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J Biomed Biotechnol. 2006; 2006: 17142. Published online 2006. doi: 10.1155/JBB/2006/17142. | PMCID: PMC1479888 |
LINE-1 Hypomethylation in a Choline-Deficiency-Induced Liver
Cancer in Rats: Dependence on Feeding Period Kiyoshi Asada,1, 2 Yashige Kotake,1 Rumiko Asada,1 Deborah Saunders,1 Robert H. Broyles,1, 3 Rheal A. Towner,1 Hiroshi Fukui,2 and Robert A. Floyd1, 3* 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 June 8, 2005; Revised November 28, 2005; Accepted December 4, 2005. 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. When rats are chronically fed a diet devoid of a methyl-donor source (choline,
methionine, folic acid, and vitamin B 12), they spontaneously develop hepatocellular carcinomas (HCCs) [ 1– 5].
This is a unique carcinogenesis model in which no known carcinogen
is involved. After the initial discovery [ 6] of this model,
many questions were raised concerning its validity including the
possibility of carcinogenic contaminants in the diet. Later, it
was definitively demonstrated that diets lacking in methionine and
choline and containing no detectable level of carcinogens acted as
a complete carcinogen [ 7, 8]. In spite of extensive
phenomenological studies, the mechanism by which dietary
methyl-donor deficiency causes HCC formation is not understood,
but it is suggested that various concurrent carcinogenic pathways
may be involved [ 4]. Oxidative stress appears to play a
major role in this model [ 4] because there is a
significant increase in oxidized DNA (8-hydroxy-deoxyguanosine)
levels from day 1 of feeding [ 4] and because antioxidant
cofeeding inhibits cancer formation [ 9]. Nevertheless, no
direct connection between oxidative stress and carcinogenesis has
been elucidated. Genome-wide demethylation of 5-methylcytosine has been regarded as
a common epigenetic event in malignancies and may play a crucial
role in carcinogenesis. In the rat methyl-donor deficiency
models, promoter CpG islands in several cancer-related genes are
known to be aberrantly methylated, as noted by changes in DNA
5-methylcytosine content [ 10]. Methyl donors including
choline and methionine are required for S-adenosyl methionine
(SAM) biosynthesis [ 11], and SAM is the substrate for DNA
cytosine methyltransferase, the enzyme responsible for
maintaining DNA cytosine methylation. Therefore, it is
possible to speculate that the dysregulation of Dnmt activity is a
cause of the genome-wide decrease of 5-methylcytosine and aberrant
methylation of specific genes. However, the selective nature of
the presence of aberrantly methylated genes suggests that other
factors are also involved. More than 90% of all 5-methylcytosines
lie within the CpG islands in the transposons, including
long/short interspersed nucleotide elements (LINE and SINE). The
presence of 5-methylcytosine in LINE-1 is considered to limit the
ability of retrotransposons to be activated and transcribed;
therefore, loss of 5-methylcytosine in LINE-1 could result in an
increase in retrotransposon activity, leading to propagation of
aberrant methylation to other genes [ 12, 13]. Genome-wide
instability inevitably results from hypomethylation. LINE-1
methylation status has also been shown to be a good indicator of
genome-wide methylation [ 14, 15]. In humans, LINE-1
hypomethylation was demonstrated in patients having various
cancers [ 16– 18]. In the present study, using the combined
bisulfite restriction analysis (COBRA) method [ 14, 19], we
evaluated the amount of cytosine/5-methylcytosine in the LINE-1
repetitive sequence in rats fed a choline-deficient (CD) diet for
various times. Animals and diets Rats were treated strictly following the animal use protocol
approved by the Institutional Animal Care and Use Committee in the
Oklahoma Medical Research Foundation. Weaned male Fisher 344 rats
were obtained from Charles River (Indianapolis, IN, USA) and
divided into 2 groups each containing 3–5 animals and fed a
choline sufficient (CS) or a choline deficient (CD) diet (Dyets
Inc Bethlehem, IN, USA). Feeding periods were 1, 4, 24, and 56
