Introduction

Lysophosphatidic acid (LPA) is a bioactive mediator that induces several cellular effects, including regulation of cell proliferation, differentiation, transcellular migration, morphogenesis, and protection from apoptosis (Furui et al. 1999; Goetzl et al. 1999; Contos et al. 2000a, b, 2002; Fang et al. 2002). In human cancer cells, LPA can stimulate not only cell proliferation but also migration, invasion, and production of angiogenic factors, suggesting that LPA may play an important role in the pathogenesis of tumor cells (An et al. 1998; Bandoh et al. 1999; Furui et al. 1999; Goetzl et al. 1999; Fang et al. 2002; Fujita et al. 2003). LPA interacts with at least six G-protein-coupled transmembrane receptors, LPA receptor-1 (LPA1), LPA2, LPA3, LPA4, LPA5, and LPA6 (An et al. 1998; Bandoh et al. 1999; Noguchi et al. 2003; Lee et al. 2006; Pasternack et al. 2008; Shimomura et al. 2008; Lin et al. 2010). Previously, aberrant expressions of LPA receptors have been reported in human several tumors, indicating that alterations of LPA receptor gene expression might be also involved in the malignant transformation of tumor cells as well as LPA per se (Schulte et al. 2001; Fujita et al. 2003; Shida et al. 2003, 2004; Tsujino et al. 2010). In rodents, we have reported that the loss of LPA1 expression is due to its aberrant DNA methylation in RH7777 rat HCC cells (Tsujiuchi et al. 2006b).

It is well known that liver tumors associated with cirrhosis can be induced by prolonged feeding of rats with a choline-deficient (CD) diet that does not contain any established carcinogens (Shinozuka et al. 1986; Lombardi 1988). A choline-deficient l-amino acid-defined (CDAA) diet used in this study is semisynthetic and provides stronger carcinogenic effects than the CD diet in rats (Nakae et al. 1990, 1992). Possible mechanisms underlying hepatocarcinogenesis by the choline deficiency have been proposed to include the following; liver necrosis associated with subsequent regeneration, induction of oxidative DNA damage and lipid peroxidation, and generation of genetic alterations (Giambarresi et al. 1982; Perera et al. 1985; Rushmore et al. 1986). Moreover, disturbance of DNA methylation status is also considered as important factor in hepatocarcinogenesis induced by methyl donor deficiency (Poirier 1994; Christman 1995). We have previously reported the hypomethylation of c-myc gene in rat HCCs resulting from the CDAA diet (Tsujiuchi et al. 1999). By contrast, despite methyl donor deficiency, E-cadherin and connexin26 genes were methylated in those tumors (Tsujiuchi et al. 2006a). Furthermore, we have reported that gene-specific changes of DNA methylation patterns occurred in livers of rats after short-term feeding of the CDAA diet (Shimizu et al. 2007).

Recently, frequent mutations of LPA1 gene were found in HCCs induced by the CDAA diet in rats, suggesting that the LPA1 gene may be involved in rat hepatocarcinogenesis by the CDAA diet (Obo et al. 2009). Therefore, in the present study, to evaluate whether DNA methylation status of LPA receptor genes is disturbed in the livers of rats by feeding the CDAA diet, we measured their DNA methylation patterns and expression levels in the livers of rats fed the CDAA diet and resultant HCCs, comparing with normal livers.

Materials and methods

Animals and treatment

A total of nineteen F344 male rats, 5 week old, were purchased from Japan SLC Inc. (Shizuoka, Japan) and were housed three per plastic cage containing white flake bedding, in an air-conditioned room, at a constant temperature of 25°C, and a 12-h light–dark cycle. Food and water were available ad libitum throughout the study. After a 1-week acclimation period on basal diet in pellet form (CF-2 Diet; Clea Japan, Tokyo, Japan), 16 animals received the CDAA diet (product number 518753; Dyets Inc., Bethlehem, PA), consisting of ingredients as previously described (Nakae et al. 1990, 1992). Subgroups of three rats were killed by exsanguination from the abdominal aorta, under light ether anesthesia, at 7 days and 2, 12 and 20 weeks after the beginning of the experiment. In order to produce HCCs, four rats were given the CDAA diet until 75 weeks. To obtain normal liver tissues, three rats were also killed at 6 week of age without the CDAA diet feeding.

