Portal de Periódicos da CAPES

Hypomethylation of Chromosome 1 Heterochromatin DNA Correlates with q-Arm Copy Gain in Human Hepatocellular Carcinoma

2001; Elsevier BV; Volume: 159; Issue: 2 Linguagem: Inglês

10.1016/s0002-9440(10)61718-x

1525-2191

Nathalie Wong, Wai-Chun Lam, Paul B.S. Lai, Elizabeth Pang, Wan-Yee Lau, Philip E. Johnson,

Cholangiocarcinoma and Gallbladder Cancer Studies

Using comparative genomic hybridization (CGH) analysis, we, and others, have shown that there is a high and consistent incidence of chromosome 1q copy gain in human hepatocellular carcinoma (HCC). Chromosome 1 rearrangements, that involved peri-centromeric breakpoints, have also been frequently reported in karyotypic studies of HCC. Satellite DNA hypomethylation has been postulated as the mechanism underlying the induction of chromosome 1 peri-centromeric instability in many human cancers and in individuals with the rare recessive disorder ICF (immunodeficiency, centromeric heterochromatin instability, facial anomalies). In this study, we have investigated the role of DNA hypomethylation in 1q copy gain in HCC by examining the methylation status of chromosome 1 heterochromatin DNA (band 1q12). Thirty-six histologically confirmed samples of HCC were studied (24 paired tumor and adjacent nontumorous liver tissues, and 12 tumor only). Hypomethylation of satellite 2 (Sat2) DNA in 1q12 was analyzed by Southern blotting using methyl-sensitive enzyme digestion. In parallel, all cases were analyzed by CGH. A strong correlation between hypomethylated Sat2 sequences and 1q copy gain with a 1q12 breakpoint was found (P < 0.001). We postulate that such hypomethylation alters the interaction between the CpG-rich satellite DNA and chromatin proteins, resulting in heterochromatin decondensation, breakage and aberrant 1q formation. Spectral karyotyping further supported the presence of fragile 1q12 in HCC. Of particular interest was the finding of Sat2 DNA hypomethylation in 5 of 24 adjacent nontumorous liver tissues examined. These tissues showed no evidence of malignancy on histological examination nor did they display any CGH abnormalities. Our findings suggest a role for Sat2 demethylation in the early stages of the stepwise progression of liver carcinogenesis. Using comparative genomic hybridization (CGH) analysis, we, and others, have shown that there is a high and consistent incidence of chromosome 1q copy gain in human hepatocellular carcinoma (HCC). Chromosome 1 rearrangements, that involved peri-centromeric breakpoints, have also been frequently reported in karyotypic studies of HCC. Satellite DNA hypomethylation has been postulated as the mechanism underlying the induction of chromosome 1 peri-centromeric instability in many human cancers and in individuals with the rare recessive disorder ICF (immunodeficiency, centromeric heterochromatin instability, facial anomalies). In this study, we have investigated the role of DNA hypomethylation in 1q copy gain in HCC by examining the methylation status of chromosome 1 heterochromatin DNA (band 1q12). Thirty-six histologically confirmed samples of HCC were studied (24 paired tumor and adjacent nontumorous liver tissues, and 12 tumor only). Hypomethylation of satellite 2 (Sat2) DNA in 1q12 was analyzed by Southern blotting using methyl-sensitive enzyme digestion. In parallel, all cases were analyzed by CGH. A strong correlation between hypomethylated Sat2 sequences and 1q copy gain with a 1q12 breakpoint was found (P < 0.001). We postulate that such hypomethylation alters the interaction between the CpG-rich satellite DNA and chromatin proteins, resulting in heterochromatin decondensation, breakage and aberrant 1q formation. Spectral karyotyping further supported the presence of fragile 1q12 in HCC. Of particular interest was the finding of Sat2 DNA hypomethylation in 5 of 24 adjacent nontumorous liver tissues examined. These tissues showed no evidence of malignancy on histological examination nor did they display any CGH abnormalities. Our findings suggest a role for Sat2 demethylation in the early stages of the stepwise progression of liver carcinogenesis. Hepatocellular carcinoma (HCC) is a highly malignant tumor that is prevalent in China, Southeast Asia, and sub-Saharan Africa. Associated genetic aberrations include common loss of heterozygosity on chromosomes 4q, 8p, 13q, and 16q,1Fujimori M Tokino T Hino O Kitagawa T Imamura T Okamoto E Mitsunobu M Ishikawa T Nakagama H Harada H Yagura M Matsubara K Nakamura Y Allelotype study of primary hepatocellular carcinoma.Cancer Res. 1991; 51: 89-93PubMed Google Scholar, 2Nagai H Pineau P Tiollais P Buendia MA Dejean A Comprehensive allelotyping of human hepatocellular carcinoma.Oncogene. 