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Keywords:

  • DNA hypomethylation;
  • hepatocellular carcinoma;
  • genomic instability;
  • prognosis;
  • transgenic mice

Abstract

  1. Top of page
  2. Abstract
  3. Material and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References
  8. Supporting Information

Mounting evidence underlines the role of genomic hypomethylation in the generation of genomic instability (GI) and tumorigenesis, but whether DNA hypomethylation is required for hepatocellular carcinoma (HCC) development and progression remains unclear. We investigated the correlation between GI and DNA methylation, and influence of methionine metabolism deregulation on these parameters and hepatocarcinogenesis in c-Myc and c-Myc/Tgf-α transgenic mice and human HCCs. S-adenosyl-L-methionine/S-adenosylhomocysteine ratio and liver-specific methionine adenosyltransferase (MatI/III) progressively decreased in dysplastic and neoplastic liver lesions developed in c-Myc transgenic mice and in human HCC with better (HCCB) and poorer (HCCP) prognosis (based on patient's survival length). Deregulation of these parameters resulted in a rise of global DNA hypomethylation both in c-Myc and human liver lesions, positively correlated with GI levels in mice and humans, and inversely correlated with the length of survival of HCC patients. No changes in MATI/III and DNA methylation occurred in c-Myc/Tgf-α lesions and in a small human HCC subgroup with intermediate prognosis, where a proliferative activity similar to that of c-Myc HCC and HCCB was associated with low apoptosis. Upregulation of genes involved in polyamine synthesis, methionine salvage and downregulation of polyamine negative regulator OAZ1, was highest in c-Myc/Tgf-α HCCs and HCCP. Our results indicate that alterations in the activity of MAT/I/III, and extent of DNA hypomethylation and GI are prognostic markers for human HCC. However, a small human HCC subgroup, as c-Myc/Tgf-α tumors, may develop in the absence of alterations in DNA methylation. © 2007 Wiley-Liss, Inc.

Hepatocellular carcinoma (HCC), is the fifth most frequent human cancer, with the highest frequency in sub-Saharan Africa and far eastern Asia, where hepatitis B virus and hepatitis C virus infections are endemic and food is contaminated by Aflatoxin B1.1 HCC incidence is rising in Europe and United States, presumably due to increased incidence of hepatitis C virus infection, cirrhosis related to Type II diabetes, and alcoholic hepatitis.2 HCC is rapidly fatal, since most patients at risk are not diagnosed in time and amenable to potentially curative treatments, i.e., partial liver resection or transplantation.1, 2

Hepatocarcinogenesis is characterized by the accumulation of various alterations in oncogenes and oncosuppressor genes.3 This event is presumably due to the increased tendency of initiated cells to acquire mutations following dysregulation of the mechanisms preserving genome integrity, a condition known as genomic instability4 (GI). Indeed, a number of studies have demonstrated the progressive appearance of GI, such as point mutations, loss of heterozygosity and chromosomal alterations from preneoplastic liver to HCC.5 In addition, microsatellite instability occurs in HCC, although large variations have been reported in different studies.5

A body of evidence indicates that labile methyl groups play a pivotal role in liver carcinogenesis. Depletion of methyl groups in rats fed a lipotrope-deficient diet causes cancer or enhances liver tumor development induced by various carcinogens.6, 7 Marked decrease in S-adenosyl-L-methionine (SAM) and SAM:S-adenosylhomocysteine (SAH) ratio, associated with global DNA hypomethylation and overexpression of c-jun, c-myc and c-H-ras, was detected in preneoplastic and neoplastic liver from rats fed this diet.7 Similar changes also occur during chemically-induced hepatocarcinogenesis, in rats fed diets with adequate content of labile methyl donors,8, 9, 10 as well as in human cirrhosis and HCC.11 Taken together, these data suggest that alterations in methionine metabolism and DNA methylation are common and essential features of liver tumor development across species.

Decrease in SAM content in preneoplastic and neoplastic liver may depend on changes in methionine adenosyltransferase (MAT) isozyme pattern. In mammals, MAT1A gene, expressed only in the liver, encodes MATI/III isozymes, whereas MAT2A encodes MATII isozyme, which is widely expressed.12 Fall in MAT1A expression and MATI/III activity with concomitant upregulation of MAT2A occurs in hepatoma cell lines and rodent HCC as well as in human liver cirrhosis and HCC.13, 14 MATII isoform is inhibited by its reaction product and its upregulation does not lead to increase in SAM liver content.12 Differential expression of MAT1A and Mat2A genes influences DNA methylation and growth of human HCC.12, 15 DNA hypomethylation may generate GI during carcinogenesis.16, 17, 18, 19, 20, 21, 22 However, whether deregulation of methionine/SAM metabolism and DNA methylation are prerequisite for HCC development, and the prognostic role of these changes, remain open questions. Interestingly, increase in SAM: SAH ratio, DNA methylation, growth inhibition and prevention of HCC development were observed in preneoplastic and neoplastic liver of rats subjected to SAM administration.8, 9 SAM effects are reversed by 5-azacytidine, a hypomethylating agent and inhibitor of DNA methyltransferases.23In vitro growth of human HCC is inhibited by either the transfection of MAT1A gene or SAM addition to the culture medium.16 Both these treatments result in increased SAM level in tumor cells. Thus, the study of the relationships between the deregulation of methionine/SAM metabolism, changes in DNA methylation, GI and HCC development could be of primary importance to improve our knowledge on HCC pathogenesis as well as to identify new diagnostic and prognostic markers and therapeutic targets.

Two distinct categories of human HCC have been hypothesized. Central features of the first category are better prognosis, activation of the canonical Wnt/β-catenin pathway and low GI, whereas poorer prognosis and extensive chromosomal instability characterize the second category.24 Intriguingly, c-Myc and c-Myc/Tgf-α transgenic mouse models recapitulate the main pathogenetic mechanisms of human HCC, with c-Myc tumors resembling human HCC characterized by activated β-catenin and better prognosis, and c-Myc/Tgf-α HCC those characterized by shorter survival.25 Thus, c-Myc and c-Myc/Tgf-α transgenic mouse models as well as human HCC with different prognosis, as defined by patient's survival length, represent valid systems to investigate the pathogenetic role of alterations in methionine metabolism and DNA methylation, and their correlation with GI and HCC prognosis. In the present paper, we addressed these issues and demonstrated the influence of decrease in MAT1A levels with concomitant MAT2A upregulation as well as reduced SAM:SAH ratio on generation of DNA hypomethylation and GI in c-Myc transgenic mice and human preneoplastic liver. These parameters progressively increased in HCC, reaching the highest values in liver tumors associated with patients' shortest survival length. Furthermore, a small human HCC subgroup was identified in which, as detected in c-Myc/Tgf-α double transgenic mice, HCC develop in the presence of normal SAM, Mat1A and global DNA methylation levels, indicating that global DNA hypomethylation is not always required for HCC development.

