Glucose Metabolism in Cancer

EVIDENCE THAT DEMETHYLATION EVENTS PLAY A ROLE IN ACTIVATING TYPE II HEXOKINASE GENE EXPRESSION*

Ashish Goel, Saroj P. MathupalaDagger, and Peter L. Pedersen§

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

    ABSTRACT
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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 (-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
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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.

    EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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.


                              
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Table I
Oligonucleotides for sodium bisulfite sequencing

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 -20 °C for up to 1 month.

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 (Rdown-arrow AATTup-arrow Y) or Tsp509I (down-arrow AATTup-arrow ) (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.

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 beta -actin-specific primers for an internal control: RACTBF (5'-ATATCGCTGCGCTCGTCGTC-3', sense, position 11-30 of rat beta -actin cDNA) and RACTBR (5'-ATCCTGTCAGCGATGCCTGG-3', antisense, position 938 to 919 of rat beta -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.

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.

    RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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 (-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).

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 (-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.

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 (-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.

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 (down-arrow AATTup-arrow ) and ApoI (down-arrow AATTup-arrow 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).

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 right-arrowA) implicating the absence of methylation (Fig. 3B, lower panel).

In a more detailed analysis of the CpG island containing 90 potential methylation sites (Fig. 4, A and B), 18 sites (CpG-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.

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-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.

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).


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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 beta -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 beta -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.

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).)


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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 beta -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).

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.

    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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).


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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 gamma -globin in breast and colon adenocarcinomas (33), parathyroid hormone and catalase in colon adenocarcinoma (33), MUC1 in breast carcinoma (34), alpha -chorionic gonadotropin in benign and malignant colon polyps (35), beta -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.

    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.

    FOOTNOTES

* 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.

Dagger 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

    ABBREVIATIONS

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.

    REFERENCES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
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