Glucose Metabolism in Cancer

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. 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. type II hexokinase methylation-sensitive restriction endonuclease DNA methyltransferase 5′-azacytidine 5′-aza-2′-deoxycytidine DNA demethylase 3-(cyclohexylamino)propanesulfonic acid reverse transcriptase N-2-hydroxy-1,1-bis(hydroxymethyl)ethylglycine One of the most common biochemical phenotypes of highly malignant, poorly differentiated cancer cells is their capacity to metabolize glucose at elevated rates (1Warburg O. The Metabolism of Tumors. Arnold Constable, London1930Google Scholar, 2Aisenberg A.C. The Glycolysis and Respiration of Tumors. Academic Press, New York1961Google Scholar, 3Pedersen P.L. Prog. Exp. Tumor Res. 1978; 22: 190-274Crossref PubMed Google Scholar). 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 (4Bustamante E. Pedersen P.L. Proc. Natl. Acad. Sci. U. S. A. 1977; 74: 3735-3739Crossref PubMed Scopus (342) Google Scholar, 5Bustamante E. Morris H.P. Pedersen P.L. J. Biol. Chem. 1981; 256: 8699-8704Abstract Full Text PDF PubMed Google Scholar), particularly the type II isoform that becomes markedly elevated in rapidly growing, highly malignant hepatomas (6Nakashima R.A. Paggi M.G. Scott L.J. Pedersen P.L. Cancer Res. 1988; 48: 913-919PubMed Google Scholar,7Mathupala S.P. Rempel A. Pedersen P.L. J. Biol. Chem. 1995; 270: 16918-16925Abstract Full Text Full Text PDF PubMed Scopus (144) Google Scholar). These experimental observations are dramatic considering that liver normally expresses glucokinase (type IV “highKm” hexokinase), whereas the type II “lowKm” form is nearly silent (7Mathupala S.P. Rempel A. Pedersen P.L. J. Biol. Chem. 1995; 270: 16918-16925Abstract Full Text Full Text PDF PubMed Scopus (144) Google Scholar). In contrast, within a poorly differentiated hepatoma, the expression of HKII1 may be elevated more than 100-fold (6Nakashima R.A. Paggi M.G. Scott L.J. Pedersen P.L. Cancer Res. 1988; 48: 913-919PubMed Google Scholar), whereas the type IV enzyme is undetectable (6Nakashima R.A. Paggi M.G. Scott L.J. Pedersen P.L. Cancer Res. 1988; 48: 913-919PubMed Google Scholar, 8Parry D.M. Pedersen P.L. J. Biol. Chem. 1983; 258: 1094-10912Abstract Full Text PDF Google Scholar). 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 (9Pedersen P.L. Mathupala S. Rempel A. Geschwind J.F. Ko Y.H. Biochim. Biophys. Acta. 2002; 1555: 14-20Crossref PubMed Scopus (301) Google Scholar), 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 (10Nakashima R.A. Mangan P.S. Colombini M. Pedersen P.L. Biochemistry. 1986; 25: 1015-1021Crossref PubMed Scopus (187) Google Scholar) where it has more ready access to ATP for phosphorylating glucose (11Arora K.K. Pedersen P.L. J. Biol. Chem. 1988; 263: 17422-17428Abstract Full Text PDF PubMed Google Scholar) and is less sensitive to both product inhibition (4Bustamante E. Pedersen P.L. Proc. Natl. Acad. Sci. U. S. A. 1977; 74: 3735-3739Crossref PubMed Scopus (342) Google Scholar) and proteolytic degradation (12Rose I.A. Warms J.V.B. Arch. Biochem. Biophys. 1982; 213: 625-634Crossref PubMed Scopus (26) Google Scholar). The second advantage is that, by binding to the mitochondria, HKII acts as an antiapoptotic factor (13Pastorino J.G. Shulga N. Hoek J.B. J. Biol. Chem. 2002; 277: 7610-7618Abstract Full Text Full Text PDF PubMed Scopus (561) Google Scholar), 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 (14Chang J.P. Gibley Jr., C.W. Ichinoe K. Cancer Res. 1967; 27: 2065-2071PubMed Google Scholar, 15Smith D.F. Walborg Jr., E.F. Chang J.P. Cancer Res. 1970; 30: 2306-2309PubMed Google Scholar). This cell line exhibits the high glycolytic phenotype characteristic of aggressive tumors (4Bustamante E. Pedersen P.L. Proc. Natl. Acad. Sci. U. S. A. 1977; 74: 3735-3739Crossref PubMed Scopus (342) Google Scholar) and contains markedly elevated levels of both HKII mRNA (7Mathupala S.P. Rempel A. Pedersen P.L. J. Biol. Chem. 1995; 270: 16918-16925Abstract Full Text Full Text PDF PubMed Scopus (144) Google Scholar) and the expressed enzyme bound to the outer mitochondrial membrane (4Bustamante E. Pedersen P.L. Proc. Natl. Acad. Sci. U. S. A. 1977; 74: 3735-3739Crossref PubMed Scopus (342) Google Scholar, 11Arora K.K. Pedersen P.L. J. Biol. Chem. 1988; 263: 17422-17428Abstract Full Text PDF PubMed Google Scholar). 