1997; 14: 2927-2933Crossref PubMed Scopus (268) Google Scholar, 3Boige V Laurent-Puig P Fouchet P Flejou JF Monges G Bedossa P Bioulas S Capron F Schmitz A Olschwang S Thomas G Concerted nonsyntenic allellic losses in hyperploid hepatocellular carcinoma as determined by a high-resolution allelotype.Cancer Res. 1997; 57: 1986-1990PubMed Google Scholar and frequent gains of 8q, 17q, and 20q.4Marchio A Meddeb M Pineau P Danglot G Tiollais P Bernheim A Dejean A Recurrent chromosomal abnormalities in hepatocellular carcinoma detected by comparative genomic hybridization.Genes Chromosom Cancer. 1997; 18: 59-65Crossref PubMed Scopus (261) Google Scholar, 5Wong N Lai P Lee S-W Fan S Pang E Liew C-T Sheng Z Lau JW Johnson PJ Assessment of genetic changes in hepatocellular carcinoma by comparative genomic hybridization analysis: relationship to disease stage, tumor size, and cirrhosis.Am J Pathol. 1999; 154: 37-43Abstract Full Text Full Text PDF PubMed Scopus (257) Google Scholar, 6Kusano N Shiraishi K Kubo K Oga A Okita K Sasaki K Genetic aberrations detected by comparative genomic hybridization in hepatocellular carcinomas: their relationship to clinicopathological features.Hepatology. 1999; 29: 1858-1862Crossref PubMed Scopus (197) Google Scholar, 7Qin LX Tang ZY Sham JS Ma ZC Ye SL Zhou XD Wu ZQ Trent JM Guan XY The association of chromosome 8p deletion and tumor metastasis in human hepatocellular carcinoma.Cancer Res. 1999; 59: 5662-5665PubMed Google Scholar By comparative genomic hybridization (CGH),8Kallioniemi A Kallioniemi O-P Sudar D Rutovitz D Gray JW Waldman F Pinkel D Comparative genomic hybridization for molecular cytogenetic analysis of solid tumors.Science. 1992; 258: 818-821Crossref PubMed Scopus (2834) Google Scholar we and others have identified a high incidence of 1q copy number gain in HCC (60 to 80%).4Marchio A Meddeb M Pineau P Danglot G Tiollais P Bernheim A Dejean A Recurrent chromosomal abnormalities in hepatocellular carcinoma detected by comparative genomic hybridization.Genes Chromosom Cancer. 1997; 18: 59-65Crossref PubMed Scopus (261) Google Scholar, 5Wong N Lai P Lee S-W Fan S Pang E Liew C-T Sheng Z Lau JW Johnson PJ Assessment of genetic changes in hepatocellular carcinoma by comparative genomic hybridization analysis: relationship to disease stage, tumor size, and cirrhosis.Am J Pathol. 1999; 154: 37-43Abstract Full Text Full Text PDF PubMed Scopus (257) Google Scholar, 6Kusano N Shiraishi K Kubo K Oga A Okita K Sasaki K Genetic aberrations detected by comparative genomic hybridization in hepatocellular carcinomas: their relationship to clinicopathological features.Hepatology. 1999; 29: 1858-1862Crossref PubMed Scopus (197) Google Scholar, 7Qin LX Tang ZY Sham JS Ma ZC Ye SL Zhou XD Wu ZQ Trent JM Guan XY The association of chromosome 8p deletion and tumor metastasis in human hepatocellular carcinoma.Cancer Res. 1999; 59: 5662-5665PubMed Google Scholar Karyotypic studies on HCC, on the other hand, indicated consistent structural abnormalities of chromosome 1.9Simon D Munoz SJ Maddrey WC Knowles BB Chromosomal rearrangements in a primary hepatocellular carcinoma.Cancer Genet Cytogenet. 1990; 45: 225-260Abstract Full Text PDF Scopus (39) Google Scholar, 10Bardi G Johansson B Pandis N Heim S Mandahl N Andren-Sandberg A Hagerstrand I Mitelman F Cytogenetic findings in three primary hepatocellular carcinomas.Cancer Genet Cytogenet. 1992; 58: 191-195Abstract Full Text PDF PubMed Scopus (43) Google Scholar, 11Bardi G Rizou H Michailakis E Dietrich C Pandis N Heim S Cytogenetic findings in three primary hepatocellular carcinomas.Cancer Genet Cytogenet. 1998; 104: 165-166Abstract Full Text PDF PubMed Scopus (7) Google Scholar, 12Werner M Nolte M Gergii M Klempnauer J Chromosome 1 abnormalities in hepatocellular carcinoma.Cancer Genet Cytogenet. 1993; 66: 130Abstract Full Text PDF PubMed Scopus (20) Google Scholar, 13Chen HL Chen YC Chen DS Chromosome 1p aberrations are frequent in human primary hepatocellular carcinoma.Cancer Genet Cytogenet. 1996; 86: 102-106Abstract Full Text PDF PubMed Scopus (36) Google Scholar, 14Parada LA Hallen M Transberg KG Hagerstarnd I Bondeson L Mitelman F Johansson B Frequent rearrangements of chromosomes 1, 7, and 8 in primary liver cancer.Genes Chromosom Cancer. 