Material and methods

  1. Top of page
  2. Abstract
  3. Material and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References
  8. Supporting Information

Tissue specimens

Generation of Alb/c-myc (c-Myc) in CBA/JxC56BL/6J, and Alb/c-myc/MT/TGF-α (c-Myc/TGF-α) transgenic mice in (CBA/JxC56BL/6J) × CD1 genetic background has been previously described.26 Intact animals of both strains were used as normal controls. In the absence of interstrain differences for all parameters studied in normal liver, the results found in both strains are presented together. Animal study protocols were conducted according to the National Institutes of Health guidelines for animal care. Normal, dysplastic (16 weeks) and neoplastic (32 and 68–70 weeks for c-Myc/Tgf-α and c-Myc transgenics, respectively) liver was collected from male mice. Some c-Myc/TGF-α mice, starting from weaning, were divided in 2 groups fed, respectively, a NIH-31 based chow without (untreated mice), and with vitamin E (2,000 units/kg of diet) up to being killed (at 9 and 26 weeks).

Five normal human livers, 52 HCCs and corresponding surrounding tissues were used. Liver samples were kindly provided by Dr. Snorri S. Thorgeirsson (Laboratory of Experimental Carcinogenesis, National Cancer Institute, Bethesda, MD) and were obtained from a previous study.25 Clinicopathological features of the patients are shown in Supplementary Table I. Institutional Review Board approval was obtained at participating hospitals and the National Institutes of Health.

Samples were processed for hematoxylin and eosin staining, and proliferating cell nuclear antigen (PCNA) immunohistochemistry as published,27 using a mouse anti-PCNA monoclonal antibody (Santa Cruz Biotechnology, Santa Cruz, CA). Nuclear changes of apoptotic cells were evaluated in sections stained with the ApoTag peroxidase in situ apoptosis detection kit (Millipore, Billerica, MA). The percentage of PCNA-positive nuclei and apoptotic cells (PCNA and apoptotic index, respectively) was calculated by counting at least 2,000 hepatocytes per sample.

S-Adenosylmethionine and S-Adenosylhomocysteine assay

Liver MatI/III and MatII activities were determined as published.13, 28 Liver SAM and SAH content in perchloric acid extracts was determined by isocratic high-performance liquid chromatography as reported.29 For quantitative analysis, the area of the peaks in the tissue extracts was compared with that of standard solutions.

DNA and RNA isolation

DNA was isolated as described.27 Total RNA was isolated, purified and treated with DnaseI (Amersham Biosciences, Piscataway, NJ), as published27 and cDNA was synthesized by the High Capacity cDNA Archive Kit (Applied Biosystems, Foster City, CA).

Quantitative real-time reverse transcription-polymerase chain reaction

Primers for S-Adenosylhomocysteine hydrolase (Ahcy), Cystathionine beta-synthase (Cbs), Betaine-homocysteine methyltransferase (Bhmt), Methionineadenosyltransferases 1A and 2A (Mat1A, Mat2A), Methylthioadenosinephosphorylase (Mtap), 5-Methyltetrahydrofolate-homocysteine methyltransferase (Mtr), Ornithine antizyme 1 (Oaz1), Ornithine decarboxylase (Odc), SAM decarboxylase (Amd), Spermidine synthase (Smr), Spermine synthase (Sms) genes and RNR-18 were chosen with the assistance of the “Assay-on-Demand™ Products” (Applied Biosystems, Foster City, CA). PCR reactions were performed with 75–300 ng of cDNA, using an ABI Prism 7000 Sequence Detection System and the TaqMan Universal PCR Master Mix (Applied Biosystems) as described.27 Quantitative values were calculated by using the PE Biosystems Analysis software.

Immunoblot analysis

Hepatic tissue samples were homogenized in lysis buffer as published.27 Protein concentrations were determined with the Bio-Rad Protein Assay Kit (Bio-Rad, Hercules, CA), using bovine serum albumin as standard. Aliquots of 100 μg were denatured, separatedby SDS-PAGE, and transferred onto nitrocellulose membranes by electroblotting.27 Membranes were probed with anti-ODC, MTAP and OAZ1 goat polyclonal antibodies (Santa Cruz Biotechnology), followed by incubation with HRP-secondary antibody, and revealed with Luminol Reagent (Santa Cruz Biotechnology). Densities of the protein bands were normalized to actin levels and calculated by ImageQuant Software. For co-immunoprecipitation studies, 350 mg of wild-type, preneoplastic and neoplastic liver from c-Myc and c-Myc/TGF-α transgenic mice were processed as reported.30 Immunoprecipitations were carried out with 6 μg of rabbit polyclonal anti-ODC (Santa Cruz Biotechnology) antibody. Immunoprecipitated proteins were subjected to western blotting and membranes were probed with anti-OAZ1 goat polyclonal antibody.

Global DNA methylation

Two μg of genomic DNA samples were pre-treated with 10-fold excess of HpaII endonuclease (New England Biolabs, Beverly, MA) and subjected to the cytosine-extension assay as described.31Msp I, an isoschizomer of HpaII insensitive to methylation status, was used as a control for efficiency of enzyme digestion. Relative incorporation of [3H]dCTP was compared between the samples. Results of the cytosine-extension assay were validated by comparing them with those obtained with the methyl acceptance assay in the same collection of samples. Methyl acceptance assay was performed as previously described.32 The percentage of methyl cytosine per μg of DNA was also determined in acid extracts of some normal and preneoplastic liver as well as HCC from transgenic mice by HPLC, as previously reported.9 No significant differences in the methylation pattern of the tissues evaluated by the 3 methods were found, and only the results of the cytosine-extension assay are shown.

Random amplified polymorphic DNA analysis

Twenty-two GC-rich arbitrary primers33 (Supplementary Table II) were used to score genomic alterations in preneoplastic and neoplastic mouse lesions and human HCCs. Random amplified polymorphic DNA (RAPD) reactions were performed as previously described.34 Every experiment included fingerprinting for 3 different concentrations of genomic DNA to evaluate the method reproducibility. The frequency of altered RAPD profiles was calculated for each liver lesion.34 RAPD analysis represents a sensitive and reliable method allowing the quantitative evaluation of a wide number of genetic alterations, ranging from large deletions or genomic rearrangements to single nucleotide mutations.35

Statistical analysis

Data are means ± standard deviation (SD). Student's t and Tukey–Kramer tests were used to evaluate statistical significance. Fisher's exact test was used for comparative analysis of the survival of HCC patient subgroups. Values of p < 0.05 were considered to be significant.

