Japanese Journal of Clinical Oncology 30:306-309 (2000)
© 2000 Foundation for Promotion of Cancer Research
Hypomethylation of LINE1 Retrotransposon in Human Hepatocellular Carcinomas, but Not in Surrounding Liver Cirrhosis
1Carcinogenesis Division, National Cancer Center Research Institute, Tokyo, Japan and 2Department of Surgery, Hammersmith Hospital, Royal Postgraduate Medical School, London, UK
 |
ABSTRACT |
|---|
 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONAcknowledgmentsREFERENCES |
|---|
Background: Cytosine methylation of LINE1 (L1) elements, some of which are capable of retrotransposition in human cells, is known to play important roles in transcriptional repression of these retrotransposons. We have previously identified consistent hypomethylation of L1 elements in mouse liver tumors by a genome-wide search technique for aberrant methylations. In this study, we analyzed the methylation status of the L1 elements in human hepatocellular carcinomas (HCCs).
Methods: Nine pairs of an HCC and its surrounding tissue were obtained from clinical cases. Genomic DNA was digested with HpaII, a methylation-sensitive restriction enzyme, and hybridized with a probe derived from the promoter region of the L1 elements.
Results: Hypomethylation of the L1 elements was detected in eight of the nine HCCs, but never in the surrounding liver tissues, whether or not liver cirrhosis was present.
Conclusion: Specific occurrence of the hypomethylation of the L1 elements in the HCCs indicated its diagnostic value for malignancy. The hypomethylation could also lead to increased incidence of retrotransposition and resultant genomic instability in HCCs.
|
INTRODUCTION |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONAcknowledgmentsREFERENCES |
|---|
The long interspersed nuclear element (LINE or L1) is one of the repetitive sequences in the genome. A complete form of an L1 element spans
6 kb and has two open reading frames, ORF1 and ORF2, which encode an RNA-binding protein and a protein with reverse transcriptase and endonuclease activities (1–3). There are 105 copies of L1 elements in a haploid human genome and L1 elements account for 5% of the total human genome (4). Although most L1 elements exist in truncated or mutated forms and are therefore inactive, it has been estimated that 30–60 active L1 elements are present per diploid genome (5). These L1 elements in intact forms can be transcribed into RNA, reverse-transcribed into cDNA and integrated into another location of the genomic DNA in mammalian cells (1,5). Germline mutations where retrotransposition of L1 elements abrogated the functions of important genes are known in several diseases, such as the factor VIII gene in hemophilia A (6), dystrophin in Duchenne muscular dystrophy (DMD) (7), fukutin in Fukuyama-type congenital muscular dystrophy (FCMD) (8) and cytochrome b558 heavy-chain gene in chronic granulomatous disease (9). Also, somatic mutations caused by retrotransposition of an L1 element are known in APC in a colon cancer (10) and in c-MYC in a breast cancer (11). This evidence clearly shows that retrotransposition of an L1 element is deleterious to the genome. Methylation of CpG sites in an L1 promoter is considered to be one of the major mechanisms which represses retrotransposition of L1 elements (12,13). CpG sites in an L1 promoter are heavily methylated in the normal state (14) and demethylation of essential CpG sites leads to increased levels of L1 transcription (12).
Previously, we applied a genome-scanning method for aberrant methylations, methylation-sensitive-representational differential analysis (MS-RDA) (15), to a mouse hepatocellular carcinoma (HCC) induced by 2-amino-3,4-dimethylimidazo[4,5-f]quinoline (MeIQ). It was shown that the methylations of L1 elements and some specific genes were reduced in the HCC. Analysis of nine additional HCCs induced by MeIQ revealed that L1 elements were consistently hypomethylated in all the HCCs examined. It was indicated that hypomethylation of L1 elements had a diagnostic value for HCCs.