1998; 23: 26-35Crossref PubMed Scopus (65) Google Scholar In our recent spectral karyotyping (SKY) analysis on short-term cultured HCCs, unbalanced translocations of 1q that involved fusion at the centromeric heterochromatin region were found to be the most frequent karyotypic abnormality.15Wong N Lai P Pang E Leung TW-T Lau JW-Y Johnson PJ A comprehensive karyotypic study on human hepatocellular carcinoma by spectral karyotyping.Hepatology. 2000; 32: 1060-1068Crossref PubMed Scopus (85) Google Scholar Current cytogenetic evidence therefore confers considerable importance of numerical 1q imbalance in liver carcinogenesis. In cultures of normal human cell, DNA methylation inhibitors have been reported to induce peri-centromeric rearrangements of chromosome 1 at a very high frequency.16Hernandez R Frady A Zhang XY Varela M Ehrlich M Preferential induction of chromosome 1 multibranched figures and whole-arm deletions in a human pro-B cell line treated with 5-azacytidine or 5-azadeoxycytidine.Cytogenet Cell Genet. 1997; 76: 196-201Crossref PubMed Scopus (54) Google Scholar, 17Ji 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: 33-41Crossref PubMed Scopus (159) Google Scholar This may suggest that DNA demethylation activity is selective and preferentially targets the centromeric region of chromosome 1. Furthermore, classical satellite 2 (Sat2) hypomethylation has been detected in individuals with the rare recessive ICF syndrome (immunodeficiency, centromeric heterochromatin instability, facial anomalies), in which the chromosome 1 peri-centromeric rearrangements in mitogen-stimulated lymphocytes are characteristic. The Sat2 DNA is the major sequence of the unusually long heterochromatic region adjacent to the centromere of chromosome 1. Several other human cancers also exhibit frequent chromosome 1 rearrangements that fuses in the vicinity of the heterochromatic region (1q12). The occurrence of chromosome 1q aberration in many different cancers suggests the likelihood of a common underlying mechanism. Hypomethylation of the constitutive heterochromatic region on chromosome 1 has been postulated as the cause for such chromosome 1 instability. Although the molecular mechanism that causes satellite DNA demethylation is still unclear, studies in breast cancer and Wilms’ tumor supported the hypothesis that common heterochromatin breakage on 1q12 is attributable to satellite hypomethylation and that this is likely to be the precursor to subsequent whole chromosome 1q translocations. In this study, we investigated the role of heterochromatin DNA hypomethylation in the formation of aberrant 1q in HCC and its possible involvement in the stepwise progression of HCC development. Tumorous liver tissues from 36 HCC patients (age 30 to 74 years; 75% male), who underwent surgical resection with curative intent and 24 paired adjacent nontumorous liver tissues were collected. Thirty-five patents were chronic carriers of viral hepatitis (97%), 32 cases of type B (HBV) and 3 cases of type C (HCV), with 81% arising from a cirrhotic liver. The disease stage of each case was classified according to the TNM staging criteria.18Beahrs OH Henson DE Hutter RVP Kennedy BJ Handbook for Staging of Cancer. J. B. Lippincott, Philadelphia1993Google Scholar Of the 36 patients recruited, 3 cases (8%) were classified as stage I (T1N0M0), 27 (75%) as stage II (T2N0M0), 4 (11%) as stage III (T3N0M0), and 2 (6%) as stage IV (T4N0M0). An experienced liver pathologist confirmed the diagnosis of HCC and the nonmalignant status of adjacent liver tissues. The macroscopic and microscopic features of resected specimens were also reviewed for the presence or absence of underlying liver cirrhosis and the maximum diameter of each tumor was recorded. Satellite DNA hypomethylation was examined by the Southern blot analysis described by Narayan and colleagues.19Narayan A Ji W Zhang X-Y Marrogi A Graff JR Baylin SB Ehrlich M Hypomethylation of peri-centromeric DNA in breast adenocarcinoma.Int J Cancer. 