates That the Methylation Pattern of the
Hepatocyte HKII Promoter and Associated First Exon and Intron Is
Different from That of the Hepatoma Model--
The above analysis
identifying a CpG island ( Bisulfite Modification/Sequence Analyses of the HKII CpG
Island Reveals Significant Methylation in Hepatocytes While Detecting
None in the Hepatoma Model--
The observations noted above provided
the impetus for subjecting the HKII CpG island (
Results presented in Fig. 3, A
and B, provide examples of how these analyses were conducted
for the HKII CpG island in hepatocyte and hepatoma DNA, whereas Fig.
4 provides a complete accounting of
methylated and unmethylated sites in these two cases. Specifically, data presented in Fig. 3A verify that the efficiency of
sodium bisulfite treatment of hepatocyte and hepatoma DNA is nearly
100% ("Experimental Procedures"). Thus, examination of lanes
1-6 show that HKII DNA when untreated with bisulfite exhibits a
single band (lane 1), which is unaffected by digestion with
restrictions enzyme, Tsp509I (
Results presented in Fig. 3B provide examples of the
sequencing data following bisulfite treatment. Here certain cytosines in the hepatocyte HKII CpG island remained unmodified by bisulfite (Fig. 3B, upper panel), implicating methylation,
whereas the corresponding regions in the hepatoma CpG island were
modified (G/C
In a more detailed analysis of the CpG island containing 90 potential
methylation sites (Fig. 4, A and B), 18 sites
(CpG
In addition to the above, two other observations of potential interest
emerged from these analyses. First, in the hepatocyte CpG island, the
CpG sites downstream to the transcription initiation site
(CpG+387, CpG+540, CpG+560,
CpG+572, and CpG+717) showed higher degrees of
DNA methylation (26-75%) as compared with the CpG sites upstream of
the transcription initiation site, except for CpG
In summary, these data show that those CpG sites indicated above that
lie within a CpG island are methylated in hepatocytes where the
expression of HKII is nearly silent and are unmethylated in the model
hepatoma AS-30D where this enzyme is markedly elevated.
In Hepatocytes, DNMT Inhibitors Increase the Basal Level of HKII
Expression at Both the mRNA and Protein Level--
To determine to
what extent methylation may be involved in silencing HKII expression in
hepatocytes, we treated the hepatocyte cell line "clone 9" with
different concentrations of DNMT inhibitors (5'azaC or 5'azadC) and
then monitored the expression of HKII mRNA and protein (see
"Experimental Procedures"). Clone 9 cells, as liver hepatocytes,
express predominantly glucokinase (27) and down-regulate other
hexokinase types.
Results presented in Fig. 5A
show, relative to the untreated control (lane 1),
that HKII mRNA expression in the hepatocyte cell line (clone 9) is
activated both by 5'azaC and 5'azadC treatment (lanes 2-9
versus lane 1). Here mRNA from AS-30D
hepatoma cells was used as a positive control (lane 10).
Quantitation of HKII mRNA expression in Fig. 5A by
densitometric scanning showed that maximal activation was about 5-fold
with 5'azaC (5 µM) and 5.8-fold with 5'azadC (2.5 µM) (Fig. 5B).
In order to determine whether the increase in HKII mRNA expression
was reflected also by an increase in protein expression, cell lysates
from the hepatocytes (clone 9 cells) were treated for 120 h with
5'azaC and 5'azadC and then subjected to SDS-PAGE (Fig.
5C, lower panel) followed by Western analysis
(Fig. 5C, upper panel). The resultant immunoblot
obtained with a polyclonal HKII antibody revealed that both 5 µM 5'azaC (lane 3) and 2.5 µM
5'azadC (lane 4) showed significant induction of HKII
protein compared with the untreated control (lane 1). This
was a consistent finding in a number of different experiments.
In experiments not reported here, we tested lysates of 5'azaC-treated
clone 9 cells also for the induction of hexokinase activity using a
spectrophotometric glucose-6-phosphate dehydrogenase-coupled assay
(12). The treated cells exhibited a maximal specific hexokinase activity of about 3 nmol of glucose 6-phosphate formed per min/mg of
protein, whereas untreated cells exhibited no detectable activity. This
result was very dependent both on obtaining fresh cells from the
supplier and assaying for hexokinase activity in the exponential growth phase.