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 (7Mathupala S.P. Rempel A. Pedersen P.L. J. Biol. Chem. 1995; 270: 16918-16925Abstract Full Text Full Text PDF PubMed Scopus (144) Google Scholar, 16Mathupala S.P. Heese C. Pedersen P.L. J. Biol. Chem. 1997; 272: 22776-22780Abstract Full Text Full Text PDF PubMed Scopus (209) Google Scholar, 17Rempel A. Mathupala S.P. Pedersen P.L. FEBS Lett. 1996; 385: 233-237Crossref PubMed Scopus (47) Google Scholar, 18Mathupala S.P. Rempel A. Pedersen P.L. J. Biol. Chem. 2001; 276: 43407-43412Abstract Full Text Full Text PDF PubMed Scopus (306) Google Scholar). These include hypoxia, glucose, dibutyryl cAMP, a phorbol ester, mutated p53, and the opposing hormones insulin and glucagon. Furthermore, fluorescencein 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 (19Rempel A. Mathupala S.P. Griffin C.A. Hawkins A.L. Pedersen P.L. Cancer Res. 1996; 56: 2468-2471PubMed Google Scholar). 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 (7Mathupala S.P. Rempel A. Pedersen P.L. J. Biol. Chem. 1995; 270: 16918-16925Abstract Full Text Full Text PDF PubMed Scopus (144) Google Scholar). These findings, and the recent progress in the study of the role of epigenetic factors in the silencing and activation of genes (20Attwood J.T. Yung R.L. Richardson B.C. Cell. Mol. Life Sci. 2002; 59: 241-257Crossref PubMed Scopus (331) Google Scholar, 21Bird A. Genes Dev. 2002; 16: 6-21Crossref PubMed Scopus (5448) Google Scholar, 22Leonhardt H. Cardoso M.C. J. Cell. Biochem. 2000; 35 (suppl.): 78-83Crossref Google Scholar), 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. 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 (23Freshney R.I. Culture of Animal Cells: A Manual of Basic Technique. 2nd Ed. Alan R. Liss, Inc., New York1987Google Scholar), 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 in a with The cells were in the at with at AS-30D hepatoma cells were grown in the peritoneal cavity of rats and were from the ascites as described earlier (6Nakashima R.A. Paggi M.G. Scott L.J. Pedersen P.L. Cancer Res. 1988; 48: 913-919PubMed Google Scholar). The in sodium bisulfite sequencing and experiments were by for sodium bisulfite indicate to accession number indicate to accession number in a The DNA was obtained from freshly isolated rat hepatocytes and AS-30D hepatoma cells a DNA to the The rat hepatocyte and hepatoma DNA were to with methylation-sensitive restriction and to the The DNA was on a and for for and pH for The was in sodium pH for and a membrane the following the DNA was the membrane by and with the HKII promoter and and (7Mathupala S.P. Rempel A. Pedersen P.L. J. Biol. Chem. 1995; 270: 16918-16925Abstract Full Text Full Text PDF PubMed Scopus (144) Google Scholar). The HKII promoter (7Mathupala S.P. Rempel A. Pedersen P.L. J. Biol. Chem. 1995; 270: 16918-16925Abstract Full Text Full Text PDF PubMed Scopus (144) Google Scholar) was to the to the The was with at for The was with a HKII at for at with with the methylation-sensitive restriction of the HKII promoter were to the bisulfite to in DNA conditions where the Thus, amplification and sequencing that were methylated in the DNA The DNA was isolated from freshly isolated rat hepatocyte and AS-30D hepatoma cells a DNA to the DNA was to by at and a DNA The bisulfite was for at pH on DNA from either rat hepatocytes or AS-30D cells DNA to the The DNA was in of mm mm pH and at for to were the DNA was amplified in a of the of mm and of DNA were with the The of the with the for the amplification of sodium DNA are in