In this study, we analyzed the methylation status of L1 elements in human hepatocellular carcinomas that were obtained in the UK and associated with various etiologies.
|
MATERIALS AND METHODS |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONAcknowledgmentsREFERENCES |
|---|
Clinical Materials
Nine specimens with a good quality of DNA were obtained from patients who were enrolled, with written informed consent, over 4 years (August 1990 to November 1993). They were diagnosed through ultrasonography, enhanced CT and angiography. All the cases were tested in their sera for the presence/absence of hepatitis B surface antigen and antibody to HCV. They were also interviewed about alcohol consumption. They underwent partial hepatectomy at Hammersmith Hospital, London, UK and histological diagnosis was obtained for their HCCs and presence or absence of liver cirrhosis in the surrounding tissues. All tissue samples were handled as potentially biohazardous throughout the analyses whether or not they were positive for virus markers.
DNA Extraction and Southern Blot Analysis
Genomic DNA was extracted by serial phenol–chloroform extraction and ethanol precipitation (16). The genomic DNA (3 µg each) was digested with 30 units of HpaII or MspI (New England Biolabs, Beverly, MA). Digestion of genomic DNA with HpaII, which is methylation-sensitive, is known to reflect the methylation status of the HpaII sites (17) and the HpaII sites analyzed can be regarded as representative of the genomic region around the HpaII sites (17). Restricted DNA was purified by phenol–chloroform extraction and ethanol precipitation and was electrophoresed in a 1.3% agarose gel at 25 V overnight. After denaturation in 0.5 M NaOH, DNA was capillary-transferred to a nylon membrane (Hybond N; Amersham-Pharmacia, Uppsala, Sweden). After cross-linking DNA to the membrane using UV light, the membrane was pre-hybridized in 50% formamide and 0.65 M NaCl at 42°C and then hybridized for 8–12 h in the same buffer containing 10% dextran sulfate and 50 ng of probe DNA labeled with [
-32P]dCTP using a Multiprime DNA labeling kit (Amersham-Pharmacia). The filter was washed for 20 min in 0.1xSSC and 0.1% SDS at 50°C four times and exposed to Kodak XAR film at –80°C.
To prepare a probe for the human L1 promoter region, PCR was performed using human genomic DNA as a template with a forward primer, 5'-CGGGTGATTTCTGCATTTCC-3', and a reverse primer, 5'-GACATTTAAGTCTGCAGAGG-3'. The PCR product was cloned into pGEM-T Easy (Promega, Madison, WI) and transformed into XL1-Blue E.coli (Toyobo, Tokyo). The plasmid clones obtained were sequenced and a clone with a correct L1 element, corresponding to nucleotides –729 to –189 upstream of L1 ORF1, was selected as a probe for Southern blot analysis.
|
RESULTS |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONAcknowledgmentsREFERENCES |
|---|
The clinicopathological features of the nine cases are summarized in Table 1. The ages of the cases ranged from 48 to 73 years and the average age was 59.9 years. Liver cirrhosis was present in the surrounding non-tumorous liver tissue in three cases. Two of the three cases had a history of excessive alcohol consumption and one had a persistent infection with hepatitis B and hepatitis C viruses.
|
To determine the methylation status of the L1 promoter in these cases, we performed Southern blot analysis with a methylation-sensitive restriction enzyme, HpaII, with a probe covering the L1 promoter region. In eight of the nine HCC cases, hypomethylation of L1 was observed (Fig. 1). Hypomethylation of L1 was absent in the surrounding non-cancerous liver tissue, even in tissues with liver cirrhosis. No obvious relationship between the clinical data and the extent of L1 hypomethylation was observed (Table 1).
|
|
DISCUSSION |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONAcknowledgmentsREFERENCES |
|---|
In this study, we revealed the presence of L1 hypomethylation in eight of nine human HCCs and its absence in the surrounding tissues, even if liver cirrhosis was present. The very high incidence of L1 hypomethylation in HCCs was comparable to its 100% incidence in MeIQ-induced HCCs. The human HCC without L1 hypomethylation was obtained from a case without any history of alcohol dependence or viral infection. The very high incidence of L1 hypomethylation indicated that the hypomethylation could have a diagnostic value for HCCs even in the background of liver cirrhosis. The clear contrast of the presence of L1 hypomethylation in the HCCs and the absence in liver cirrhosis might be due to a failure in the maintenance system of an accurate methylation pattern in the HCCs. High incidences of L1 hypomethylation have also been reported in human urothelial carcinomas (18) and testicular cancers (19). Since antibodies to the products of ORFs in the L1 element have been established (19), staining of L1 products can be expected to help histological diagnosis with regard to the presence of malignant lesions in histological specimens.