Hepatocytes Stably Transfected with dMTase Also Exhibit Increased
Basal Levels of HKII mRNA and Protein--
In order to test more
directly whether DNA methylation plays a role in silencing HKII
expression in hepatocytes, these cells (clone 9) were stably
transfected with dMTase (see "Experimental Procedures") and
monitored for induction of HKII mRNA and protein. As shown in Fig.
6A, cells stably transfected
with dMTase showed severalfold higher expression of HKII mRNA
(2nd lane) than untreated cells (1st lane). The
total cell lysate prepared from the same cells and subjected to
SDS-PAGE (Fig. 6B, lower panel) followed by
Western analysis (Fig. 6B, upper panel) showed
also an increased level of hexokinase protein (2nd lane)
relative to the control (1st lane). (It will be noted that
two bands are observed here for HKII expression at the protein level
consistent with an earlier report of a slightly larger precursor form
of HKII in the AS-30D hepatoma (28).)
Taken together, the last two experiments described here are consistent
with a role for methylation events in down-regulating the expression of
HKII in hepatocytes.
The study reported here was undertaken to test the hypothesis that
methylation events may be involved in down-regulating HKII gene
expression in normal hepatocytes, whereas demethylation events may
be contributing in part to its activation during tumor formation or
progression. Considering that the high glycolytic/high hexokinase phenotype is one of the most commonly observed among highly malignant tumors (1-3, 9), and that it is used worldwide via positron emission tomography scanning to detect many human cancers (29), the
hypothesis tested here takes on added significance. Specifically, as it
relates to the rat hepatocyte/AS-30D hepatoma experimental system
studied, the data obtained provide substantial support for the
hypothesis tested. Thus, we have identified within the HKII gene a
single CpG island that encompasses the transcription start site, first
exon, and part of the first intron (Fig. 1), and we have gone on to
show that this island is significantly methylated in hepatocytes (Figs.
2-4) but completely devoid of methylation in the AS-30D hepatoma (Fig.
4). Finally, in other experiments we have shown that demethylation
agents like 5'azaC, 5'azadC, and dMTase activate the basal level of
expression of HKII mRNA and protein in hepatocytes (Figs. 5
and 6).
It will be noted that demethylation agents cause modest increases in
HKII mRNA and protein expression levels in hepatocytes rather than
the high increases observed in AS-30D cells (Figs. 5 and 6) (7, 10,
11). Therefore, we envision as shown in Fig.
7 that in the hepatocyte to hepatoma
transformation (or progression) program, the demethylation events that
occur may be necessary to prepare the HKII gene for interaction with
its preferred transcriptional regulators and therefore maximal
activation. In support of this view, we have reported previously (7,
16-18) that the hepatoma HKII promoter, shown here to have a
completely unmethylated CpG island (Figs. 3 and 4), is activated by a
number of metabolically related agents or conditions in reporter gene
assays only in hepatoma cells (7).
350 to +781 bp) in the
hepatocyte gene but none in that of the hepatoma. In addition,
treatment of a hepatocyte cell line with the DNA methyltransferase
inhibitors, 5'-azacytidine and 5'-aza-2'-deoxycytidine, activated basal
expression levels of HKII mRNA and protein. Finally, stably
transfecting the hepatocyte cell line with DNA demethylase also
resulted in activating the basal expression levels of HKII mRNA and
protein. These novel observations indicate that one of the initial
events in activating the HKII gene during either transformation or
tumor progression may reside at the epigenetic level.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
Oligonucleotides for sodium bisulfite sequencing
20 °C for up to 1 month.
AATT
Y) or
Tsp509I (
AATT
) (New England Biolabs) that cut only
modified DNA. These restriction enzyme sites are only generated when
cytosine residues are modified to thymidine residues.
Subsequently, the efficiency of bisulfite conversion is assessed by
complete digestion of a PCR fragment by ApoI or
Tsp509I.
-actin-specific primers for an internal control: RACTBF
(5'-ATATCGCTGCGCTCGTCGTC-3', sense, position 11-30 of rat
-actin
cDNA) and RACTBR (5'-ATCCTGTCAGCGATGCCTGG-3', antisense,
position 938 to 919 of rat
-actin cDNA), and 1×
RT-TITANIUMTM TaqEnzyme mix (containing MMLV-RT
mutant, TITANIUMTM TaqDNA polymerase and
TaqStart antibody). The PCR cycling parameters were 50 °C for 1 h × 1 cycle; 94 °C for 5 min × 1 cycle; 94 °C for 30 s, 65 °C for 30 s, 68 °C for 1 min × 30 cycles;
68 °C for 2 min × 1 cycle. PCR products were electrophoresed
on 2% agarose gels, stained with ethidium bromide, and photographed.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
350 to
+781 bp) shown in Fig. 1B contains 58.5% GC content with CGobs/CGexp ratio >0.8 and fits the criteria
attributed to a classical CpG island (26). This finding applies
to both normal liver and the AS-30D model hepatoma as they show >99%
sequence identity (GenBankTM accession numbers AY082375 and
U19605).