The program for the was as for 15 for or for for for The amplified from rat hepatocyte and AS-30D hepatoma DNA were to the The were sequenced in the and Department of The Johns Hopkins University School of Medicine. the of bisulfite the DNA was for HKII and with the restriction that These restriction enzyme sites are when are to Subsequently, the of bisulfite is by of a by hepatocyte cells that high glucokinase were in this These cells were at a of and in DMEM/Ham's F-12 (1:1) and 10% fetal bovine serum as The of cells were with either and or and The cells were and of and was isolated to the was to the from was for in a mm mm mm of and of the of HKII and to of HKII of rat for an of rat and to of rat and and The were for for for for for for were on with and cells were at a of and with and and and for as described cell from was by 10% Subsequently, the on the were in the a membrane in mm 10% pH at The were for at with in mm mm pH with at for by of with a Finally, HKII was by an to the The DNA demethylase a from Dr. M. been in the expression for of S. N. M. PubMed Scopus Google Scholar). cells were in DMEM/Ham's F-12 (1:1) 10% fetal bovine as described The cells were in at a of The expression was well to the for the cells were in DMEM/Ham's F-12 (1:1) 10% fetal bovine serum and The cells were on for and were for The accession for the rat HKII promoter sequence from normal liver and hepatoma cells are As an initial of our hypothesis that methylation/demethylation events may be involved in regulating HKII gene expression in normal liver and hepatoma cells, we a for CpG Proc. Natl. Acad. Sci. U. S. A. 2002; PubMed Scopus Google Scholar). frequently methylated in genes (20Attwood J.T. Yung R.L. Richardson B.C. Cell. Mol. Life Sci. 2002; 59: 241-257Crossref PubMed Scopus (331) Google Scholar, 21Bird A. Genes Dev. 2002; 16: 6-21Crossref PubMed Scopus (5448) Google Scholar, 22Leonhardt H. Cardoso M.C. J. Cell. Biochem. 2000; 35 (suppl.): 78-83Crossref Google Scholar). Significantly, a high of CpG was found in a response the This (−350 to +781 bp) shown in contains with and the to a CpG island M. M. J. Mol. Biol. PubMed Scopus Google Scholar). This to both normal liver and the AS-30D model hepatoma as they sequence (GenBankTM accession The above analysis a CpG island (−350 to +781 bp) in the HKII promoter the as to this and of the promoter are methylated in hepatocytes and hepatoma this we DNA obtained from freshly isolated hepatocytes and AS-30D cells to with methylation-sensitive restriction The DNA was to hybridization a the HKII promoter with and (7Mathupala S.P. Rempel A. Pedersen P.L. J. Biol. Chem. 1995; 270: 16918-16925Abstract Full Text Full Text PDF PubMed Scopus (144) Google Scholar). one the results obtained showed more in the restriction hepatoma DNA than hepatocyte DNA and and and the were in in Thus, of the HKII promoter in hepatocyte DNA as with hepatoma hepatoma DNA showed much more than the of hepatocyte DNA with our earlier HKII gene amplification in the AS-30D hepatoma cell line (19Rempel A. Mathupala S.P. Griffin C.A. Hawkins A.L. Pedersen P.L. Cancer Res. 1996; 56: 2468-2471PubMed Google Scholar). The observations above provided the for the HKII CpG island (−350 to +781 bp) to sodium bisulfite bisulfite to in DNA conditions amplification and that are the that were originally in provide of were conducted for the HKII CpG island in hepatocyte and hepatoma whereas provides a of methylated and sites in two in that the of sodium bisulfite treatment of hepatocyte and hepatoma DNA is nearly Thus, of that HKII DNA when with bisulfite exhibits a single which is by with and when the DNA is with completely the DNA to a nearly of to this in an identical type of for the hepatoma HKII and In experiments where a of were bisulfite the restriction enzyme also completely the but the In to DNA sequence the of bisulfite was with sequencing when was or of methylation analysis of the CpG island located in the rat