Hypomethylation of the L1 promoter is known to lead to increased expression of two ORFs in the L1 element and probably to retrotransposition (2). Active retrotransposition of L1 can lead to inactivation of tumor suppressor genes or to activation of oncogenes when an L1 was inserted next to an oncogene and the promoter of the L1 enhanced the expression of the oncogene (20). Therefore, it is expected that hypomethylation of L1 is involved in instability of the genome (20). The molecular mechanism that causes hypomethylation of L1 elements and also of other repetitive sequences is almost totally unknown. Investigations into this mechanism are also important.
A reduced content of methylated cytosine is one of the characteristics of tumor cells (21). Recent techniques of genome scanning for differences in DNA methylation indicate that ‘global hypomethylation’ is not homogeneous over the genome, but hypomethylated regions are scattered in the genome. Hypomethylation of the repetitive sequences, such as L1, subtelomeric repeats, alphoid repeats and Alu, seem to constitute the major part of the ‘global hypomethylation’ of the genome in tumors.
|
Acknowledgments |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONAcknowledgmentsREFERENCES |
|---|
This study was supported by a Grant-in-Aid from the Ministry of Education, Science, Sports and Culture, Japan. D.T. is a recipient of a Research Resident Fellowship from the Foundation for Promotion of Cancer Research.
|
FOOTNOTES |
|---|
+ For reprints and all correspondence: Toshikazu Ushijima, Carcinogenesis Division, National Cancer Center Research Institute, 1–1 Tsukiji 5-chome, Chuo-ku, Tokyo 104-0045, Japan. E-mail: tushijim@ncc.go.jp
|
REFERENCES |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONAcknowledgmentsREFERENCES |
|---|
1 Moran JV, Holmes SE, Naas TP, DeBerardinis RJ, Boeke JD, Kazazian HH Jr. High frequency retrotransposition in cultured mammalian cells. Cell 1996;87:917–27.[Web of Science][Medline]
2 Fen Q, Moran JV, Kazazian HH Jr, Boeke JD. Human L1 retrotransposon encodes a conserved endonuclease required for retrotransposition. Cell 1996;87:905–16.[Web of Science][Medline]
3 Hohjoh H, Singer MF. Sequence-specific single-strand RNA binding protein encoded by the human LINE-1 retrotransposon. EMBO J 1997;16:6034–43.[Web of Science][Medline]
4 Fanning TG, Singer MF. LINE-1: a mammalian transposable element. Biochim Biophys Acta 1987;910:203–12.[Medline]
5 Sassaman DM, Dombroski BA, Moran JV, Kimberland ML, Naas TP, DeBerardinis RJ, et al. Many human L1 elements are capable of retrotransposition. Nature Genet 1997;16:37–43.[Web of Science][Medline]
6 Kazazian HH Jr, Wong C, Youssoufian H, Scott AF, Phillips DG, Antonarakis SE. Haemophilia A resulting from de novo insertion of L1 sequences represents a novel mechanism for mutation in man. Nature 1988;332:164–6.[Medline]
7 Narita N, Nishio H, Kitoh Y, Ishikawa Y, Minami R, Nakamura H, et al. Insertion of a 5' truncated L1 element into the 3' end of exon 44 of the dystrophin gene resulted in skipping of the exon during splicing in a case of Duchenne muscular dystrophy. J Clin Invest 1993;91:1862–7.