Tissue preparation

Upon sacrifice, whole livers were immediately excised and frozen in liquid nitrogen, and stored at −80°C until analysis. Grossly apparent tumors were also dissected from their surrounding tissue and frozen. A part of samples were fixed in 10% neutrally buffered formalin at 4°C and routinely processed for hematoxylin and eosin staining, and histopathologically evaluated according to diagnostic criteria previously described (Nakae et al. 1990, 1992). All experiments and procedures carried out on the animals were approved by the Animal Care Committee of All experiments, and procedures carried out on the animals were approved by the Animal Care Committees of Kinki University.

Bisulfite sequencing

Genomic DNA was extracted with a DNeasy tissue kit (QIAGEN, Hilden, Germany) from pooled liver samples of three rats in each subgroup and each HCC of four rats, and treated with an EpiTect Bisulfite Kit (QIAGEN). For bisulfite sequencing, PCR was performed with the following primer sets: LPA1 (NCBI accession number; NW_047713.2) BS-F: 5′-GTGATAGAGGTGGGTGTGTTTGAT-3′, BS-R: 5′-CACTATACTAAAAAACAAAAATCACA-3′; LPA2 (NCBI accession number; NW_047470.1) BS-F: 5′-GGGGGAGGTTAGGGGAGGAGG-3′, BS-R: 5′-CCCCCAAAAAAATTCCACCCC-3′; and LPA3 (NCBI accession number; NW_047633.2) BS-F: 5′-GGTTTGGATGTTATTAGTAGGAAA-3′, BS-R: 5′-CATCCACTTATCATAATAACACTC-3′. PCR products were subcloned with a DynaExpress TA PCR cloning kit (BioDynamics Laboratory Inc., Tokyo, Japan) and sequenced with a BigDye terminator v3.0 cycle sequencing ready reaction kit (Applied Biosystems Japan Ltd.) and an ABI PRISM 310 genetic analyzer (Applied Biosystems Japan Ltd.). For each sample, eight clones were sequenced (Tsujiuchi et al. 2006b).

Semiquantitative reverse transcription (RT)-polymerase chain reaction (PCR) amplification for LPA receptor gene expressions

Total RNA was extracted from each sample, using ISOGEN (Nippon Gene, Inc. Toyama, Japan), and first-strand cDNA was synthesized from 0.5 μg samples with Transcriptor First Strand cDNA Synthesis Kit (Roche Diagnostics Co. Ltd., Mannheim, Germany). To eliminate possible false positives caused by residual genomic DNA, all samples were treated with DNase.

Semiquantitative RT-PCR analysis was performed as described previously (Tsujiuchi et al. 2006b). PCR amplification was carried out in a reaction volume of 10 μl containing 1 μM of each gene primer, 200 μM of each dNTP, 1× PCR buffer, 0.5 U of AmpliTaq Gold (Applied Biosystems Japan Ltd., Tokyo, Japan), and 0.5 μl of synthesized cDNA mixture. Primer pairs were as follows: for LPA1 (NCBI accession number; NC_005104), F: 5′-CGGGATTGGTCTTGCTACTG-3′, R: 5′-CATCTCTTTGTCGCGGTAGG-3′ (annealing temperature: 64°C); for LPA2 (NCBI accession number; NC_005115), F: 5′-AAAGGCTGGTTCCTGCGACA-3′, R: 5′-TGCTCTGCCATGCGTTCAAC-3′ (annealing temperature: 69°C); and for LPA3 (NCBI accession number; NC_005101), F: 5′-CTCGTACAAGGACGAGGACAT-3′, R: 5′-TGAGACAGGCAAGGACTCTTA-3′ (annealing temperature: 62°C). The rat glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene was used as an internal control to adjust the amounts of template. For each gene, multiple cycles of PCR amplification were tested. The cycle at which a sample having the highest expression reached an amplification plateau was determined, and a cycle number smaller than this was adopted for the analysis. The amplified products were then separated on 2% agarose gels containing 0.05 μg/ml ethidium bromide. The bands were quantitated with image analysis software (NIH Image, Bethesda, MD), and peak intensities for PCR products derived for LPA receptor genes were divided by those for GAPDH.

Results

Histopathologically, in the livers of rats fed the CDAA diet, diffuse fatty change was observed at 7 days. Fatty change widely expanded in all areas of livers at 2 weeks and the extension of collagen fibers were observed from Glisson’s sheaths. Borderline cirrhosis resulted by 12 weeks, and frank cirrhosis developed by 20 weeks. Four HCCs induced by the CDAA diet in four rats were all well differentiated (Nakae et al. 1990, 1992).