Results

  1. Top of page
  2. Abstract
  3. Material and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References
  8. Supporting Information

General findings

Human tumors were classified as HCC with better (HCCB) and poorer (HCCP) prognosis on the basis of the survival time, which ranges higher and lower than 36 months for HCCB and HCCP, respectively. Mean values ± SD of survival length were: 75.27 ± 20.85 (n = 15) for HCCB, and 22.58 ± 6.29 (n = 28) for HCCP (HCCB vs. HCCP, p < 0.001; Supplementary Table I). On the basis of the length of patients' survival, PCNA and apoptosis indices and DNA methylation features (see later) a 3rd human HCC subgroup was identified, with mean survival of 43 ± 27.07 months (n = 9; HCCI vs. HCCB and HCCP, p < 0.001 and < 0.01, respectively). Fisher's exact test comparison of means, and comparison of Kaplan–Meyer survival curves (Supplementary Fig. 1) by log-rank test showed significant differences among the 3 survival subgroups (p < 0.0001). No significant differences among groups were observed for average age at the time of diagnosis, sex, tumor size and differentiation and alpha-fetoprotein serum levels. Cirrhosis was present in about 87 and 65% of HCCB and HCCP, respectively, whereas all HCCI developed in the absence of cirrhosis. No significant differences were detected in any of the parameters investigated between noncirrhotic and cirrhotic surrounding liver tissues.

S-Adenosylmethionine/S-adenosylhomocysteine ratio and methionine adenosyltransferase isozymes

SAM content underwent 44% decrease in c-Myc HCC, when compared with normal liver, but slightly increased in neoplastic liver of c-Myc/Tgf-α transgenic mice (Fig. 1). Consequently, SAH level increased 70% in dysplastic and neoplastic lesions of c-Myc transgenics, whereas it did not change and decreased 59% in corresponding lesions from c-Myc/Tgf-α transgenics. SAM/SAH ratio sharply decreased in dysplastic and neoplastic liver of c-Myc transgenics, and rose 207% in HCC of double transgenics.

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Figure 1. SAM and SAH content, and Mat isozymes activity in wild-type (control), dysplastic and neoplastic liver of c-Myc and c-Myc/Tgf-α transgenic mice. Data are means ± SD of 6–8 mice per time point. Specific activity of methionine adenosyltransferases: nmol of labeled SAM/min, mg protein. Statistical analysis: *different from control for at least p < 0.05. different from c-Myc transgenic mice for p < 0.001; Abbreviations: C, control liver; D, dysplastic liver; H, HCC; M, c-Myc transgenic mice; MT, c-Myc/Tgf-α transgenic mice.

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Decrease in SAM/SAH ratio was associated with reduced MatI/III and increased MATII activity in HCC of c-Myc transgenic mice, with consequent 32-fold decrease in MatI/III:MatII activity ratio (Fig. 1). Less pronounced changes were detected in c-Myc dysplastic liver. In dysplastic and neoplastic lesions of c-Myc/Tgf-α transgenics, the levels of MatI/III did not change when compared with wild-type livers, whereas a progressive induction of MatII activity (although at lower extent than in c-Myc corresponding lesions) occurred. As a consequence, MatI/III:MatII ratio, although lower in dysplastic and neoplastic liver of c-Myc/Tgf-α transgenic mice than in normal liver, was still 2- to 11-fold higher than in c-Myc corresponding lesions.

In human liver samples, a significant decrease in SAM content was detected in HCCB (Fig. 2) and, at a lower extent, in surrounding livers (SLB and SLP), with respect to control liver. SAM decrease was associated with increased SAH levels and consequent fall in SAM:SAH ratio in HCCB, SLB and SLP. An even more pronounced fall in SAM content occurred in HCCP with a concomitant rise in SAH levels, leading to a marked decrease in SAM:SAH ratio, up to values 22-fold lower than those of HCCB (p < 0.0001). No change in these parameters occurred in SLI and HCCI, with respect to control. The alterations in SAM and SAH content were paralleled by decrease in MATI/III and rise in MATII activities in the lesions with better and poorer prognosis, with consequent sharp reduction of MATI/III:MATII ratio. No significant changes in these parameters occurred in SLI and HCCI when compared with normal livers.

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Figure 2. SAM and SAH content and activity of MAT isozymes in human HCC with different prognosis. Data are means ± SD of 15 HCCB ad SLB, 9 HCCI and SLI and 28 HCCP and SLP. Specific activity of MATI/III and MATII: nmol of labeled SAM/min, mg of protein. Statistical analysis: *different from control for at least p < 0.05; different from HCCB for p < 0.001; different from HCCI for at least p < 0.05. Abbreviations: C, normal (control liver); HCCB, HCCI and HCCP; human HCC with better, intermediate, and poorer prognosis, respectively; SLB, SLI and SLP, surrounding liver of HCCB, HCCI and HCCP, respectively.

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The changes in MATs activity were paralleled by decrease in Mat1A and increase in Mat2A gene expression (Fig. 3) in HCC of c-Myc transgenic mice, and increase in Mat1A and, at a lower level, in Mat2A in HCC of double transgenics. Analogous but lower changes occurred in dysplastic liver of the 2 transgenic lines. In human liver lesions (Fig. 4), a sharp decrease in MAT1A expression occurred in HCC and surrounding liver with better and poorer prognosis when compared with normal livers, with the lowest levels being detected in HCCP. MAT1A decline was associated with an increase in MAT2A expression in HCC, particularly in HCCP. In contrast, these parameters did not change in SLI and HCCI.

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Figure 3. Expression of genes involved in methionine, glutathione and polyamine metabolism in wild-type (control), dysplastic and neoplastic liver of c-Myc and c-Myc/Tgf-α transgenic mice. NT = 2math image, where ΔCt value of each sample was calculated by subtracting the average Ct value of the target gene from the average Ct value of the RNR-18 (housekeeping) gene. Data are means ± SD of N target (NT) of 5–13 mice. Statistical analysis: *different from control for at least p < 0.05. Different from c-Myc transgenic mice for at least p < 0.05. Abbreviations: C, control liver; D, dysplastic liver; H, HCC; M, c-Myc transgenics; MT, c-Myc/Tgf-α transgenics; Mat, methionine adenosyltransferase; Ahcy, S-adenosylhomocysteine hydrolase; Cbs, cystathionine beta-synthase; Bhmt, betaine-homocysteine methyltransferase; Mtr, 5-methyltetrahydrofolate-homocysteine methyltransferase; Amd, S-adenosylmethionine decarboxylase; Odc, ornithine decarboxylase; Smr, spermidine synthase; Sms, spermine synthetase; Oaz1, Ornithine antizyme-1; Mtap, methylthioadenosine phosphorylase.

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Figure 4. Expression of genes involved in methionine, glutathione and polyamine metabolism in human HCC with different prognosis. NT = 2math image, where ΔCt value of each sample was calculated by subtracting the Ct value of the target gene from the average Ct value of the RNR-18 gene. Data are means ± SD of N target (NT) of 15 HCCB and SLB, 9, HCCI and SLI and 28 HCCP and SLP. Statistical analysis: *different from control for at least p < 0.05; different from HCCB for at least p < 0.05; different from HCCI for at least p < 0.05. Abbreviations: C, normal (control liver); HCCB, HCCI, and HCCP; human HCC with better, intermediate, and poorer prognosis, respectively; SLB, SLI, and SLP, surrounding liver of HCCB, HCCI, and HCCP, respectively; MAT, methionine adenosyltransferase; AHCY, S-adenosylhomocysteine hydrolase; CBS, cystathionine beta-synthase; BHMT, betaine-homocysteine methyltransferase; MTR, 5-methyltetrahydrofolate-homocysteine methyltransferase; AMD, S-adenosylmethionine decarboxylase; ODC, ornithine decarboxylase; SMR, spermidine synthase; SMS, spermine synthetase; OAZ1, Ornithine antizyme-1; MTAP, methylthioadenosine phosphorylase.

