LINE-1 hypomethylation in a choline-deficiency-induced liver cancer in rats: dependence on feeding period

Abstract

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.

Figure 1

Genomic structure of LINE-1 in rats. Total length is 6 kb which is composed of three portions, 5' UTR, ORF I, ORF II, and 3'UTR. Bisulfite PCR was conducted in the 5'UTR sequence. The primer sequences are shown with arrows.

Figure 2

Typical COBRA for LINE-1 cytosine methylation status in the livers of rats fed CD or CS diet for 4 weeks. Numbers shown at the bottom of each lane indicate the percentage of unmethylated cytosine (115 bp band + 48 bp band) versus total cytosine (163 bp band). Panel A: CS livers (#1–#5) after 4-week feeding; Panel B: CD livers (#1–#5) after 4-week feeding. Isolated genomic DNA was treated with sodium bisulfite and PCR-amplified with LINE-1 primers and divided into two portions. The one portion was digested with RsaI (designated with +), the other was not digested (designated with −), and then both were run on the gel and stained with ethidium bromide. The band at 163 bp was identified as the LINE-1 sequence and those at 115 bp and 48 bp were identified as RsaI-digested LINE-1. The numbers on the top of each pair of lanes indicate the rat identification numbers. Lanes designated with M-control are COBRA treated samples obtained from SssI-hypermethylated rat DNA.

Figure 3

Percentage of unmethylated cytosine versus total (methylated plus unmethylated) cytosine in LINE-1 sequences in the livers of CSAA- and CDAA-diet fed rats. Actual data are shown in the table under the graph. Symbols: (a) significantly different from CSAA at the same feeding period; (b) significantly different from CDAA at 1 week; (c) significantly different from CSAA at 1 and 4 weeks; (d) significantly different from CDAA at 1 and 4 weeks. %±SE; standard error

Figure 4

Percentage of unmethylated cytosine in total (methylated plus unmethylated) cytosine in LINE-1 sequences in tumor or nontumor liver tissue in CD-diet fed rats. Actual data are shown in the table under the graph. Statistically significant differences (*) are seen between nontumor and tumor at 56 weeks.