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Fig. 1.
Analysis of the rat HKII promoter and
associated first exon and intron for CpG dinucleotide frequency.
A, schematic representation of 4.3-kb HKII promoter and
associated first exon and intron showing the frequency of CpG
dinucleotides. The putative binding sites for some of the common
transcription factors have also been marked. The arrow
denotes the transcription start site. Each filled circle
above the line represents one CpG dinucleotide.
The CpG island has been marked by a black line
below the schematic representation of the HKII promoter.
B, the sequence of the CpG island of the rat HKII gene
(GenBankTM accession number AY082375). The putative motifs
for binding of different transcription factors as well as all the CpG
dinucleotides (filled circles) have been marked in this
1131-bp region ( 350 to +781 bp) straddling the transcription
initiation site (denoted by +1).
350 to +781 bp) in the HKII promoter raised
the question as to whether this segment and perhaps other regions of
the promoter are differentially methylated in hepatocytes and hepatoma
cells. For this reason, we subjected genomic DNA obtained from freshly
isolated hepatocytes and AS-30D cells to digestion with several
methylation-sensitive restriction enzymes (BstUI,
HhaI, HpaII, EagI, and
ClaI). The fully digested genomic DNA was subjected to
Southern blot hybridization using a probe containing the 4.3-kb HKII
promoter with first exon and intron (7). With only one exception
(EagI), the results obtained (Fig.
2) clearly showed more bands in the lanes
containing restriction enzyme-digested hepatoma DNA than hepatocyte DNA
(compare lanes 1 and 2; 3 and
4; 5 and 6; and 9 and
10). Moreover, the bands were in different positions in all
cases. Thus, these findings strongly implicate hypermethylation of the
HKII promoter in hepatocyte genomic DNA as compared with hepatoma DNA.
Also, lanes containing digested hepatoma DNA showed much more intense
bands than lanes containing the same amount of hepatocyte DNA
consistent with our earlier work showing HKII gene amplification in the
AS-30D hepatoma cell line (19).
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Fig. 2.
MSRE analysis of the HKII promoter and
associated first exon and intron. The genomic DNA isolated from
rat hepatocyte (lanes 1, 3, 5,
7, and 9) and hepatoma (AS-30D) cells (lanes
2, 4, 6, 8, and 10)
was subjected to complete digestion using various methylation-sensitive
restriction endonucleases including BstUI (lanes
1 and 2), HhaI (lanes 3 and
4), HpaII (lanes 5 and 6),
EagI (lanes 7 and 8), and
ClaI (lanes 9 and 10). The digested
DNA was run on a 1% agarose gel, transferred onto a nylon membrane,
and probed with the 4.3-kb rat HKII promoter and associated first exon
and first intron, as detailed under "Experimental Procedures." The
arrows mark the bands showing different patterns of
digestion of the promoter in rat hepatocytes and hepatoma cells.
350 to +781 bp) to
sodium bisulfite modification/sequence analyses. Sodium bisulfite
converts cytosine to uracil in single-stranded DNA under conditions
whereby 5-methylcytosine remains non-reactive. After PCR amplification
and sequencing, all cytosines that remain are the ones that were
originally methylated.
AATT
) and
ApoI (
AATT
Y) (lanes 2 and 3).
However, when the DNA is modified with bisulfite, Tsp509I
completely cuts the DNA to give a smaller fragment (lane 6, arrow), showing nearly 100% efficiency of conversion to this site
in bisulfite-modified DNA. Lanes 7-12 present an identical
type of control experiment for the hepatoma HKII DNA, compare
lanes 10 and 12 (arrow). In experiments not presented here, where a different set of PCR products were generated after bisulfite treatment, the other selected
restriction enzyme (ApoI) also completely cleaved the
modified but not the unmodified DNA. In all experiments, prior to
performing any DNA sequence analysis, the efficiency of bisulfite
conversion was assessed, with sequencing being performed only when
conversion was complete or very near completion.
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Fig. 3.