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Proliferation, genomic instability and DNA methylation

RAPD analysis (Fig. 5) showed highest GI in the lesions of double transgenic mice, with significantly higher values in HCC than dysplastic liver of both transgenic lines. Similarly, PCNA index was ∼6 times higher in dysplastic lesions and 11–15 times higher in HCC of both transgenic mice than in control liver, with a relatively small difference between HCCs and no difference between dysplastic lesions of c-Myc and c-Myc/Tgf-α transgenics. A significant correlation occurred between RAPD and PCNA values in the tissues tested (r = 0.909; p < 0.0001).

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Figure 5. RAPD analysis, PCNA index and DNA methylation levels in wild-type (control), dysplastic, and neoplastic liver from c-Myc and c-Myc/Tgf-α transgenic mice. RAPD is expressed as the percentage of primers showing altered RAPD profiles in preneoplastic or neoplastic mouse lesions when compared to normal, wild-type liver. PCNA index is expressed as the percentage of PCNA-positive nuclei with respect to total cells counted. Global DNA methylation was evaluated by determining [3H]dCTP incorporation into DNA. The incorporation values are inversely proportional to DNA methylation. The correlation between DNA hypomethylation and GI does not include the data from control livers and lesions from c-Myc/TGF-α transgenic mice, since no hypomethylation was detected in double transgenics. Data are means ± SD of 6–8 mice. Statistical analysis: *different from control for p < 0.001. Different from c-Myc transgenic mice for at least p < 0.01. Abbreviations: C, control liver; D, dysplastic liver; H, HCC; M, c-Myc transgenics; MT, c-Myc/Tgf-α transgenics.

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A progressive rise in DNA hypomethylation was observed in dysplastic and neoplastic liver of c-Myc transgenic mice, with no changes detected in liver lesions from c-Myc/Tgf-α transgenics (Fig. 5). Therefore, evaluation of the correlation between DNA methylation and GI was only possible in c-Myc transgenics. Increase in dCTP incorporation into DNA, inversely proportional to global DNA methylation, exhibited a strong and highly significant correlation with GI (r = 0.957; p < 0.0001).

Since c-Myc/Tgf-α transgenic mice exhibit strong oxidative damage,36 we evaluated the effect of vitamin E on GI and DNA methylation of early (9 weeks) and late (16 weeks) dysplastic lesions in these mice. Vitamin E treatment had no effect on DNA methylation levels in c-Myc/TGF-α liver lesions ([3H]dCTP incorporation in untreated animals: 5.6 ± 0.3, 9.8 ± 0.3 and 16.8 ± 1.6, in normal liver, dysplastic liver at 9 and 26 weeks, respectively; [3H]dCTP incorporation following vitamin E administration: 6.3 ± 0.4, 10.1 ± 0.7 and 18.0 ± 0.9, respectively; means ± SD, n = 8). However, about 70% decrease in GI occurred at 9 and 26 weeks in dysplastic liver of vitamin E-treated mice (RAPD values of early and late dysplasia: 19.62 ± 2.56 and 21.12 ± 3.33, respectively, without vitamin E, and 5.81 ± 1.1 and 6.37 ± 1.53, following vitamin E treatment, respectively; means ± SD, n = 8; without versus with vitamin E, p < 0.001).

In the absence of normal liver controls corresponding to single HCCs, RAPD analysis of human lesions was performed by comparing each HCC with corresponding non-neoplastic surrounding liver (Fig. 6). GI was 2 times higher in HCCP and 2 times lower in HCCI than in HCCB. Similarly, PCNA index was highest in HCCP and lowest in HCCI, whereas no significant differences among surrounding liver subgroups were detected. A strong correlation between RAPD and PCNA was found (r = 0.92; p < 0.0001). DNA hypomethylation progressively increased from nontumorous surrounding liver tissues to HCC, with the highest values detected in HCCP. No changes in global DNA methylation occurred in HCCI and SLI. A strong correlation occurred between GI and global DNA methylation (r = 0.95; p < 0.0001). The results in Figure 6 also show significant negative correlations of RAPD and PCNA with survival rate (r = −0.453 and −0.582, respectively; p < 0.0001). A negative correlation of labeled cytosine incorporation into DNA and survival rate (r = −0.463; p < 0.0005) was also found, indicating that global DNA hypomethylation negatively correlates with survival length.

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Figure 6. RAPD analysis, PCNA index, and DNA methylation levels in human HCC with different prognosis. RAPD is expressed as the percentage of primers showing altered banding profiles in each HCC compared to its surrounding liver. PCNA index is expressed as percentage of PCNA-positive nuclei with respect to total cell counted. Global DNA methylation is expressed as [3H]dCTP incorporation into DNA, inversely proportional to DNA methylation. Data are means ± SD of 15 HCCB, 9 HCCI and 28 HCCP and corresponding surrounding liver. Correlation analysis of GI (as measured by RAPD) and DNA hypomethylation does not include HCCI since no DNA hypomethylation was found in this HCC subtype. Statistical analysis: *Different from control for p < 0.001; Different from HCCB for p < 0.05; Different from HCCI for p < 0.001. Abbreviations: HCCB, HCCI, and HCCP; human HCC with better, intermediate, and poorer prognosis, respectively; SLB, SLI, and SLP, surrounding liver of HCCB, HCCI, and HCCP, respectively.

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Apoptosis

In HCCI, the presence of GI and PCNA values lower than those of HCCB and HCCP apparently contrasts with the observation that the survival of HCCI patients is intermediate between that of HCCB and HCCP patients. Thus, we evaluated whether changes in apoptosis were responsible for the reduced survival of HCCI patients when compared with HCCB patients. For comparison, apoptosis was also determined in dysplastic lesions and HCCs of c-Myc and c-Myc/Tgf-α transgenic mice. As shown in Figure 7(a), the apoptotic index increased more than 200-folds in dysplastic and neoplastic lesions from c-Myc transgenic mice with respect to normal liver. A lower apoptosis increase occurred in c-Myc/Tgf-α dysplastic liver, whereas it declined sharply in HCC, reaching values ∼10-fold lower than those of c-Myc HCC. Rise in apoptosis was also found in all human HCC subgroups and corresponding SL when compared with normal livers [Fig. 7(b)]. Importantly, HCCI and HCCP showed apoptotic indices ∼4-fold lower than HCCB. A positive correlation between apoptosis and survival rate was also found (r = 0.743; p < 0.0001). Taking into account the PCNA data (Figures 5 and 6), these results assign a role to apoptosis in the determination of HCC aggressiveness and prognosis.