weeks. In these experiments, protein in the diet was substituted
by a defined amino acid diet (CSAA or CDAA diet), because the CDAA
diet has been shown to considerably accelerate carcinogenesis
without altering cancer pathology as compared with the
conventional CD (Lombardi's CD diet) [ 20]. Sequence of LINE-1 The LINE-1 sequence of interest in this study is illustrated in
. The consensus
sequence revealed that LINE-1 elements have a 5' untranslated
region (UTR) with internal promoter activity, two open reading
frames (ORFs), a 3'UTR that ends in an AATAAA polyadenylation
signal, and a poly A tail [ 21, 22]. Combined bisulfite restriction analysis COBRA is a simple method of CpG methylation analysis which
utilizes the cleaving ability of the restriction enzyme RsaI
specifically at bisulfite-modified CpG sites [ 14]. Genomic
DNA from rat liver tissues was extracted using a Quiagen's genomic
DNA extraction kit (Qiagen, Valencia, CA, USA). Bisulfite
modification of genomic DNA was performed as follows: 3  μg
of DNA, digested with the restriction enzyme EcoRI, was incubated
with 0.3 N NaOH in a volume of
20 μL for 15 minutes, and then combined with a
120 μL portion of 3.6 M sodium bisulfite (Sigma, St
Louis, MO, USA)/0.6 mM hydroquinone (Sigma)
(adjusted to pH 5.0 with NaOH). The bisulfite reaction was
performed by utilizing a thermocycler (Perkin Elmer 9600, Boston,
MA, USA) with 15 cycles of 95°C for 30 seconds followed by
50°C for 15 minutes. The samples were desalted with a
Wizard DNA Clean-Up System (Promega, Madison, WI, USA) and
desulfonated with 5 minutes incubation in 0.3 N NaOH. Bisulfite modified DNA was PCR-amplified with
custom-synthesized primers (Molecular Biology Resource Facility,
University of Oklahoma Health Sciences Center, Oklahoma City, OK,
USA). The primer sequences which correspond to the nucleotides in
the regulatory region of LINE-1 sequence (GenBank: U87600) are as
follows: L1bisF, 5'-TTT GGT GAG TTT GGG ATA-3'; L1bisR, 5'-CTC AAA
AAT ACC CAC CTA AC-3'. The PCR conditions were 30 cycles of
94°C 30 seconds, 55°C 30 seconds, 72°C 30
seconds. Subsequently, the PCR product was digested with
10 unit of RsaI at 37°C, for 5 hours, isolated
with ethanol-chloroform precipitation, and subjected to
polyacrylamide gel electrophoresis. The gel was stained with
ethidium bromide, and the band intensity in the fluorogram was
analyzed with an imaging workstation (NucleoTech Corp, Hayward,
CA, USA). The data for densitometric analysis is presented as mean
± SE Tests for statistical significance were evaluated using
Student t test. RNA extraction and Semiquantitative RT-PCR Total RNA was extracted from liver tissues of CS- or CD-diet fed
rats with Qiagen RNeasy kit (Qiagen, Crawley, UK). After RNA
samples were treated with DNase I (Invitrogen, Carlsbad, CA, USA),
RT-PCR was performed with SuperScript III One-Step RT-PCR System
(Invitrogen, Carlsbad, CA, USA). Common GenBank accession number
of ORF I and ORF II sequences in LINE-1 is DQ100473. PCR was
performed under the following conditions: annealing temperatures
were 60°C for ORF I and 55 °C for ORF II, cycle
number was 23, and primer sequences were ORF I forward; 5′-AAG AAA
CAC CTC CCG TCA CA-3′, ORF I reverse; 5′-CCT CCT TAT GTT GGG CTT
TAC C-3′, ORF II forward; 5′-CCC ACT CTC TCC CTA CTT A-3′, and ORF II
reverse; 5′-TAT AGA GGA AGG CAA CTG AT-3′. The expression of the
glyceraldehydes phosphate dehydrogenase gene (GAPDH) was used to
normalize the transcript band intensity. LINE-1 COBRA in artificially methylated DNA To confirm the accuracy of COBRA, control genomic DNA obtained
from rats fed a regular diet was enzymatically methylated with
SssI methylase (Sigma) in the presence of the substrate
S-adenosylmethionine, and its LINE-1 promoter CpG island
methylation was determined with COBRA. The results indicate that
there was 5.9–6.1% undigested DNA (, 163 kb band in the lanes marked with M-control +), suggesting
that the amount of mutated DNA plus SssI methylation resistant
cytosine is within this level. LINE-1 COBRA in CD livers LINE-1 methylation was analyzed with COBRA in CD and CS livers
after various feeding periods, including 1, 4, 24, and 56 weeks.