Sodium bisulfite modification of the rat HKII
promoter. The genomic DNA was isolated from rat hepatocyte and
hepatoma (AS-30D) cells and modified by sodium bisulfite as detailed
under "Experimental Procedures." A, example of an
evaluation of sodium bisulfite modification efficiency. The genomic DNA
modified by sodium bisulfite was PCR-amplified using modified primers
HKIIMF (5'-GGTTTGTGATTATGTGTTTTTTATTT-3', sense, 336 to
311
bp, rat HKII promoter) and HKIIMR (5'-AAATCTCCTAAACAAAATAACTCCC-3',
anti-sense, +54 to +31 bp, rat HKII promoter) for 40 cycles of
amplification using the following cycling parameters: 94 °C for 1 min, 60 °C for 1 min, and 72 °C for 1 min. The PCR product
was purified and digested to completion using ApoI
(A) and Tsp509I (T). The digested
samples were electrophoresed on a 1.5% agarose gel and visualized by
ethidium bromide staining. The unmodified genomic DNA from hepatocyte
and hepatoma cells amplified using HKIIWTF
(5'-TCCGTGATCACGCGCCCCCCACCC-3', sense) and HKIIWTR
(5'-GGGTCTCCTAAGCGGGATAACTCC-3', anti-sense) were used as negative
controls (lanes 1-3 and 7-9). The
arrowheads mark the 390-bp PCR amplified product
(lanes 1-5 and 7-11), and the arrows
mark the fully digested product (lanes 6 and 12).
B, example of sodium bisulfite sequencing of the HKII CpG
island. The genomic DNA was isolated from rat hepatocyte and hepatoma
(AS-30D) cells, bisulfite-treated, PCR-amplified, and cloned as
detailed under "Experimental Procedures." The electrophoretograms
show the differences in methylated CpGs in hepatocyte (upper
panel) and AS-30D hepatoma cells (lower panel). The
arrowheads mark the CpG dinucleotide sites that are
methylated only in the promoter region of rat hepatocyte HKII.
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Fig. 4.
Summary of methylation analysis of the CpG
island located in the rat HKII gene in the region encompassing the
transcription initiation site. Each strand of sodium
bisulfite-treated genomic DNA from rat hepatocyte and hepatoma (AS-30D)
cells was PCR-amplified using appropriate sets of modified PCR primers
as detailed under "Experimental Procedures." Subsequently, the PCR
products were cloned in pCR® 2.1 vector and sequenced using a M13R
primer from the vector backbone. Upper panel, schematic
representation of hypermethylated CpG dinucleotides in the rat HKII CpG
island. The primary sequence of the HKII CpG island is shown with all
CpG dinucleotides marked as vertical lines. The putative
motifs for binding of different transcription factors in this CpG
island are also shown. Information about the primers used for the
amplification of bisulfite-treated DNA and the region amplified are
given in Table I. The black circles represent the CpG
dinucleotides that show different methylation patterns in the
hepatocyte and hepatoma HKII CpG island. The position of CpG
dinucleotide with respect to the transcription initiation site has been
marked below each black circle. The bent
arrow denotes the transcription initiation site. Middle
and lower panels, the methylation profile of the HKII gene
within the CpG island found in rat hepatocytes and hepatoma cells. The
methylation profile of 15 individual, bisulfite-treated clones from
hepatocytes (middle panel) and hepatomas (lower
panel) is shown. Only the CpG dinucleotides that show differences
in methylation between hepatocyte and hepatoma cells have been depicted
in the figure. The remaining CpG sites do not show any differences in
their methylation pattern between rat hepatocyte and hepatoma cells
(data not shown). The open circles represent unmethylated
CpGs, and the black circles represent methylated CpGs. The
degree of methylation is indicated by a + number ( , 0%; +, 1-25%;
++, 26-50%; +++, 51-75%; ++++, 76-100%). Each row represents a
single clone. The methylated CpG dinucleotide mapping of the rat HKII
CpG island:
350 to +1 bp (A) and +1 to +781 bp
(B).
A) implicating the absence of methylation (Fig.
3B, lower panel).
294, CpG
291, CpG
266,
CpG
226, CpG
202, CpG
164,
CpG
70, CpG
55, CpG+101,
CpG+142, CpG+155, CpG+167,
CpG+172, CpG+387, CpG+540,
CpG+560, CpG+572, and CpG+717) were
found to be methylated to varying degrees in hepatocytes. In sharp
contrast, no methylation was observed in the entire CpG island of
hepatoma HKII. The remaining 72 CpG sites in the CpG island of the HKII
gene showed no methylation in hepatocytes or hepatoma cells.
