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Journal of Biomedicine and Biotechnology
Volume 2006 (2006), Article ID 17142, 6 pages
Research Article

LINE-1 Hypomethylation in a Choline-Deficiency-Induced Liver Cancer in Rats: Dependence on Feeding Period

1Free Radical Biology and Aging Research Program, Oklahoma Medical Research Foundation, Oklahoma City, Ok 73104, USA
2Third Department of Internal Medicine, Nara Medical University, Kashihara, Nara 634-8521, Japan
3Department of Biochemistry and Molecular Biology, University of Oklahoma Health Sciences Center, Oklahoma City, OK 73104, USA

Received 8 June 2005; Revised 28 November 2005; Accepted 4 December 2005

Copyright © 2006 Kiyoshi Asada et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.


Chronic feeding of methyl-donor (methionine, choline, folic acid, and vitamin B12) deficient diet induces hepatocellular carcinoma formation in rats. Previous studies have shown that promoter CpG islands in various cancer-related genes are aberrantly methylated in this model. Moreover, the global genome in methyl-donor-deficient diet fed rats contains a lesser amount of 5-methylcytosine than control livers. It is speculated that more than 90% of all 5-methylcytosines lie within the CpG islands of the transposons, including the long/short interspersed nucleotide elements (LINE and SINE). It is considered that the 5-methylcytosines in LINE-1 limit the ability of retrotransposons to be activated and transcribed; therefore, the extent of hypomethylation of LINE-1 could be a surrogate marker for aberrant methylation in other tumor-related genes as well as genome instability. Additionally, LINE-1 methylation status has been shown to be a good indicator of genome-wide methylation. In this study, we determined cytosine methylation status in the LINE-1 repetitive sequences of rats fed a choline-deficient (CD) diet for various durations and compared these with rats fed a choline-sufficient (CS) diet. The methylation status of LINE-1 was assessed by the combined bisulfite restriction analysis (COBRA) method, where the amount of bisulfite-modified and RsaI-cleaved DNA was quantified using gel electrophoresis. Progressive hypomethylation was observed in LINE-1 of CD livers as a function of feeding time; that is, the amount of cytosine in total cytosine (methylated and unmethylated) increased from 11.1% (1 week) to 19.3% (56 weeks), whereas in the control CS livers, it increased from 9.2% to 12.9%. Hypomethylation in tumor tissues was slightly higher (6%) than the nontumorous surrounding tissue. The present result also indicates that age is a factor influencing the extent of cytosine methylation.