Results of bisulfite sequencing analysis for LPA receptor genes are shown in Figs. 1, 2, 3 and 4. The livers of rats fed the CDAA diet were unmethylated in LPA1 and LPA2 genes as well as normal livers (Figs. 1 and 2). In LPA3 gene, although normal livers and the livers at 7 days were unmethylated, those at 2 and 12 weeks gradually methylated. Moreover, the livers at 20 weeks markedly methylated (Fig. 3).

Fig. 1
figure 1

Results of the methylation analysis of the 5′ upstream region of the rat LPA1 gene evaluated by bisulfite sequencing. For each sample from pooled liver tissues of three rats, eight clones were sequenced. The primer pair used for the bisulfite sequencing is indicated by arrow heads. Methylated CpG sites are shown by closed circles, and unmethylated CpG sites are shown by open circles. The first base of exon1 was defined +1

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Fig. 2
figure 2

Results of the methylation analysis of the 5′ upstream region of the rat LPA2 gene evaluated by bisulfite sequencing. For each sample from pooled liver tissues of three rats, eight clones were sequenced. The primer pair used for the bisulfite sequencing is indicated by arrow heads. Methylated CpG sites are shown by closed circles, and unmethylated CpG sites are shown by open circles. The first base of exon1 was defined +1

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Fig. 3
figure 3

Results of the methylation analysis of the 5′ upstream region of the rat LPA3 gene evaluated by bisulfite sequencing. For each sample from pooled liver tissues of three rats, eight clones were sequenced. The primer pair used for the bisulfite sequencing is indicated by arrow heads. Methylated CpG sites are shown by closed circles, and unmethylated CpG sites are shown by open circles. The first base of exon1 was defined +1

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Fig. 4
figure 4

Expressions of LPA receptor genes by semiquantitative RT-PCR analysis. NRL normal liver

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Expression levels of LPA receptor genes in the livers of rats fed the CDAA diet were measured by semiquantitative RT-PCR analysis. Representative results are shown in Fig. 4. No changes in the expression levels of LPA1 and LPA2 genes were found in the livers of rats fed the CDAA diet. By contrast, while LPA3 gene expressed in the livers at 7 days and 2 and 12 weeks as well as normal livers, reduced expression was found in those at 20 weeks.

Since DNA methylation patterns and expression levels of LPA3 gene were found, we next investigated these of LPA3 gene in HCCs induced by the feeding of the CDAA diet for 75 weeks. Four HCCs were highly methylated in the LPA3 gene. The surrounding liver tissue was also highly methylated (Fig. 5a). No expressions of the LPA3 gene were detected in four HCCs and the surrounding liver tissue of HCCs (Fig. 5b).

Fig. 5
figure 5

a Results of the methylation analysis of the 5′ upstream region of the rat LPA3 gene evaluated by bisulfite sequencing in HCCs. For each sample, eight clones were sequenced. Methylated CpG sites are shown by closed circles, and unmethylated CpG sites are shown by open circles. b Representative results of expressions of LPA receptor genes in HCCs by semiquantitative RT-PCR analysis. non-HCC; surrounding liver tissue of HCC

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Discussion

The present study indicated that DNA methylation status of the LPA3 gene was disturbed in the livers of rats fed the CDAA diet and established HCCs, but not LPA1 and LPA2 genes (summarized in Table 1). Recently, we have reported that frequent mutations (41.7% incidence) of the LPA1 gene were detected in rat HCCs induced by the CDAA diet, suggesting that the LPA1 gene mutations may play an important role in the development of tumor cells (Obo et al. 2009). Taken together, it is suggested that alterations of the LPA receptor genes might be involved during rat hepatocarcinogenesis induced by the CDAA diet.

Table 1 Summary for the expression level and DNA methylation status of LPA receptor genes
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Previously, it has been reported that genome-wide hypomethylation of liver DNA occurred during hepatocarcinogenesis induced by a CD diet (Locker et al. 1986). Since the CD diet lacks multiple methyl donors, DNA hypomethylation is considered as one of the mechanisms underlying rat hepatocarcinogenesis resulting from the CD diet (Locker et al. 1986; Poirier 1994; Christman 1995). As far, regional hypomethylation of c-fos, c-myc, and c-Ha-ras genes was detected in livers of rats after short-term feeding with the CD diet (Christman et al. 1993). In our previous report, hypomethylation of c-myc gene was found in HCCs induced by the CDAA diet in rats (Tsujiuchi et al. 1999). By contrast, we have also reported that regional methylation in the 5′ upstream regions of E-cadherin and connexin 26 genes in rat HCCs as a result of the CDAA diet (Tsujiuchi et al. 2006a). Moreover, we also detected that E-cadherin, connexin 26, and Rassf1a genes were methylated in the early phase of hepatocarcinogenesis induced by the CDAA diet (Shimizu et al. 2007). In the present study, although the livers of rats fed the CDAA diet were not methylated in the LPA1 and LPA2 genes, the LPA3 gene showed methylated status, demonstrating that gene-specific methylation changes in LPA receptor genes were also found during hepatocarcinogenesis induced by the CDAA diet.