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Figure 7. Apoptotic index in control, dysplastic, and neoplastic liver from c-Myc and c-Myc/Tgf-α transgenic mice (a) and human HCC with different prognosis (b). Data are means ± SD of 5–13 mice or 15 HCCB, 9 HCCI, and 28 HCCP and the corresponding surrounding livers. Statistical analysis: Different from c-Myc transgenic mice or HCCB for p < 0.001. Different from HCCI for p < 0.001. Abbreviations: C, control liver; D, dysplastic liver; H, HCC; M, c-Myc transgenics; MT, c-Myc/Tgf-α transgenics; HCCB, HCCI and HCCP, human HCC with better, intermediate, and poorer prognosis, respectively. SLB, SLI and SLP, surrounding liver of HCCB, HCCI, and HCCP, respectively.

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S-Adenosylmethionine-related metabolic pathways

SAM-related pathways are controlled by genes involved in reduced glutathione, methionine and polyamine synthesis. We comparatively evaluated the expression of some key genes of these pathways. In transgenic mice (Fig. 3), Ahcy levels increased in HCC of c-Myc transgenic mice as well as in dysplastic liver and HCC of the double transgenics, suggesting a relatively high production of homocysteine. Presumably, this was not associated with rise in reduced glutathione synthesis because of decreased Cbs levels in dysplastic and neoplastic tissues of the 2 transgenic models. Bhmt and Mtr control methionine synthesis. Bhmt expression decreased in dysplastic and neoplastic liver of both transgenic lines. Mtr expression did not change in c-Myc lesions, showing a slight increase in dysplastic liver and a 400% rise in HCC of double transgenics. As concerns polyamine synthesis, a progressive increase in Amd, Odc, Smr and Sms mRNAs was detected in dysplastic and neoplastic lesions of both transgenic models, with highest levels being detected in the lesions of c-Myc/Tgf-α transgenics. An increase in Oaz1, much more evident in the lesions of c-Myc transgenics, occurred in dysplastic and neoplastic lesions of both mouse models. Finally, Mtap, encoding a key enzyme for methionine re-synthesis through the salvage pathway, was overexpressed only in the lesions of double transgenics.

Some genes related to polyamine synthesis and salvage pathway were also evaluated at the protein level in transgenic mice (Fig. 8). Odc and Oaz1 expression in the different tissues closely resembled that of specific mRNAs. Relatively low increase in Odc and high rise in Oaz1 was associated with the formation of relative high amounts of Odc/Oaz1 complexes in the lesions of c-Myc transgenics. In contrast, high Odc levels and low production of Oaz1 led to the formation of relatively low amounts of Odc/Oaz1 complexes in double transgenic mice. These changes were more evident in HCC than dysplastic lesions. The high expression of Mtap gene, in the lesions of double transgenics, was also confirmed at the protein level.

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Figure 8. Detection by Western blot of Odc, Oaz1, and Mtap levels in control (C), dysplastic (D), and neoplastic (H) liver of c-Myc (M) and c-Myc/Tgf-α (MT) transgenic mice. Upper panel: Representative western blot analysis. Abbreviations: IB, immunoblotting; IP, immunoprecipitation. Lower panel: Densitometric analysis showing mean values ± SD of 6–8 mice, normalized to actin levels and expressed in arbitrary units. Statistical analysis: *Different from control (C) for at least p < 0.01. Different from c-Myc transgenic mice for at least p < 0.01.

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A slight increase or no change in AHCY expression (Fig. 4) in human HCCs with better and poorer prognosis and their corresponding surrounding liver, with respect to control liver, contrasted with AHCY sharp increase in HCC with intermediate prognosis (HCCI) and surrounding liver (SLI). Cbs mRNA underwent a marked decrease in HCC and SL of all subgroups. As concerns the genes encoding key enzymes of methionine synthesis, BHMT gene expression decreased in liver lesions of all subgroups, MTR showed a consistent increase only in SLI and HCCI, and MTAP, a key gene for methionine synthesis through the salvage pathway, decreased in HCCI and in HCCP. Finally, the levels of genes encoding key enzymes for polyamine synthesis, including AMD, ODC, SMR and SMS, were progressively increased from non-neoplastic surrounding liver tissues to HCC, with the highest values being detected in HCCP. OAZ1 was overexpressed both in HCC and surrounding liver from all subgroups, with HCCP showing the lowest values.

Discussion

  1. Top of page
  2. Abstract
  3. Material and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References
  8. Supporting Information

Several researches showed the role of low SAM levels in the pathogenesis of oxidative damage, inflammation, fibrosis and carcinogenesis of rodent and human liver.7, 8, 9, 10, 11, 12, 13, 14, 37 Knockout mice lacking Mat1A gene exhibit low hepatic levels of SAM, spontaneous oxidative stress, hepatic hyperplasia, steatohepatitis and HCC.37 Furthermore, transfection of Hu-H7 cells with MAT1A and antisense oligonucleotides against MAT2A results in sizable decrease in DNA synthesis and increase in SAM level, SAM:SAH ratio, and DNA methylation.15 Analogous changes occur after supplementation of nontransfected Hu-H7 cells with SAM. In preneoplastic and neoplastic rat liver lesions, DNA synthesis inversely correlates with MATI/III activity, SAM content, SAM:SAH ratio and DNA methylation.9, 10 These observations suggest a causative role of decreased Mat1A expression and MATI/III activity in liver carcinogenesis. Here, we show similar changes of MATs mRNAs and activity associated with Mat1A:Mat2A switch in dysplastic and neoplastic liver of c-Myc transgenic mice and human HCCs with better and poorer prognosis. Most pronounced fall in SAM:SAH, MATI/III:MATII ratio and MAT1A and MAT2A expression ratio was detected in Myc HCC and HCCP, with lower changes being present in mouse dysplastic liver and human nontumorous surrounding liver. The alterations of MAT1A:MAT2A ratio, associated with the reduction of methionine synthesis through the pathway controlled by BHMT gene and, in the case of human HCC, also through the salvage pathway, may contribute to the decrease in SAM synthesis, high SAH content, and decrease in SAM:SAH ratio and DNA methylation in c-Myc transgenics and human HCCs (Fig. 9). A fall in SAM liver content may enhance tumorigenesis through multiple mechanisms, including spontaneous oxidative stress, eventually enhanced by a fall in GSH synthesis, and DNA hypomethylation.7, 9, 37 Attention has been focused on the role of DNA hypomethylation in generation of chromosome instability.16, 17, 18, 19 GI and DNA hypomethylation are often early events in carcinogenesis, and a link between DNA hypomethylation and chromosome instability is shown by the finding that the demethylating agents 5-azacytidine and 5-aza-2-deoxycytidine induce pericentromeric rearrangement.18, 19 Our findings are in agreement with these observations, and the presence of abnormal altered RAPD bands in 45% of the primers used in this study indicates a relatively high number of genomic abnormalities in murine and human liver lesions, implying a relatively high GI. Notably, global DNA hypomethylation correlates with GI and DNA synthesis both in c-Myc transgenic mice and human HCCs. In the latter, DNA hypomethylation and GI are higher in HCCP than HCCB and inversely correlated with the length of patients' survival. These observations strongly suggest that changes in methionine and SAM metabolism and in DNA hypomethylation may have an important prognostic value in human hepatocarcinogenesis. A correlation of global DNA hypomethylation with poor prognosis has been reported for ovary38 and squamous cell lung39 cancers, whereas the impact of aberrant DNA methylation on breast cancer is controversial.40, 41