illustrates typical COBRA fluorograms
obtained from livers of rats fed either CD or CS diet for 4 weeks.
There was a clear tendency for the CD diet to promote
hypomethylation in LINE-1 during the entire feeding period (1 week
to 56 weeks). The amount of unmethylated cytosine in LINE-1 ranged
from 9.2% at 1 week to 12.9% at 56 weeks in CS livers, while
in the CD livers, it ranged from 11.1% at 1 week to 19.3% at 56
weeks (). There was a statistical significant
difference between CS and CD livers at 4 and 56 weeks of feeding
(). LINE-1 hypomethylation in tumor and nontumor tissues After 24 weeks of feeding the CD diet, rats began to have tumor
nodules which were histologically identified as adenomas (19), and
at 56 weeks, most of these tumor nodules had developed into HCC.
COBRA of LINE-1 DNA for tumor and nontumor tissues showed that
there is a tendency for the DNA in tumor tissues to be more
hypomethylated than nontumor tissues, however, a statistically
significant difference was obtained only in the 56-week fed
animals (). LINE-1 transcript expression The expression of LINE-1 (ORF1 and ORF2) gene transcript was
assessed with semiquantitative RT-PCR. Densitometry-analysis
indicated (in arbitrary unit) (1) for ORF I CS(8
weeks →16 weeks): 1.60 ± 0.42 → 1.35 ± 0.49, and CD (8 weeks → 16 weeks):
1.40±0.42 → 1.90±0.28, and (2) for ORF II CS(8 weeks
→ 16 weeks): 1.45±0.21 → 1.35±0.07, and CD (8 weeks → 16 weeks): 0.75±0.21 → 0.70±0.14. The comparison of the numbers of 8 weeks and 16
weeks in each group indicates that there was no increase in LINE-1 transcript expression.
However, these results may have marginal statistical
significance because of the small number of samples (N = 2 for each group). Chronic CD diet is hepatocarcinogenic in male rats and global
hypomethylation has been shown to exist from early feeding times
[ 10, 23]. In many cancers, global hypomethylation as well as
hypo- or hypermethylation in specific genes are widely accepted
epigenetic changes [ 24]; however, which gene or DNA region
responsible for aberrant methylation, especially in the
methyl-deficient diet models, is not clear. Using a rat CD model,
we applied the COBRA method to analyze LINE-1 methylation.
COBRA requires small amount of DNA samples and was previously
employed to analyze human LINE-1 hypomethylation [ 18]. We showed that the LINE-1 promoter was hypomethylated in the
livers of rats fed a CD diet from as early as 4-weeks' feeding
(14.4%) as compared to CS livers of the same feeding period
(9.1%), and that hypomethylation increased as a function of
feeding period up to 19.3% at 56 weeks ().