1998; 77: 833-838Crossref PubMed Scopus (211) Google Scholar Two μg of DNA were digested in 20 U of CpG methyl-sensitive restriction enzyme Bst BI (New England Biolabs, Beverly, MA) for 16 hours. Complete DNA digestion was indicated with the aid of an internal DNA control, λHin dIII. Fractionated DNA blotted on Hybond N membrane (Amersham-Pharmacia, Arlington, Heights, IL) was probed against satellite 2 (Sat2), a major DNA component of the chromosome 1 heterochromatin. The Sat2 probe used was a single-stranded oligonucleotide of 18 mer that has a consensus sequence of 5′-TCGAGTCCATTCGATGAT-3′. Blotting hybridization in Rapid-Hyb buffer (Amersham-Pharmacia) was performed at 50°C using 5′-[γ-32P]-end radiolabeled dATP Sat2 probe. In each blot, normal liver and sperm DNAs were included as the methylated and hypomethylated standards, respectively. Posthybridization washes were performed in 5× sodium saline citrate/0.1% sodium dodecyl sulfate for 30 minutes at room temperature, followed by 1× sodium saline citrate/0.1% sodium dodecyl sulfate for 30 minutes at 50°C. Using a phosphoimager (Instantimager; Packard, Australia), the approximate extent of hypomethylation in each lane was quantitated by comparing the ratio intensities of hybridized fragments 4 kb molecular weight. DNA samples with ratios 1.1 were considered to be extensively hypomethylated. Ratio values between 0.7 to 1.1 were considered to display a moderate level of hypomethylation. The cut off value of 0.7 for the presence of hypomethylation was assigned based on the degree of methylation obtained from four normal liver tissues (mean plus 1 SD). These tissues were neither cirrhotic nor viral infected, and had no apparent malignant morphology on histological examination. The ratio of 1.1 for extensive hypomethylation was established from four positive sperm controls (mean minus 1 SD). Ratio values for moderate hypomethylation were those that were between the normal level of methylation and extensive hypomethylation. Statistical analysis for the association between 1q copy gain and Sat2 hypomethylation was performed by the Fisher's exact test. The CGH protocol was performed according to the method of Kallioniemi and colleagues8Kallioniemi A Kallioniemi O-P Sudar D Rutovitz D Gray JW Waldman F Pinkel D Comparative genomic hybridization for molecular cytogenetic analysis of solid tumors.Science. 1992; 258: 818-821Crossref PubMed Scopus (2834) Google Scholar with modifications described in Wong and colleagues.5Wong N Lai P Lee S-W Fan S Pang E Liew C-T Sheng Z Lau JW Johnson PJ Assessment of genetic changes in hepatocellular carcinoma by comparative genomic hybridization analysis: relationship to disease stage, tumor size, and cirrhosis.Am J Pathol. 1999; 154: 37-43Abstract Full Text Full Text PDF PubMed Scopus (257) Google Scholar Briefly, differentially labeled tumor and normal DNA with biotin-16-dUTP (Boehringer Mannheim, Mannheim, Germany) and dig-11-dUTP (Boehringer Mannheim) were co-hybridized onto normal metaphase chromosomes. After hybridization, biotin signals were detected through avidin conjugated-fluorescein isothiocyanate antibody (Sigma, St. Louis, MO), and dig-labeled DNA visualized by tetramethylrhodamine isothiocyanate-conjugated antibody (Sigma). Chromosomes counterstained with 4′,6-diamidino-2-phenylindole (DAPI) were captured through a cooled charge-coupled device camera mounted on a Leitz DM RB (Leica, Wetzlar, Germany) fluorescence microscope. Three band-pass filter sets (DAPI, fluorescein isothiocyanate, and tetramethylrhodamine isothiocyanate) arranged in an automated filter-wheel were used for image acquisition. CGH software ver 3.1 on Cytovision (Applied Imaging Ltd., Sunderland, UK) was used for digital image analysis of fluorescence intensity. Average ratio profiles of 10 to 12 metaphases were calculated based on chromosome identification of the inverted DAPI.3Boige V Laurent-Puig P Fouchet P Flejou JF Monges G Bedossa P Bioulas S Capron F Schmitz A Olschwang S Thomas G Concerted nonsyntenic allellic losses in hyperploid hepatocellular carcinoma as determined by a high-resolution allelotype.Cancer Res. 1997; 57: 1986-1990PubMed Google Scholar Thresholds for gains and losses were defined as the theoretical value of 1.25 and 0.75, respectively. SKY analysis was performed on the aberrant metaphases of cases H25, H26, H29, H32, and H34. The short-term culture of primary tumors was performed according to the procedure described in Wong and colleagues,15Wong N Lai P Pang E Leung TW-T Lau JW-Y Johnson PJ A comprehensive karyotypic study on human hepatocellular carcinoma by spectral karyotyping.Hepatology. 2000; 32: 1060-1068Crossref PubMed Scopus (85) Google Scholar and subsequent SKY analysis according to the method described by Schröck and colleagues.20Schröck E du Manoir S Veldman T Schoell B Wienberg J Ferguson-Smith M Ning Y Ledbetter DH Bar-Am I Soenksen Y Garini T Ried T Multicolor spectral karyotyping of human chromosomes.Science. 