226.
Second, the methylated CpG sites fell in some of the confirmed and
putative sites for binding of different transcription factors, e.g. CpG
294 in cAMP-response element;
CpG
55, CpG
70, and CpG+717 in GC
boxes; CpG+155 in the activator protein-2-binding site;
CpG+540 in the E-box, and CpG+560 in the NF-1
binding site.
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Fig. 5.
Effect of DNMT inhibitors 5'azaC and 5'azadC
on HKII expression. A, effect of DNMT inhibitors on rat
HKII gene expression. Total RNA was isolated from rat "hepatocyte"
cell line (clone 9), treated for 96 h (lanes 2-5) and
120 h (lanes 6-9) with different concentrations of
DNMT inhibitors: 2.5 µM 5'azaC (lanes 2 and
6); 5 µM 5'azaC (lanes 3 and
7); 2.5 µM 5'azadC (lanes 4 and
8); 5 µM 5'azadC (lanes 5 and
9). Total RNA (1 µg) from clone 9 cells treated with DNMT
inhibitors was reverse-transcribed and PCR-amplified simultaneously
using gene-specific primers for HKII and -actin (internal control),
as detailed under "Experimental Procedures." Total RNA (1 µg)
isolated from untreated clone 9 cells and subjected to multiplex RT-PCR
was used as a control for HKII expression (lane 1). Clone 9 RNA amplified without reverse transcription was used as a control for
DNA contamination (lane 11). Total RNA (1 µg) isolated
from rat hepatoma (AS-30D) cells and subjected to multiplex RT-PCR was
used as a positive control (lane 10). M, 1-kb
plus DNA ladder. B, quantitation of rat HKII gene
expression. The multiplex RT-PCR samples from clone 9 cells treated
with DNMT inhibitors were electrophoresed on 2% agarose gels and
stained with ethidium bromide, and bands were densitometrically
quantified using AlphaEaseFCTM analysis software
(FluoroChemTM, Alpha Innotech Corp.). The HKII mRNA
fold activation of cells treated with DNMT inhibitors was calculated
relative to untreated cells after normalization for equal amounts of
starting RNA using
-actin (internal control). The values are
depicted as mean ± S.D. (n = 4). C,
effect of DNMT inhibitors on HKII protein expression in clone 9 cells.
Upper panel, identical amounts (100 µg) of total cell
lysates from clone 9 cells treated for 120 h with 2.5 and 5 µM 5'azaC (lanes 2 and 3) and 2.5 and 5 µM 5'azadC (lanes 4 and 5)
were separated by 10% SDS-PAGE. The proteins were transferred onto a
polyvinylidene difluoride membrane and probed with a goat anti-HKII
polyclonal antibody. The cell lysate from untreated clone 9 cells was
taken as a negative control (lane 1) and lysate from AS-30D
hepatoma cells as a positive control (lane 6). Lower
panel, SDS-PAGE profile in the 45- to >200-kDa range of the total
cell lysates (100 µg) from clone 9 cells treated with DNMT
inhibitors.
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Fig. 6.
Effect of dMTase on HKII expression. The
DNA demethylase expression construct was stably transfected into
clone 9 cells to make a new cell line "CRL1439dM" as detailed under
"Experimental Procedures." A, analysis of rat HKII gene
expression. Total RNA was isolated from clone 9 (1st lane),
CRL1439dM (2nd lane), and AS-30D hepatoma cells (3rd
lane). Equal amounts (1 µg each) of total RNA was
reverse-transcribed and PCR-amplified using simultaneously
gene-specific primers for HKII and -actin (internal control) as
detailed under "Experimental Procedures." The RT-PCR products were
electrophoresed on 2% agarose gel and visualized by ethidium
bromide staining. B, analysis of rat HKII protein
expression. Upper panel, equal amounts (75 µg) of total
cell lysates from clone 9 (1st lane), CRL1439dM (2nd
lane), and AS-30D hepatoma cells (3rd lane) were
separated by 10% SDS-PAGE, transferred onto polyvinylidene difluoride
membrane, and probed using a rabbit anti-HKII polyclonal antibody. The
arrowhead marks the band showing HKII (100 kDa). Lower
panel, SDS-PAGE profile in the 45- to >200-kDa range of the total
cell lysates (75 µg) from clone 9 (1st lane), CRL1439dM
(2nd lane), and AS-30D hepatoma cells (3rd
lane).