8 Kobayashi K, Nakahori Y, Miyake M, Matsumura K, Kondo-Iida E, Nomura Y, et al. An ancient retrotransposal insertion causes Fukuyama-type congenital muscular dystrophy. Nature 1998;394:388–92.[Medline]
9 Kumatori A, Faizunnessa NN, Suzuki S, Moriuchi T, Kurozumi H, Nakamura M, et al. Nonhomologous recombination between the cytochrome b558 heavy chain gene (CYBB) and LINE-1 causes an X-linked chronic granulomatous disease. Genomics 1998;53:123–8.[Web of Science][Medline]
10 Miki Y, Nishisho I, Horii A, Miyoshi Y, Utsunomiya J, Kinzler KW, et al. Disruption of the APC gene by a retrotransposal insertion of L1 sequence in a colon cancer. Cancer Res 1992;52:643–5.
11 Morse B, Rotherg PG, South VJ, Spandorfer JM, Astrin SM. Insertional mutagenesis of the myc locus by a LINE-1 sequence in a human breast carcinoma. Nature 1988;333:87–90.[Medline]
12 Hata K, Sakaki Y. Identification of critical CpG sites for repression of L1 transcription by DNA methylation. Gene 1997;189:227–34.[Web of Science][Medline]
13 Woodcock DM, Lawler CB, Linsenmeyer ME, Doherty JP, Warren WD. Asymmetric methylation in the hypermethylated CpG promoter region of the human L1 retrotransposon. J Biol Chem 1997;272:7810–6.
14 Bender J. Cytosine methylation of repeated sequences in eukaryotes: the role of DNA pairing. Trends Biochem Sci 1998;23:252–6.[Web of Science][Medline]
15 Ushijima T, Morimura K, Hosoya Y, Okonogi H, Tatematsu M, Sugimura T, et al. 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;94:2284–9.
16 Sambrook J, Fritsch EF, Maniatis T. Molecular Cloning. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory Press 1989;9.14–9.23.
17 Bickmore WA, Bird AP. Use of restriction endonucleases to detect and isolate genes from mammalian cells. Methods Enzymol 1992;216:224–44.[Web of Science][Medline]
18 Jurgens B, Schmitz-Drager BJ, Schulz WA. Hypomethylation of L1 LINE sequences prevailing in human urothelial carcinoma. Cancer Res 1996;56:5698–703.
19 Bratthauer GL, Fanning TG. Active LINE-1 retrotransposons in human testicular cancer. Oncogene 1992;7:507–10.[Web of Science][Medline]
20 Florl AR, Lower R, Schmitz-Drager BJ, Schulz WA. DNA methylation and expression of LINE-1 and HERV-K provirus sequences in urothelial and renal cell carcinomas. Br J Cancer 1999;80:1312–21.[Web of Science][Medline]
21 Gama-Sosa MA, Slagel VA, Trewyn RW, Oxenhandler R, Kuo KC, Gehrke CW, et al. The 5-methylcytosine content of DNA from human tumors. Nucleic Acids Res 1983;11:6883–94.
Received December 27, 1999; accepted May 8, 2000.
CiteULike 
Connotea 
Del.icio.us What's this?
This article has been cited by other articles:
|
|
 |
|
  S. H. Tang, D. H. Yang, W. Huang, H. K. Zhou, X. H. Lu, and G. Ye Hypomethylated P4 Promoter Induces Expression of the Insulin-Like Growth Factor-II Gene in Hepatocellular Carcinoma in a Chinese Population. Clin. Cancer Res., July 15, 2006; 12(14): 4171 - 4177. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 V. P. Belancio, D. J. Hedges, and P. Deininger LINE-1 RNA splicing and influences on mammalian gene expression Nucleic Acids Res., March 22, 2006; 34(5): 1512 - 1521. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 A. N. Carnell and J. I. Goodman The Long (LINEs) and the Short (SINEs) of It: Altered Methylation as a Precursor to Toxicity Toxicol. Sci., October 1, 2003; 75(2): 229 - 235. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 N. Wong, W.-C. Lam, P. B.-S. Lai, E. Pang, W.-Y. Lau, and P. J. Johnson Hypomethylation of Chromosome 1 Heterochromatin DNA Correlates with q-Arm Copy Gain in Human Hepatocellular Carcinoma Am. J. Pathol., August 1, 2001; 159(2): 465 - 471. [Abstract] [Full Text]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||