HKII gene in the the of sodium DNA from rat hepatocyte and hepatoma cells was of as Subsequently, the were in and sequenced a from the of CpG in the rat HKII CpG The sequence of the HKII CpG island is shown with CpG marked as The for binding of factors in this CpG island are also about the for the amplification of DNA and the amplified are in The the CpG that methylation patterns in the hepatocyte and hepatoma HKII CpG The of CpG with to the has been marked below The the the methylation of the HKII gene within the CpG island found in rat hepatocytes and hepatoma The methylation of 15 from hepatocytes and hepatomas is the CpG that differences in methylation hepatocyte and hepatoma cells have been in the The CpG sites differences in their methylation rat hepatocyte and hepatoma cells The and the methylated The of methylation is by a number a single The methylated CpG of the rat HKII CpG to and to +781 in provide of the sequencing following bisulfite in the hepatocyte HKII CpG island by bisulfite whereas the in the hepatoma CpG island were the of methylation In a more analysis of the CpG island methylation sites A and 18 sites and were found to be methylated to in In contrast, methylation was in the CpG island of hepatoma HKII. The CpG sites in the CpG island of the HKII gene showed methylation in hepatocytes or hepatoma In to the two observations of from in the hepatocyte CpG the CpG sites to the and showed higher of DNA methylation as with the CpG sites of the for the methylated CpG sites in of the and sites for binding of in and in in the in the and in the binding In that CpG sites above that within a CpG island are methylated in hepatocytes where the expression of HKII is nearly silent and are in the model hepatoma AS-30D where this enzyme is markedly to methylation may be involved in silencing HKII expression in hepatocytes, we the hepatocyte cell line with of or and the expression of HKII mRNA and cells, as liver hepatocytes, glucokinase R. Cancer Res. Google Scholar) and down-regulate hexokinase in to the that HKII mRNA expression in the hepatocyte cell line (clone 9) is activated both by and treatment versus mRNA from AS-30D hepatoma cells was as a of HKII mRNA expression in by showed that activation was about 5-fold with and with In to the in HKII mRNA expression was also by an in cell from the hepatocytes (clone were for with and and to by analysis The obtained with a HKII revealed that both and showed of HKII with the This was a in a number of In experiments we tested of cells also for the of hexokinase a (12Rose I.A. Warms J.V.B. Arch. Biochem. Biophys. 1982; 213: 625-634Crossref PubMed Scopus (26) Google Scholar). The cells a hexokinase of about of glucose of whereas cells This was dependent both on cells from the and for hexokinase in the In to more DNA methylation plays a role in silencing HKII expression in hepatocytes, cells (clone 9) were stably with and for of HKII mRNA and protein. As shown in cells stably with showed higher expression of HKII mRNA than cells The cell prepared from the cells and to by analysis showed also an of hexokinase to the be that two are for HKII expression at the with an earlier of a form of HKII in the AS-30D hepatoma M.G. M. C. A. Biochem. Biophys. Res. PubMed Scopus Google the two experiments described are with a role for methylation events in the expression of HKII in The study was to the hypothesis that methylation events may be involved in HKII gene expression in normal hepatocytes, whereas events may be in to its activation during tumor or that the high hexokinase phenotype is one of the most highly malignant tumors (1Warburg O. The Metabolism of Tumors. Arnold Constable, London1930Google Scholar, 2Aisenberg A.C. The Glycolysis and Respiration of Tumors. Academic Press, New York1961Google Scholar, 3Pedersen P.L. Prog. Exp. Tumor Res. 1978; 22: 190-274Crossref PubMed Google Scholar, P.L. Mathupala S. Rempel A. Geschwind J.F. Ko Y.H. Biochim. Biophys. Acta. 2002; 