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Figure 9. Schematic representation of hepatic methionine metabolism showing the interconversion of methionine and SAM, and its connection to polyamine and glutathione metabolism. 1, Methionine adenosyl transferase; 2, SAM-dependent methyltransferases; 3, S-adenosylhomocysteine hydrolase; 4, Cystathionine beta-synthase; 5, Betaine-homocysteine methyltransferase; 6, 5-Methyltetrahydrofolate-homocysteine methyltransferase; 7, 5,10-methylenetetrahydrofolate reductase; 8, SAM decarboxylase; 9, Ornithine decarboxylase; 10, Spermidine synthase; 11, Spermine synthase. 12, Methylthioadenosine phosphorylase. Abbreviations: DMG, dimethylglycine; GSH, reduced glutathione; MTA, methylthioadenosine; MTR-1-P, methylthioribose-1-phosphate; PUTR, putrescine; SAM, S-adenosylmethionine; SAH, S-adenosylhomocysteine; SPD, spermidine; SPE, spermine.

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A completely different situation occurred in c-Myc/Tgf-α double transgenics, where absence of Mat1A:Mat2A switch was associated with relatively high SAM content and SAM:SAH ratio, and elevated expression of genes regulating methionine synthesis (at least through Mtr), and methionine recovery through the salvage pathway. These observations are in agreement with the absence or very low decrease of DNA methylation in c-Myc/Tgf-α double transgenics. Thus, these findings indicate that DNA hypomethylation does not play a role in the generation of high GI and proliferation, associated with low apoptosis, in dysplastic lesions and HCCs from c-Myc/Tgf-α transgenic mice. In this respect, liver lesions of the double transgenics differ from HCCB and HCCP, which instead exhibit decrease in MATI/III:MATII and SAM:SAH ratios leading to DNA hypomethylation, high GI and elevated growth rate. The observation that all these changes are significantly higher in HCCP than in HCCB strongly suggests their prognostic role. In contrast, the behavior of c-Myc/Tgf-α double transgenics closely corresponds to that of a small human HCC subgroup, representing about 17% of total HCCs examined, with intermediate survival length between HCCB and HCCP. HCCI are characterized by the absence of both MAT1A:MAT2A switch and DNA hypomethylation, and the presence of relatively low GI. Similar to HCCB, HCCI exhibit relatively low DNA synthesis, but an apoptotic index as low as that of HCCP. These parameters categorize HCCI. The low apoptosis, in the presence of relatively low proliferation, might be responsible in HCCI of a progression rate intermediate between that of HCCB and HCCP. Recent observations indicate a proapoptotic effect of the inhibition of AHCY.42 This suggests a link between high AHCY gene expression and low apoptosis, both in HCCI and c-Myc/Tgf-α HCC. The molecular mechanisms whereby AHCY suppresses apoptosis are still unclear. To the best of our knowledge, this is the first report of a HCC subgroup, both in rodents and humans, showing barely detectable changes in DNA methylation, a situation that excludes a role played by DNA hypomethylation in the high GI characteristic of c-Myc/Tgf-α double transgenic mice. In these mice, GI and HCC development are strongly inhibited by vitamin E.36, 43 High production of reactive oxygen species, associated with lipid peroxidation, increased nitric oxide synthase and nitric oxide-mediated oxidative damage, and disruption of DNA repair mechanisms36, 44 may concur to elevated GI, proliferative activity, and HCC aggressiveness in double transgenics. Overproduction of nitric oxide also occurs in human HCC.45 However, different from c-Myc/Tgf-α mice, human HCCI exhibit relatively low GI, implying that diverse molecular mechanisms underlie the phenotypic behavior of c-Myc/Tgf-α HCC and HCCI. Further studies are needed to analyze these differences.

An intriguing finding of this study is the absence of cirrhosis in all HCCI patients (Supplementary Table I). Cirrhosis was instead present in the majority of HCCB and HCCP cases. Since various alterations present in human HCC may also be found, although at a lower extent, in cirrhotic liver,3, 5 it cannot be excluded the influence of the lesions present in precancerous liver on HCC subtypes. However, the absence of cirrhosis in 45% of HCCP patients, excludes a major role of this alteration in the phenotypic behavior of HCCI.

The expression features of key enzymes in polyamine synthesis indicate a rise in polyamine production in dysplastic and neoplastic lesions of c-Myc and c-Myc/Tgf-α transgenic mice, as well as in human HCC and nontumorous surrounding liver. OAZ1 expression may control polyamine synthesis by counterbalancing ODC activity. We found that OAZ1 expression is inversely correlated to HCC aggressiveness in transgenic mice models, and to HCC prognosis in humans. SAM decarboxylation is a key reaction of polyamine synthesis, and in the presence of low SAM liver levels, polyamine synthesis is metabolically conserved at the expense of methylation reactions,3 This may explain the apparent scarce influence of the alterations of methionine metabolism on polyamine synthesis, both in transgenic mice and human HCC.

The molecular mechanisms underlying the changes in expression of genes involved in methionine and SAM metabolism in c-Myc and c-Myc/Tgf-α transgenic mice remain unknown. Promoter methylation has been reported as a mechanism responsible for downregulation of the MAT1A gene in HCCs.46 However, the relationships between the overexpression of the c-Myc transgene either alone or in combination with Tgf-α and MAT1A expression levels are unclear. Both c-Myc and TGF-α are upregulated in human HCCs,3, 5 but at a lower extent than in double transgenic mice. Further work is required to address the molecular mechanisms underlying different dysregulation of methionine metabolism in different HCC subtypes.

Collectively, our results indicate that early changes in methionine/SAM metabolism and global DNA methylation may have a prognostic value for hepatocarcinogenesis in the majority of individuals, probably acting through a modulation of GI. Furthermore, we identified a HCC subtype that may develop both in human and mouse liver independent of changes in DNA methylation. Interestingly, global DNA hypomethylation was found to promote early lesions in the colon and liver in a mouse model of intestinal carcinogenesis, while suppressing later stages of tumorigenesis.21 Conversely, DNA hypomethylation in ApcMin/+ mice suppresses tumor formation,47, 48, 49 implying that DNA hypomethylation may exert paradoxically opposite roles on carcinogenesis depending on the cell type, tumor stage or the model used. These findings are in keeping with our observation in double transgenic mice and HCCI, in which tumor development can also occur in the absence of global DNA hypomethylation. This indicates, different from previous evidence,9, 13, 15 that molecular alterations linked to SAM metabolism and DNA methylation are necessary for the development of the majority, but not all, human HCCs. These observations may have some relevance to chemoprevention of liver tumors by SAM,8, 9, 10 at least in HCC subtypes in which DNA hypomethylation plays a pathogenetic role. Furthermore, our finding that a rise in DNA hypomethylation is associated with increased aggressiveness of HCC and decreased patient survival calls for caution in using demethylating agents as anticancer drugs.