It is not unexpected that there was a significant increase in
unmethylated cytosine (or a decrease in 5-methylcytosine) in
LINE-1 of control CS livers as a function of feeding period
(12.9% at 56 weeks) because it has been shown that aging is a
major cause of genome-wide hypomethylation in mice and rats
[ 25, 26]. For example, using Southern blotting, Mays-Hoopes et
al estimated that there is an 8% decrease in LINE-1's
5-methylcyotsine content in the livers of 27-month old mice
[ 26]. Furthermore, it is possible that the age-dependent
increase of mutation in LINE-1 could provide false signals of
hypomethylation in this assay. In humans, LINE-1 hypomethylation
was detected only in HCC but not in nontumor liver cirrhosis
[ 16]. Also, a recent report has indicated that in some
cancers, such as lymphoma, renal cell carcinoma, and papillary
carcinoma of the thyroid, the LINE-1 hypomethylation level is not
significantly different from normal tissues [ 18]. The authors
suggested that human cancers may be classified into two groups, a
low (0–3.4%) LINE-1 hypomethylation group and a moderately high
(6.8–9.5%) group [ 18]. We speculate that HCC in this model
may mimic the situation in the low group. The same study also
showed a linear correlation of methylation levels analyzed by
COBRA with levels determined by using semiquantitative
conventional Southern blotting hybridization analysis [ 18]. Florl et al reported that in human urothelial cancer, there were
coordinate changes of LINE-1 and HERV-K DNA methylation,
suggesting that hypomethylation affects a variety of retroelements
to similar extents [ 12]. Thus, LINE-1 hypomethylation is
thought to be one of the important surrogate markers of global
hypomethylation [ 14, 15]. In the present study, LINE-1
hypomethylation in CD livers increased in a time-dependent fashion
from 11.1% at 1 week to 19.3% at 56 weeks. The cytosine
(demethylated 5-methylcytosine) level in LINE-1 in this experiment
was much lower than that of the global genome as determined by
using an HpaII/MspI-based cytosine extension assay [ 23],
which resulted in cytosine content increasing from 46% at 9 weeks
up to 54% at 36 weeks. However, another study using HPLC
analysis indicated that genome-wide unmethylated cytosine
increased from 6% at 8 weeks to 11–14% at 22 weeks [ 10]. The cause of discrepancies in the three methods is unknown. LINE-1 promoter methylation is thought to play an important role
in transcriptional activation of retrotransposons [ 27].
Active retrotransposition can cause the movement of LINE-1 to
anywhere within one chromosome and, as such, could disrupt tumor
suppressor genes and/or activate oncogenes [ 28, 29]. Indeed,
human colon cancer has been shown to be associated with APC
retrotransposon activity [ 28]. In addition, aberrant LINE-1
methylation may cause specific gene modification as well as
genomic instability [ 13, 30]. In rat methyl-deficiency models,
CpG island aberrant methylation has been seen in several oncogenes
and tumor-suppressor genes, such as alpha-fetoprotein (AFP)
[ 31], c-Ha-ras [ 32], c-Ki-ras [ 32], c-fos
[ 32], c-myc [ 33], Dnmt [ 34], glutathione
S-transferase pi (GSTP) [ 35], p16 [ 36], and protein
tyrosine phosphatase receptor O gene (PTPRO) [ 37]. Although
LINE-1 hypomethylation should be followed by the increase in
LINE-1 transcripts, our RT-PCR assessment did not show the
increasing tendency. Previously, the similar discrepancy was
reported in human liver cancer tissues [ 38]. In conclusion, the degree of hypomethylation in promoter CpG
islands in LINE-1 repetitive sequences in the livers of rats fed a
CD diet progresses as a function of feeding period. The level of
LINE-1 hypomethylation was similar to that found in previous HPLC
analysis for genome-wide hypomethylation. These results suggest
that genome-wide hypomethylation occurs because of the choline
deficiency diet and that LINE-1 methylation status is a good
indicator of such. Moreover, because LINE-1 hypomethylation can
activate its retrotransposon activity, it may be the root cause of
aberrant methylation in several cancer-related genes in this
model. This notion is yet to be proven. Support from the National Cancer Institute, National Institutes of
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