1555: 14-20Crossref PubMed Scopus (301) Google Scholar), and that it is to J. 2002; 30: Google Scholar), the hypothesis tested on as it to the rat hepatoma experimental the obtained provide substantial support for the hypothesis Thus, we have within the HKII gene a single CpG island that the and of the and we have on to that this island is significantly methylated in hepatocytes but completely of methylation in the AS-30D hepatoma Finally, in experiments we have shown that agents and the basal of expression of HKII mRNA and in hepatocytes be that agents in HKII mRNA and expression levels in hepatocytes than the high in AS-30D cells and (7Mathupala S.P. Rempel A. Pedersen P.L. J. Biol. Chem. 1995; 270: 16918-16925Abstract Full Text Full Text PDF PubMed Scopus (144) Google Scholar, R.A. Mangan P.S. Colombini M. Pedersen P.L. Biochemistry. 1986; 25: 1015-1021Crossref PubMed Scopus (187) Google K.K. Pedersen P.L. J. Biol. Chem. 1988; 263: 17422-17428Abstract Full Text PDF PubMed Google Scholar). we as shown in that in the hepatocyte to hepatoma transformation the events that may be to the HKII gene for with its transcriptional and In support of this we have (7Mathupala S.P. Rempel A. Pedersen P.L. J. Biol. Chem. 1995; 270: 16918-16925Abstract Full Text Full Text PDF PubMed Scopus (144) Google S.P. Heese C. Pedersen P.L. J. Biol. Chem. 1997; 272: 22776-22780Abstract Full Text Full Text PDF PubMed Scopus (209) Google Scholar, 17Rempel A. Mathupala S.P. Pedersen P.L. FEBS Lett. 1996; 385: 233-237Crossref PubMed Scopus (47) Google Scholar, 18Mathupala S.P. Rempel A. Pedersen P.L. J. Biol. Chem. 2001; 276: 43407-43412Abstract Full Text Full Text PDF PubMed Scopus (306) Google Scholar) that the hepatoma HKII shown to have a completely CpG island and is activated by a number of related agents or conditions in gene in hepatoma cells (7Mathupala S.P. Rempel A. Pedersen P.L. J. Biol. Chem. 1995; 270: 16918-16925Abstract Full Text Full Text PDF PubMed Scopus (144) Google Scholar). The that the CpG island within the HKII promoter the the of the promoter as a major role in regulating the expression of this studies S.P. Rempel A. Pedersen P.L. J. Biol. Chem. 2001; 276: 43407-43412Abstract Full Text Full Text PDF PubMed Scopus (306) Google Scholar, A. S. M. C.W. J. 2000; PubMed Google Scholar) conditions that markedly type II hexokinase also that at least this response be to the of the promoter that contains response and of which a CpG that is methylated to in hepatocytes As sites are in the hepatoma model they may the high glycolytic tumor phenotype by binding their thus of the HKII Although much has on the role of of genes in particularly tumor genes H. Cardoso M.C. J. Cell. Biochem. 2000; 35 (suppl.): 78-83Crossref Google Scholar, 2001; PubMed Scopus (26) Google Scholar, J.G. 2001; PubMed Scopus Google Scholar), less has been to that are the glycolytic related gene described is a rapidly growing of genes where has been These include for in and S. R. J. Cell. Biochem. 56: PubMed Scopus Google Scholar), and in S. R. J. Cell. Biochem. 56: PubMed Scopus Google Scholar), in carcinoma S. M. J. 1995; PubMed Scopus Google Scholar), in and malignant PubMed Scopus Google Scholar), in Mol. PubMed Scopus Google Scholar), in cell M. A. E. PubMed Google Scholar), and in S. K. PubMed Scopus Google Scholar), and in carcinoma S. J. PubMed Scopus Google Scholar). genes is of with recent studies Thus, the levels of with the of CpG sites in the genes in M. H. K. Cancer Lett. 2001; PubMed Scopus Google Scholar, M. H. J. Res. 2001; PubMed Scopus Google Scholar). the basis of the above and results in this it is that during transformation of hepatocytes to highly malignant hepatoma cells, the levels of may be elevated or the enzyme may be by or This in to a of the HKII gene and ultimately to the of a more of the thus factors to and are to Dr. Dr. and for and to Dr. Ko for with the hexokinase are also to Dr. for providing the expression for DNA