Acknowledgements

  1. Top of page
  2. Abstract
  3. Material and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References
  8. Supporting Information

We thank Dr. Snorri S. Thorgeirsson (Laboratory of Experimental Carcinogenesis, National Cancer Institute, Bethesda, MD, USA) for providing the c-Myc and c-Myc/Tgf-α transgenic mice and the human liver tissue samples.

References

  1. Top of page
  2. Abstract
  3. Material and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References
  8. Supporting Information
  • 1
    Feitelson MA, Sun B, Satiroglu Tufan NL, Liu J, Pan J, Lian Z. Genetic mechanisms of hepatocarcinogenesis. Oncogene 2000; 221: 2593604.
  • 2
    Bosch FX, Ribes J, Diaz M, Cleries R. Primary liver cancer: worldwide incidence and trends. Gastroenterology 2004; 127: S5S16.
  • 3
    Feo F, Pascale RM, Simile MM, De Miglio MR, Muroni MR, Calvisi DF. Genetic alterations in liver carcinogenesis. Crit Rev Oncog 2000; 11: 1962.
  • 4
    Loeb LA, Loeb KR, Anderson JP. Multiple mutations and cancer. Proc Natl Acad Sci USA 2003; 100: 77681.
  • 5
    Thorgeirsson SS, Grisham JW. Molecular pathogenesis of human hepatocellular carcinoma. Nat Genet 2002; 31: 33946.
  • 6
    Ghoshal AK, Farber E. The induction of liver cancer by dietary deficiency of choline and methionine without added carcinogens. Carcinogenesis 1984; 5: 136770.
  • 7
    Wainfan E, Poirier LA. Methyl groups in carcinogenesis: effects on DNA methylation and gene expression. Cancer Res 1992; 52: 2071s77s.
  • 8
    Garcea R, Daino L, Pascale RM, Simile MM, Puddu M, Ruggiu ME, Seddaiu MA, Satta G, Sequenza MJ, Feo F. Protooncogene methylation and expression in regenerating liver and preneoplastic liver nodules induced in the rat by diethylnitrosamine: effect of variations of S-adenosylmethionine:S-adenosylhomocysteine ratio. Carcinogenesis 1989; 10: 118392.
  • 9
    Garcea R, Daino L, Pascale RM, Simile MM, Puddu M, Frassetto S, Cozzolino P, Seddaiu MA, Gaspa L, Feo F. Inhibition of promotion and persistent nodule growth by S-adenosyl-L-methionine in rat liver carcinogenesis: role of remodeling and apoptosis. Cancer Res 1989; 49: 18506.
  • 10
    Pascale RM, Marras V, Simile MM, Daino L, Pinna G, Bennati S, Carta M, Seddaiu MA, Massarelli G, Feo F. Chemoprevention of rat liver carcinogenesis by S-adenosyl-L-methionine: a long-term study. Cancer Res 1992; 52: 497986.
  • 11
    Avila MA, Berasain C, Torres L, Martin-Duce A, Corrales FJ, Yang H, Prieto J, Lu SC, Caballeria J, Rodes J, Mato JM. Reduced mRNA abundance of the main enzymes involved in methionine metabolism in human liver cirrhosis and hepatocellular carcinoma. J Hepatol 2000; 33: 90714.
  • 12
    Mato JM, Corrales FJ, Lu SC, Avila MA. S-Adenosylmethionine: a control switch that regulates liver function. FASEB J 2002; 16: 1526.
  • 13
    Cai J, Sun WM, Hwang JJ, Stain SC, Lu SC. Changes in S-adenosylmethionine synthetase in human liver cancer: molecular characterization and significance. Hepatology 1996; 24: 10907.
  • 14
    Martínez-Chantar ML, García-Trevijano ER, Latasa MU, Perez-Mato I, Sanchez del Pino MM, Corrales FJ, Avila MA, Mato JM. Importance of a deficiency in S-adenosyl-L-methionine synthesis in the pathogenesis of liver injury. Am J Clin Nutr 2002; 76: 1177S82S.
  • 15
    Cai J, Mao Z, Hwang JJ, Lu SC. Differential expression of methionine adenosyltransferase genes influences the rate of growth of human hepatocellular carcinoma cells. Cancer Res 1998; 58: 144450.
  • 16
    Eden A, Gaudet F, Waghmare A, Jaenisch R. Chromosomal instability and tumors promoted by DNA hypomethylation. Science 2003; 300: 455.
  • 17
    Rodriguez J, Frigola J, Vendrell E, Risques RA, Fraqa MF, Morales C, Moreno V, Esteller M, Capellá G, Ribas M, Peinado MA. Chromosomal instability correlates with genome-wide DNA demethylation in human primary colorectal cancers. Cancer Res 2006; 66: 84628.
  • 18
    Kokalj-Vokac N, Almeida A, Viegas-Pequignot E, Jeanpierre M, Malfoy B, Dutrillaux B. Specific induction of uncoiling and recombination by azacytidine in classical satellite-containing constitutive heterochromatin. Cytogenet Cell Genet 1993; 63: 1115.
  • 19
    Ji W, Hernandez R, Zhang XY, Qu GZ, Frady A, Varela M, Ehrlich M. DNA demethylation and pericentromeric rearrangements of chromosome 1. Mutat Res 1997; 379: 3341.
  • 20
    James SJ, Pogribny IP, Pogribna M, Mille BJ, Jernigan S, Melnyk S. Mechanisms of DNA damage, DNA hypomethylation, and tumor progression in the folate/methyl-deficient rat model of hepatocarcinogenesis. J Nutr 2003; 133: 3740S7S.
  • 21
    Yamada Y, Jackson-Grusby L, Linhart H, Linhart H, Meissner A, Eden A, Lin H, Jaenisch R. Opposing effects of DNA hypomethylation on intestinal and liver carcinogenesis. Proc Natl Acad Sci USA 2005; 102: 135805.
  • 22
    Gaudet F, Hodgson JG, Eden A, Jackson-Grusby L, Dausman J, Gray JW, Leonhardt H, Jaenisch R. Induction of tumors in mice by genomic hypomethylation. Science 2003; 300: 48992.
  • 23
    Pascale RM, Simile MM, Ruggiu ME, Seddaiu MA, Satta G, Sequenza MJ, Daino L, Vannini MG, Lai P, Feo F. Reversal by 5-azacytidine of the S-adenosyl-L-methionine-induced inhibition of the development of putative preneoplastic foci in rat liver carcinogenesis. Cancer Lett 1991; 56: 25965.
  • 24
    Laurent-Puig P, Legoix P, Bluteau O, Belghiti J, Franco D, Binot F, Monges G, Thomas G, Bioulac-Sage P, Zucman-Rossi J. Genetic alterations associated with hepatocellular carcinomas define distinct pathways of hepatocarcinogenesis. Gastroenterology 2001; 120: 176373.
  • 25
    Lee JS, Chu IS, Mikaelyan A, Calvisi DF, Heo J, Reddy JK, Thorgeirsson SS. Application of comparative functional genomics to identify best-fit mouse models to study human cancer. Nat Genet 2004; 36: 130611.