1996; 273: 494-496Crossref PubMed Scopus (1471) Google Scholar Briefly, tumorous liver tissues digested by collagenase (type II) were seeded in RPMI 1640 medium supplemented with 16% fetal bovine serum, 35 U/ml penicillin, 35 μg/ml streptomycin, 10 ng/ml selenium, 10 μg/ml transferrin, and 10 μg/ml insulin. At 80% confluency, which took 3 to 5 days, cells were harvested for metaphase chromosomes by colchicine. Labeled SKY probe mixture (Applied Spectral Imaging Ltd., Migdal Haemek, Israel) was applied onto the tumor metaphases. After posthybridization washes, indirectly labeled probes were visualized using fluorescence-conjugated antibodies. Chromosomes counterstained in DAPI was acquired using a SD200 Spectracube (Applied Spectral Imaging,) mounted on a Leica DMRXA microscope (Leica). Spectral information obtained on each chromosome was analyzed by the SkyView software ver 1.6. Sat2 DNA hypomethylation was detected in 25 of 36 HCC tissues studied (69%) (Table 1). Examples of hypomethylated Sat2 cases are shown in Figure 1a. Twenty-two of the 25 cases with Sat2 hypomethylation displayed 1q copy number gain as suggested from CGH analysis (88%). Hybridized chromosome from CGH indicated a consistent breakpoint within the heterochromatic region, band 1q12, in these 22 cases (Figure 1b). Although chromosome 1 instability was not suggested in the remaining three cases (H9, H27, and H29), the finding of extensive heterochromatin hypomethylation prompted us to undertake further karyotypic investigation (Table 1). SKY analysis on H29 indicated an unbalanced translocation of chromosome 1p and 1q, t(1;17)(p10;p10) and t(1;3)(q10;p10), respectively (Figure 1b), suggesting no net gain or loss of chromosome 1 material. Of particular interest, SKY analysis in cases H25, H26, and H29 indicated copies of chromosome 1 with much extended heterochromatic region, suggesting fragility of the 1q12 segment (Figure 1b).Table 1Heterochromatin Hypomethylation and CGH Finding on HCC and Adjacent Nontumorous Liver TissuesCGH analysisSat2 hypomethylationCase no.Age/sexViral statusCirrhosisTumor diameter (cm)TNM stageTumorAdjacent tissueTumorAdjacent tissue†Moderate hypomethylation with ratio values ranging from 0.7 to 0.9 was detected in five adjacent nontumorous livers (H1 to H5).H169 /MHBV−11.0T2N0M0+1q, +2,−4,+5q31-qter,+6pter-p22,−9,+10p,+14q,−15q,+16, −17pter-p13,+17q11-q21,+17q22-qter,+20, −21qBalanced+++H237 /MHBV−6.9T2N0M0+1q, +2q21-q24,+3p,+4p,−4q,+5,−6q22-q26,+7p22-p11.2,+7q21-qter, −8pter-p12,+8p11.2-qter,−9pter-p21,+9p13-p12,+10q,−11q14-qter, −12pter-p12,+12p11.2-q22,−13q,−14q,−17p,+18p,−18q21-qter, +Y,+Xq13-qterBalanced+++H359 /MHBV+4.0T3N0M0−1p36, +1q,+3pter-p13,−4q13-qter,+6pter-p22,+8q,−12q24-qter, −16q13,−16q24Balanced++H458 /MHBV−4.5T2N0M0+1q12-q32, +3q12-q23,−4q,−5q,+8q,+10pter-q21,−11q21-qter,−13q12-q21, +13q32,+14q11.2,+17p12-q21,+18q11.2,+19p13-q13.2, +20q12-q13.2Balanced++H558 /FHBV−7.0T2N0M0−1p35-p21, +1q,−3p,−4q12-q21,−6p12-qter,−9q13-q34,+11q12-q13, −13q,−14q22-qter,+15q,+20,+21q,−Y,+XBalanced++H630 /FHBV+8.0T2N0M0+1q, −2q32-qter,−4q28-qter,−7q32-qter,−8p,+8q,+9q32-qter,+10p, −10q,−11q,−12p,−13q12-q31,−14q,−16q,+16pter-p11.2, −Xpter-q22Balanced++−H774 /FHCV+3.0T2N0M0+1q, +11q12-q14Balanced++−H869 /FHBV+8.0T2N0M0−1pter-p35, +1q,+2pter-p21,−4q,−5q,+7pter-q33,+7q21-q31, −9pter-p13,−12p,+13q22-qter,−16q, −Xpter-p21Balanced++−H951 /MHBV+7.0T2N0M0+7p, −8p22-p12,+8p11-qter,+11pter-p14,−14q,−15q,−17pter-q11, +17q12-qter,−YBalanced++−H1061 /MHCV+2.5T2N0M0+1q, −2q11-q32,−4q13-qter,+5q31-qter,+6p,−6q12-q22,−8p, +10pter-p12,−16q,+17q22-qter,+20q11.2-q13.2, +Xq21-q27Balanced++−H1151 /MHCV+4.4T2N0M0+1q12-q42, +8q,+XqBalanced++−H1255 /MHBV+5.8T3N0M0+1q, +3q22-qter,−4q,−5q,+6p,−8p,−14q22-qterBalanced+−H1355 /MHBV+7.0T2N0M0+1q, −4,+5pter-q33,+8q,+10pter-q21,−13q21-qter,−14q21-qter,−16q +18q21-qter,+20p,+20q,+XBalanced+−H1451 /MN/A+3.5T2N0M0+1q, +5q15-qter,+7,−8pter-p12,+8q21-qter,−10q23-qter,−16q,−21q, −22q,+XBalanced+−H1561 /FHBV+11.0T2N0M0+1q, +8q13-qterBalanced+−H1669 /MHBV+2.0T1N0M0+1q, +5q14-qter,−8p21-q13,+8q14-qter,−17p,−22qBalanced+−H1748 /MHBV−7.0T2N0M0+1q12-q25, +2pter-p12,−4q21-q28,−10q25-qter,−13q14-q21, +XBalanced+−H1847 /MHBV+3.9T2N0M0+1q, −6q24-qter,+12q15-q24.1,−13q,−15q13-qter,−17p,+18q12-q21, −19p,−22q,+XBalanced+−H1951 /MHBV+8.5T2N0M0−4q12-q32, −5q11-q14,+6q25-qter,+8q22-qter,−10p11-qter,−11q14-qter, +12q12-q21,−12q23-qter,−13q,−16q,−17p,+19q,−21q, +Xq22-qterBalanced−−H2034 /FHBV+3.2T2N0M0+1q25-qter, +5,−6q12-q24,+7q21-q31,−8pter-q21.3,−9pter-p22,+12q24.1, +15q23-qter,−17p11.2-q24,−18qBalanced−−H2152 /MHBV+4.9T4N0M0+7, −8p,+8qBalanced−−H2244 /FHBV−2.7T2N0M0−8p, +8q,−9pter-p22,−13q,−16q,−17p13,−18, +XqBalanced−−H2354 /MHBV+2.7T2N0M0−1pter-p21, +1p21-qter,+4p,−4q,+6pter-q14,−6q15-qter,−8pter-p21, +8p12-qter,−16Balanced−−H2460 /MHBV+2.4T3N0M0+1p13-qter, −4,+7q21-qter,−8p,+8q,−12q21-qter,−13q21-qter,−16q,−17p13, −19q,−20q13,−22qBalanced−−H2567 /FHBV+3.0T2N0M0+1q, +2q32-qter,+6pter-p21,+7,+17q,+XqN/A++N/AH2660 /MHBV−11.0T2N0M0+1q, +5q13-qter,−8p,+8q,+10p,−13q12-q22,+13q22-q34,−16q, +XN/A++N/AH2733 /MHBV+2.0T1N0M0−4pter-p14, −4q,+5,+6pter-p21.1,+8pter-q23,−11pter-p12,+12q13-q22, −13q12-q22,+14q13-q22,−16q,−17p,+18p,−18q,−19p, −20pter-p11.2,−22qN/A++N/AH2869 /MHBV+6.5T2N0M0+1q, −4,+6p,−8p21-p22,−9,+16p,−16q,+17pter-q11.2,+18p11.2-qter, +19q13.1,−19q13.2-qter,−Y,+Xq22-qterN/A++N/AH2940 /MHBV+5.5T4N0M0−8pter-p21, −8p12-qter,+10p,+12q,+17q,+Xpter-p21N/A++N/AH3074 /FHBV+3.6T2N0M0+1q, +5p,+8qN/A+N/AH3160 /MHBV+2.6T2N0M0−1p, +2q21-qter,−6q13-qter,+8qN/A+N/AH3239 /MHBV+8.5T2N0M0+1p13-qter, +2q14.1-q33,+5p,+8q11.2-q22,+11q14-qter,−16pN/A−N/AH3368 /MHBV+4.8T2N0M0−4p21-qter, +6q24-qter,−11q22-qter,+17q23-qterN/A−N/AH3465 /MHBV+1.4T1N0M0−1p22-pter, +3,+4p,−4q24-qter,+5p,−5q,−8p,+11,+12,−13q12-q21, +13q22-qter,−14q21-qter,−15q22-qter,−16,−17p,−18q,+19,+20, −21q,+22qN/A−N/AH3557 /MHBV+4.0T3N0M0+1p21-qter, +8q21.2-qter,+13q22-qterN/A−N/AH3671 /MHBV+6.5T2N0M0+1q, +6p,−14q13-qterN/A−N/AThirty-six tumor samples and 24 paired adjacent nontumorous liver tissues were studied by CGH and analyzed for Sat2 hypomethylation. ++, Indicates extensive hypomethylation of the Sat2 DNA in the heterochromatic region of chromosome 1; +, indicates moderate Sat2 hypomethylation; and −, indicates absence of Sat2 hypomethylation.Genetic changes detected by CGH are listed as gains and losses. Chromosome 1 imbalances are highlighted in bold and underlined.HBV, hepatitis B surface antigen positive; HCV, anti-hepatitis C positive; N/A, not determined.† Moderate hypomethylation with ratio values ranging from 0.7 to 0.9 was detected in five adjacent nontumorous livers (H1 to H5). Open table in a new tab Thirty-six tumor samples and 24 paired adjacent nontumorous liver tissues were studied by CGH and analyzed for Sat2 hypomethylation. ++, Indicates extensive hypomethylation of the Sat2 DNA in the heterochromatic region of chromosome 1; +, indicates moderate Sat2 hypomethylation; and −, indicates absence of Sat2 hypomethylation. Genetic changes detected by CGH are listed as gains and losses. Chromosome 1 imbalances are highlighted in bold and underlined. HBV, hepatitis B surface antigen positive; HCV, anti-hepatitis C positive; N/A, not determined. In 11 HCC cases with a normal level of Sat2 methylation, 4 showed no evidence of an unbalanced chromosome 1 (H19, H21, H22, and H33). Gain on chromosome 1 was detected in six cases (H20, H23, H24, H32, H35, and H36), and one case exhibited loss of regional 1p (H34). All but one case displayed a breakpoint outside the region of 1q12 (Table 1). The assignment of a breakpoint was further supported by SKY analysis, which indicated a p-arm breakage in cases H32 and H34 (Figure 1b). A 3×2 contingency statistical analysis was performed to examine the correlation between 1q gain, Sat2 hypomethylation, and the significance of the 1q12 breakpoint (Table 2). Fisher's exact test indicated a strong correlation between heterochromatin DNA hypomethylation and 1q copy gain with a breakpoint at 1q12 (P < 0.001). In 36 HCC tissues studied, no obvious relation between the degree of Sat2 hypomethylation and clinical staging, tumor size, or viral infection could be discerned.Table 2A Correlation between Heterochromatin Hypomethylation and 1q12 Breakpoint in 1q Copy Number GainChromosome 1 heterochromatin hypomethylationPositiveNegativeBreakpoint within 1q12 with evidence of 1q gain221Breakpoint outside 1q12 with evidence of 1q gain06Absence of chromosome 1q12 breakpoint with no evidence of 1q gain34Fisher's exact test, P < 0.001. Open table in a new tab Fisher's exact test, P < 0.001. Five of 24 adjacent liver tissues studied displayed a moderate level of Sat2 hypomethylation (H1 to H5). All cases were viral hepatitis B related, and arose from a noncirrhotic liver except for case H3. Histological