While global hypomethylation generally occurs in several human cancer cells, CpG islands of tumor suppressor genes are regionally hypermethylated (Laird and Jaenisch 1994). However, despite methyl donor deficiency, it is unclear why site-specific hypermethylation, such as E-cadherin, connexin 26, Rassf1a, and LPA3 genes, occurs in rat hepatocarcinogenesis by the CDAA diet (Tsujiuchi et al. 2006a; Shimizu et al. 2007). In our previous report, the increased expression of DNA methyltransferase 1 (DNMT1) gene was detected in livers of rats fed the CDAA diet (Shimizu et al. 2007). It has been reported that a folate/methyl-deficient diet induced DNA damages, including strand breaks, gaps, and abasic sites, and DNMT1 could bind with high affinity to those lesions (James et al. 2003). Inappropriate binding of the DNMT1 may induce regional hypermethylation in promoter regions of tumor suppressor genes (James et al. 2003). As another possibility, it is suggested that 8-hydroxydeoxyguanosine (8-OHdG), which is a common oxygen radical-induced guanine derivative, could inhibit DNA methylation (Weitzman et al. 1994). In livers of rats fed the CDAA diet, the 8-OHdG is significantly detectable after only 1 day and progressively accumulates at least up to 12 week (Nakae et al. 1990). Therefore, the aberrant DNA methylation pattern might be due to inappropriate binding of the DNMT1 to DNA containing damaged lesions or the accumulation of 8-OHdG in livers of rats fed the CDAA diet (Shimizu et al. 2007).

LPA1 is ubiquitously expressed in normal tissues, but the expressions of LPA2 and LPA3 are relatively restricted, suggesting these receptors may have different biological functions regarding LPA (An et al. 1998; Bandoh et al. 1999; Contos et al. 2000a). LPA could induce a wide range of cellular responses throughout LPA1 or LPA2, such as cell proliferation, phospholipase C activation, intracellular calcium mobilization, and adenylyl cyclase inhibition (Contos et al. 2000a). LPA3 had a critical function in spacing and implantation of embryos in early pregnancy (Lin et al. 2010). In human cancer cells, different expression patterns of the LPA receptor genes and different cellular responses to LPA have been reported (Furui et al. 1999; Fang et al. 2002; Fujita et al. 2003; Shida et al. 2003). While the expression levels of LPA2 and LPA3 genes in ovarian cancer cells were markedly higher than those in normal ovarian epithelial cells, LPA1 gene expression levels were various (Furui et al. 1999; Fang et al. 2002; Fujita et al. 2003). In colon cancer tissues, the expression levels of LPA1 and LPA3 genes were relatively low and the LPA2 gene was overexpressed (Shida et al. 2004). Our recent studies have also indicated various levels of LPA1 and LPA3 expressions in colon cancer cells, but well expressed LPA2, correlating with DNA methylation status (Tsujino et al. 2010). The ovarian cancer cells with high expression of LPA1 indicated lower cell proliferation, cell death through apoptosis, and anoikis to LPA, suggesting that LPA1 may act as a negative regulator for cell growth (Furui et al. 1999; Fang et al. 2002). By contrast, LPA stimulated cell migration, proliferation, and adhesion in the LPA1-expressing colon cancer cells (Shida et al. 2003). Therefore, it is suggested that the expression pattern of LPA receptors may be related to clinical characteristics of cancer cells through the different responses to LPA (Shida et al. 2003, 2004). In the present study, it is unclear why LPA3 gene was gradually methylated during the feeding of the CDAA diet but not LPA1 and LPA2 genes, and whether the loss of LPA3 expression may affect the acquisition of growth advantage of tumor cells. In this context, to better understand the biological significance of LPA3 in carcinogenesis, we are currently investigating functional analyses for LPA3 in several cancer cells with various expression levels of LPA3 gene.