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 7.
Simplified scheme depicting a possible role
for methylation/demethylation events in the regulation of HKII
expression during transformation of liver hepatocytes. Consistent
with data presented here, HKII expression is nearly silent in normal
hepatocytes, and the CpG island within the proximal region of the
promoter is significantly methylated. This may result in a closed
conformation of the surrounding chromatin that prevents accessibility
of transcription activators. Once transformation or transformation plus
further progression occurs, demethylation is depicted to occur, again
consistent with data presented here showing that the hepatoma CpG
island is no longer methylated. This may result initially in the basal
expression levels of HKII reported here and also promote an open
conformation of the chromatin such that many other transcription
factors can bind, thus allowing overexpression of the enzyme that then
promotes the high glycolytic phenotype.
The finding here that the only clearly defined CpG island within the HKII promoter encompasses the transcription start site implicates the proximal region of the promoter as playing a major role in regulating the expression of this gene. Previous studies (18, 30) involving hypoxic conditions that markedly activate type II hexokinase also show that at least half this response can be attributed to the proximal region of the promoter that contains several potential response elements, e.g. cAMP-response element, activator protein-2, E-box, and NF-1, all of which contain a CpG dinucleotide that is methylated to different degrees in hepatocytes (Fig. 4). As these sites are demethylated in the hepatoma model studied here, they may help promote the high glycolytic tumor phenotype by binding their respective transcription factors, thus enhancing transcription of the HKII gene.
Although much attention has focused on the role of
hypermethylation of certain genes in cancer, particularly tumor
suppressor genes (22, 31, 32), less attention has been given to those that are hypomethylated like the glycolytic related gene
described here. Nevertheless, there is now a rapidly growing
list of cancer-related genes where hypomethylation has been observed.
These include those for -globin in breast and colon
adenocarcinomas (33), parathyroid hormone and catalase in colon
adenocarcinoma (33), MUC1 in breast carcinoma (34),
-chorionic gonadotropin in benign and malignant colon polyps (35),
-chorionic gonadotropin in choriocarcinoma (36), BCL-2 in
human B cell chronic lymphocytic leukemia (37), H-Ras and
metallothionein (MT-1) in mouse lymphosarcoma (38), and
c-myc in colorectal carcinoma (39). How these genes become hypomethylated is of considerable interest with some recent studies implicating dMTase. Thus, the levels of dMTase correlate with the
demethylation of CpG sites in the c-erbB-2, survivin,
and lung resistance protein genes in ovarian cancers (40, 41).
On the basis of the above examples and results presented in this
report, it is conceivable that during transformation of hepatocytes to
highly malignant hepatoma cells, the levels of dMTase may be elevated
or the enzyme may be activated, e.g. by phosphorylation or
dephosphorylation. This in turn would lead to a progressive demethylation of the HKII gene and perhaps ultimately to the formation of a more accessible open conformation of the chromatin, thus allowing
various transcription factors to bind and activate transcription.
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ACKNOWLEDGEMENTS |
---|
We are grateful to Dr. Young Hee Ko, Dr. Stephen Baylin, and Min Gyu Lee for many helpful suggestions and to Dr. Ko for assistance with the hexokinase activity assays. We are also grateful to Dr. Moshe Szyf, Pharmacology Department, McGill University, Montreal, Canada, for providing the expression construct for DNA demethylase.
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FOOTNOTES |
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* This work was supported in part by National Institutes of Health Grant CA 80118 (to P. L. P.).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.
Supported as a research fellow by National Institutes of Health
Grant T32 CA 67751. Present address: Dept. of Neurological Surgery,
Wayne State University School of Medicine, Detroit, MI 48201.
§ To whom correspondence should be addressed. Tel.: 410-955-3827;Fax: 410-614-1944; E-mail: ppederse@jhmi.edu.
Published, JBC Papers in Press, February 3, 2003, DOI 10.1074/jbc.M300608200
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ABBREVIATIONS |
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The abbreviations used are: HKII, type II hexokinase; MSRE, methylation-sensitive restriction endonuclease; DNMT, DNA methyltransferase; 5'azaC, 5'-azacytidine; 5'azadC, 5'-aza-2'-deoxycytidine; dMTase, DNA demethylase; CAPS, 3-(cyclohexylamino)propanesulfonic acid; RT, reverse transcriptase; Tricine, N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine.
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