examination revealed no apparent malignant phenotype in these tissues, and CGH analysis did not indicate genomic imbalances in any of the 24 adjacent liver tissues examined (Table 1). Methylation of CpG dinucleotides functions to maintain the stability of chromosome structures.21Hori TA Induction of chromosome decondensation, sister-chromatid exchanges and endoreduplications by 5-azacytidine, an inhibitor of DNA methylation.Mutat Res. 1983; 121: 47-52Crossref PubMed Scopus (50) Google Scholar Satellite DNA in the heterochromatin, in particular, is more heavily methylated at the CpG islands than the rest of the genomic DNA.22Ehrlich M Wang RY 5-Methylcytosine in eukaryotic DNA.Science. 1981; 212: 1350-1357Crossref PubMed Scopus (543) Google Scholar, 23Feinstein SI Racaniello VR Ehrlich M Gehrke CW Miller DA Miller OJ Pattern of undermethylation of the major satellite DNA of mouse sperm.Nucleic Acids Res. 1985; 13: 3969-3978Crossref PubMed Scopus (17) Google Scholar It has been postulated that under-methylated satellite sequences confer abnormal chromatin structures and predispose to chromosomal instability by enhancing chromosome recombinations.24Engler P Haasch D Pinkert CA Doglio L Glymour M Brinster R Storb U A strain-specific modifier on mouse chromosome 4 controls the methylation of independent transgene loci.Cell. 1991; 65: 939-947Abstract Full Text PDF PubMed Scopus (177) Google Scholar A high frequency of chromosome rearrangements with peri-centromeric or heterochromatic breakpoints has been observed in many tumors, which may suggest a relationship between satellite hypomethylation, chromosome instability and carcinogenesis.25Sawyer JR Tricot G Mattox S Jagannath S Barlogie B Jumping translocations of chromosome 1q in multiple myeloma: evidence for a mechanism involving decondensation of pericentromeric heterochromatin.Blood. 1998; 91: 1732-1741Crossref PubMed Google Scholar, 26Qu G Dubeau L Narayan A Yu MC Ehrlich M Satellite DNA hypomethylation vs. overall genomic hypomethylation in ovarian epithelial tumors of different malignant potential.Mutat Res. 1999; 423: 91-101Crossref PubMed Scopus (134) Google Scholar Our current finding supported a relationship between hypomethylated Sat2 sequences and recurrent aberrant 1q formation. We investigated the methylation status of Sat2 DNA, rather than the centromeric satellite (Sat-α), as Sat2 is a more CpG rich region than Sat-α (AT-rich region). In this series, methyl-sensitive endonuclease analysis showed a reduced methylation of classical Sat2 in 76% of HCC cases that displayed 1q copy number gain. In particular, we found Sat2 hypomethylation to be strongly associated with a 1q12 breakpoint (P < 0.001) (Table 2). In our recent karyotypic study on human HCCs, SKY analysis did not identify nonrandom chromosome 1 rearrangements, but rather frequent unbalanced 1q translocations with breakage in the vicinity of 1q12. Consistent localization of breakpoints within the heterochromatic region in HCC therefore suggests an important pathogenic consequence of Sat2 hypomethylation in 1q abnormalities. Structural decondensation of 1q12 is likely to result in centromeric fragility, somatic pairing, and the formation of jumping 1q translocations. We were able to support the presence of 1q12 segment decondensation by the finding of a fragile heterochromatic region in three cases (H25, H26, and H29) that displayed extensive Sat2 hypomethylation (Figure 1b). DNA methylation patterns are often altered in cancer. In a number of human malignancies, regional hypermethylation of the promoter region of critical tumor suppressor gene(s) results in silencing of transcriptional activity, and global DNA hypomethylation leading to activation of proto-oncogenes and re-expression of provirus sequences has been described.27Fearon ER Vogelstein BA Genetic model for colorectal tumorigenesis.Cell. 1990; 61: 759-767Abstract Full Text PDF PubMed Scopus (10134) Google Scholar, 28Baylin SB Makos M Wu J Chiu Yen RW de Bustros A Vertino P Nelkin BD Abnormal patterns of DNA methylation in human neoplasia: potential consequences for tumor progression.Cancer Cells. 