  • 26
    Murakami H, Sanderson ND, Nagy P, Marino PA, Merlino G, Thorgeirsson SS. Transgenic mouse model for synergistic effects of nuclear oncogenes and growth factors in tumorigenesis: interaction of c-myc and transforming growth factor alpha in hepatic oncogenesis. Cancer Res 1993; 53: 171923.
  • 27
    Pascale RM, Simile MM, Calvisi DF, Frau M, Muroni MR, Seddaiu MA, Daino L, Muntoni MD, De Miglio MR, Thorgeirsson SS, Feo F. Role of HSP90, CDC37, and CRM1 as modulators of P16(INK4A) activity in rat liver carcinogenesis and human liver cancer. Hepatology 2005; 42: 13109.
  • 28
    Kramer DL, Sufrin JR, Porter CW. Relative effects of S-adenosylmethionine depletion on nucleic acid methylation and polyamine biosynthesis. Biochem J 1987; 247: 25965.
  • 29
    Simile MM, Banni S, Angioni E, Carta G, De Miglio MR, Muroni MR, Calvisi DF, Carru A, Pascale RM, Feo F. 5′-Methylthioadenosine administration prevents lipid peroxidation and fibrogenesis induced in rat liver by carbon-tetrachloride intoxication. J Hepatol 2001; 34: 38694.
  • 30
    Santoni-Rugiu E, Jensen MR, Thorgeirsson SS. Disruption of the pRb/E2F pathway and inhibition of apoptosis are major oncogenic events in liver constitutively expressing c-myc and transforming growth factor alpha Cancer Res 1998; 58: 12334.
  • 31
    Pogribny I, Yi P, James SJ. Sensitive new method for rapid detection of abnormal methylation patterns in global DNA and within CpG islands. Biochem Biophys Res Commun 1999; 262: 6248.
  • 32
    Balaghi M, Wagner C. DNA methylation in folate deficiency: use of CpG methylase. Biochem Biophys Res Commun 1993; 193: 118490.
  • 33
    Woodward SR, Sudweeks J, Teuscher C. Random sequence oligonucleotide primers detect polymorphic DNA products which segregate in inbred strains of mice. Mamm Genome 1992; 3: 738.
  • 34
    Luceri C, De Filippo C, Caderni G, Gambacciani L, Salvadori M, Giannini A, Dolara P. Detection of somatic DNA alterations in azoxymethane-induced F344 rat colon tumors by random amplified polymorphic DNA analysis. Carcinogenesis 2000; 21: 17536.
  • 35
    Atienzar FA, Jha AN. The random amplified polymorphic DNA (RAPD) assay and related techniques applied to genotoxicity and carcinogenesis studies: a critical review. Mutat Res 2006; 613: 76102.
  • 36
    Calvisi DF, Thorgeirsson SS. Molecular mechanisms of hepatocarcinogenesis in transgenic mouse models of liver cancer. Toxicol Pathol 2005; 33: 1814.
  • 37
    Martínez-Chantar ML, Corrales FJ, Martínez-Cruz LA, Garcia-Trevijano ER, Huang ZZ, Chen L, Kanel G, Avila MA, Mato JM, Lu SC. Spontaneous oxidative stress and liver tumors in mice lacking methionine adenosyltransferase 1A. FASEB J 2002; 16: 12924.
  • 38
    Widschwendter M, Jiang G, Woods C, Muller HM, Fiegl H, Goebel G, Marth C, Muller-Holzner E, Zeimet AG, Laird PW, Ehrlich M. DNA hypomethylation and ovarian cancer biology. Cancer Res 2004; 64: 447280.
  • 39
    Piyathilake CJ, Henao O, Frost AR, Macaluso M, Bell WC, Johanning GL, Heimburger DC, Niveleau A, Grizzle WE. Race- and age-dependent alterations in global methylation of DNA in squamous cell carcinoma of the lung. J Cancer Causes Control 2003; 14: 3742.
  • 40
    Soares J, Pinto AE, Cunha CV, Andre S, Barao I, Sousa JM, Cravo M. Global DNA hypomethylation in breast carcinoma. Correlation with prognostic factors and tumor progression. Cancer 1999; 85: 1128.
  • 41
    Bernardino J, Roux C, Almeida A, Vogt N, Gibaud A, Gerbault-Seureau M, Magdelenat H, Bourgeois CA, Malfoy B, Dutrillaux B. DNA hypomethylation in breast cancer: an independent parameter of tumor progression? Cancer Genet Cytogenet 1997; 97: 839.
  • 42
    Hermes M, Osswald H, Kloor D. Role of S-adenosylhomocysteine hydrolase in adenosine-induced apoptosis in HepG2 cells. Exp Cell Res 2007; 313: 26483.
  • 43
    Factor VM, Laskowska D, Jensen MR, Woitach JT, Popescu NC, Thorgeirsson SS. Vitamin E reduces chromosomal damage and inhibits hepatic tumor formation in a transgenic mouse model. Proc Natl Acad Sci USA 2000; 97: 2196201.
  • 44
    Calvisi DF, Ladu S, Hironaka K, Factor VM, Thorgeirsson SS. Vitamin E down-modulates iNOS and NADPH oxidase in c-Myc/TGF-alpha transgenic mouse model of liver cancer. J Hepatol 2004; 41: 81522.
  • 45
    Ikeguchi M, Ueta T, Yamane Y, Hirooka Y, Kaibara N. Inducible nitric oxide synthase and survivin messenger RNA expression in hepatocellular carcinoma. Clin Cancer Res 2002; 8: 31316.
  • 46
    Torres L, Avila MA, Carretero MV, Latasa MU, Caballeria J, Lopez-Rodas G, Boukaba A, Lu SC, Franco L, Mato JM. Liver-specific methionine adenosyltransferase MAT1A gene expression is associated with a specific pattern of promoter methylation and histone acetylation: implications for MAT1A silencing during transformation. FASEB J 2000; 14: 95102.
  • 47
    Laird PW, Jackson-Grusby L, Fazeli A, Dickinson SL, Jung WE, Li E, Weinberg RA, Jaenisch R. Suppression of intestinal neoplasia by DNA hypomethylation. Cell 1995; 81: 197205.
  • 48
    Cormier RT, Dove WF. Dnmt1N/+ reduces the net growth rate and multiplicity of intestinal adenomas in C57BL/6-multiple intestinal neoplasia (Min)/+ mice independently of p53 but demonstrates strong synergy with the modifier of Min 1(AKR) resistance allele. Cancer Res 2000; 60: 396570.
  • 49
    Eads CA, Nickel AE, Laird PW. Complete genetic suppression of polyp formation and reduction of CpG-island hypermethylation in Apc(Min/+) Dnmt1-hypomorphic Mice. Cancer Res 2002; 62: 12969.

Supporting Information

  1. Top of page
  2. Abstract
  3. Material and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References
  8. Supporting Information

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