1991; 3: 383-390PubMed Google Scholar Given the multistep nature of liver carcinogenesis, cancer-associated genetic and epigenetic alterations are probable in the putative precancerous liver lesions, the surrounding viral hepatitis-infected cirrhotic tissues. Indeed, microsatellite instability and aberrant DNA methylation of E-cadherin, p16, and c-myc have been reported in the noncancerous liver tissues of HCC.29Aiba N Nambu S Inoue K Sasaki H Hypomethylation of the c-myc oncogene in liver cirrhosis and chronic hepatitis.Gastroenterol Jpn. 1989; 24: 270-276Crossref PubMed Scopus (16) Google Scholar, 30Kanai Y Ushijima S Hui AM Ochiai A Tsuda H Sakamoto M Hirohashi S The E-cadherin gene is silenced by CpG methylation in human hepatocellular carcinomas.Int J Cancer. 1997; 71: 355-359Crossref PubMed Scopus (243) Google Scholar, 31Matsuda Y Ichida T Matsuzawa J Sugimura K Asakurs H p16INK4 is inactivated by extensive CpG methylation in human hepatocellular carcinoma.Gastroenterology. 1999; 116: 394-400Abstract Full Text Full Text PDF PubMed Scopus (188) Google Scholar, 32Kondo Y Kanai Y Sakamoto M Mizokami M Ueda R Hirohashi S Genetic instability and aberrant DNA methylation in chronic hepatitis and cirrhosis—a comprehensive study of loss of heterozygosity and microsatellite instability at 39 loci and DNA hypermethylation on 8CpG islands in microdissected specimens from patients with hepatocellular carcinoma.Hepatology. 2000; 32: 970-979Crossref PubMed Scopus (242) Google Scholar In our current series, 24 adjacent nonmalignant liver tissues had been examined for heterochromatin hypomethylation. Similar to our previous report5Wong N Lai P Lee S-W Fan S Pang E Liew C-T Sheng Z Lau JW Johnson PJ Assessment of genetic changes in hepatocellular carcinoma by comparative genomic hybridization analysis: relationship to disease stage, tumor size, and cirrhosis.Am J Pathol. 1999; 154: 37-43Abstract Full Text Full Text PDF PubMed Scopus (257) Google Scholar and that of a recent study from Taiwan,33Chen YJ Yeh SH Chen JT Wu CC Hsu MT Tsai SF Chen PJ Lin CH Chromosomal changes and clonality relationship between primary and recurrent hepatocellular carcinoma.Gastroenterology. 2000; 119: 431-440Abstract Full Text Full Text PDF PubMed Scopus (208) Google Scholar CGH aberrations were not found in any of the adjacent liver tissues. However, a moderate level of Sat2 demethylation was detected in 20% of the viral hepatitis B-related surrounding liver. A viral origin in the induction of peri-centromeric fragility in human neoplasms has been previously suggested.34Bain BJ Rooney DE Czepulkowski BH ed 2. Human Cytogenetics: A practical Approach. vol II. IRL Press, 1992: 127-128Google Scholar, 35Shabtai F Klar D Kimchi D Antebi E Hart J Halbrecht I Juxta-centromeric fragility of chromosomes 1, 2, 9, 16, and immunodeficiency. Special reference to the fragility of chromosome 2 and its oncogenic potential.Anticancer Res. 1984; 4: 235-239PubMed Google Scholar Although the role of hepatitis B infection, a DNA virus, in the demethylation of repeat sequences is unclear, gene products of cancer-associated DNA virus, such as SV40 and HPV, are known to alter cellular proteins and affect cell-cycle checkpoints, thereby inducing karyotypic instability.36Hartwell LH Kastan MB Cell cycle control and cancer.Science. 1994; 266: 1821-1828Crossref PubMed Scopus (2320) Google Scholar Genome-wide hypomethylation facilitates tumor progression. Demethylation of the repetitive sequences, such as LINE1, alphoid repeats, and Alu, constitute a major part of the global hypomethylation in tumor development. Although LINE1 hypomethylation has not been suggested in surrounding liver tissues of HCC,37Takai D Yagi Y Habib N Sugimura T Ushijima T Hypomethylation of LINE1 retrotransposon in human hepatocellular carcinomas, but not in surrounding liver cirrhosis.Jpn J Clin Oncol. 2000; 30: 306-309Crossref PubMed Scopus (84) Google Scholar methylationsensitive representational differential analysis38Ushijima T Morimura K Hosoya Y Okonogi H Tatematsu M Sugimura T Nagao M Establishment of methylation-sensitive-representational difference analysis and isolation of hypo- and hypermethylated genomic fragments in mouse liver tumors.Proc Natl Acad Sci USA. 1997; 94: 2284-2289Crossref PubMed Scopus (175) Google Scholar has indicated that global hypomethylation is not homogenous throughout the entire genome. Instead, hypomethylatedregions are scattered in the genome. Our present finding therefore suggests that heterochromatin demethylation precedes genome-wide hypomethylation, whereby heterochromatin fragility results in the clonal evolution of cells with extra copies of 1q. Chromosome 1q copy number gain may confer proliferative advantages that contribute to the natural evolution of HCC progression.